Application of ametinib in preparation of lung cancer treatment medicine
By regulating gut microbiota abundance and influencing glutamine metabolism, amitinib has revealed a novel mechanism of action in lung cancer treatment, addressing the indirect effects that have not been thoroughly explored in existing technologies. It significantly inhibits lung cancer cell growth and provides a combination therapy strategy.
Patent Information
- Application Number
- CN202511494350.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-20
- Publication Date
- 2026-02-10
AI Technical Summary
In the current technology, the indirect mechanism of action of ametinib, especially the mechanism of action mediated by other signaling pathways or gut ecology, has not been systematically and thoroughly explored, which affects its efficacy and drug resistance management in the treatment of lung cancer.
Ametinib inhibits the growth of lung cancer cells by regulating the abundance of gut microbiota and affecting glutamine metabolism. Specifically, it upregulates the abundance of species related to glutamine metabolism and downregulates glutamine levels in the host gut and blood, targeting non-small cell lung cancer cell lines carrying EGFR mutations such as PC-9 and H1975.
This study revealed a novel mechanism by which amitinib regulates glutamine metabolism through gut microbiota, providing a new approach to lung cancer treatment. Combined with 16S rRNA and LC-MS technology, the study confirmed that amitinib significantly inhibits the growth and migration of lung cancer cells and delays drug resistance.
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Figure CN121489950A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of amitinib in the preparation of lung cancer treatment drugs. Background Technology
[0002] Lung cancer is one of the leading causes of cancer-related morbidity and mortality worldwide, with non-small cell lung cancer (NSCLC) accounting for the largest proportion. Despite the availability of various treatment options for lung cancer, including surgical resection, radiotherapy, chemotherapy, targeted therapy, and immunotherapy, the incidence and mortality rates continue to rise rapidly. Almonertinib is a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) primarily used to treat advanced or metastatic NSCLC caused by EGFR mutations (T790M or L858R mutations). Clinical observations have revealed varying responses to amonertinib among patients; some patients, even those carrying EGFR-sensitive mutations, still experience unsatisfactory treatment outcomes. Therefore, it is necessary to explore other potential mechanisms to provide more treatment options for improving the efficacy of amonertinib and addressing resistance. However, current research mainly focuses on the direct anti-tumor effects of amonertinib. The direct mechanism of action involves amonertinib selectively inhibiting the tyrosine kinase activity of EGFR mutants, blocking downstream signal transduction in the EGFR signaling pathway, inhibiting cancer cell proliferation, promoting apoptosis, and preventing cancer cell invasion and metastasis. Furthermore, the occurrence and progression of tumors depend not only on the proliferation and apoptosis of cancer cells themselves, but also on a variety of indirect factors such as gut microbiota, tumor microenvironment, immune response, paracrine signaling, and metabolic reprogramming. Although the direct mechanism of action of amitinib has been well elucidated, its indirect mechanisms of action mediated by other signaling pathways or gut ecology have not yet been systematically and thoroughly explored.
[0003] In recent years, the role of gut microbiota in cancer treatment has received widespread attention, as they participate in immune regulation and functional metabolism. Secondly, to maintain rapid proliferation and survival, tumor cells undergo significant metabolic reprogramming, including enhanced glycolysis, abnormal lipid metabolism, and amino acid metabolic remodeling. Amino acid metabolic reprogramming, especially glutamine metabolism, plays a central role in the rapid proliferation and adaptive survival of tumor cells.
[0004] Exploring the role of amitinib in gut microbiota is crucial for a comprehensive understanding of its anti-tumor mechanism and can provide new insights into optimizing combination therapy strategies, delaying the onset of drug resistance, and improving patient prognosis. Therefore, in-depth analysis of amitinib's indirect mechanisms of action has significant scientific research value and clinical implications. Summary of the Invention
[0005] Technical problems to be solved: In view of the shortcomings of the prior art, this application provides an application of ametinib in the preparation of lung cancer treatment drugs, which solves the technical problem that the indirect mechanism of action of ametinib mediated by other signaling pathways or intestinal ecology has not been systematically and deeply explored in the prior art.
[0006] To achieve the above objectives, this application provides the following technical solution: An application of amitinib in the preparation of a lung cancer treatment drug, wherein amitinib inhibits lung cancer by regulating host glutamine metabolism through intestinal flora.
[0007] Furthermore, ametinib inhibits lung cancer by regulating the abundance of gut microbiota associated with glutamine metabolism.
[0008] Furthermore, ametinib acts on non-small cell lung cancer carrying EGFR mutations, upregulates the abundance of species related to glutamine metabolism, downregulates glutamine levels in the host gut and blood, thereby inhibiting the growth of glutamine-dependent lung cancer cells.
[0009] Furthermore, the EGFR mutant cell line is PC-9 and / or H1975.
[0010] Furthermore, the lung cancer mentioned is non-small cell lung cancer (NSCLC).
[0011] A drug for treating lung cancer, comprising amitinib and pharmaceutically acceptable excipients.
[0012] Furthermore, the drug for treating lung cancer is in oral or non-oral form.
[0013] Furthermore, the drug for treating lung cancer is a pharmaceutically permissible injection, powder, ointment, transdermal patch, tablet, capsule, powder, pill, granule, solution, suspension, syrup, suppository, inhaler, or spray.
[0014] Furthermore, the excipients include one or more of the following: fillers, stabilizers, diluents, adjuvants, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorbent carriers, and lubricants.
[0015] This application provides an application of amitinib in the preparation of lung cancer treatment drugs, which has the following advantages compared with the prior art: 1. To reveal a novel mechanism by which amitinib inhibits lung cancer development by regulating glutamine metabolism through gut microbiota; 2. Combining 16S rRNA, LC-MS and other technologies to conduct in vivo and in vitro experimental verification provides new ideas for the preparation of lung cancer treatment drugs. Attached Figure Description
[0016] Figure 1 Volcano plot of differential metabolites between the ametinib treatment group and the subcutaneous tumor model group in this application; Figure 2 This is a KEGG pathway enrichment map of differentially expressed metabolites between the amitinib treatment group and the subcutaneous tumor model group in this application. Figure 3 LEfSe analysis diagram of significantly different microbial communities between the amitinib treatment group and the subcutaneous tumor model group in this application; Figure 4 This application provides a heatmap of the abundance distribution of differentially enriched metabolites in the aminoacyl-tRNA biosynthetic pathway across groups, based on KEGG annotations. Figure 5 Spearman correlation analysis diagram of the differentially enriched microbiota and differentially enriched metabolites in the aminoacyl-tRNA biosynthesis pathway in the amitinib group of this application. Figure 6 This is a graph showing the survival rate of PC-9 lung cancer cells detected by CCK8 under the condition of single amino acid deficiency in this application. Figure 7 This is a graph showing the survival rate of H1975 lung cancer cells detected by CCK8 under the condition of single amino acid deficiency according to this application. Figure 8 This application describes the detection of glutamine (Gln) levels in tumor tissues and serum of mice in the subcutaneous tumor model group and the amitinib group using ELISA. Figure 9 This application describes the detection of glutaminase (GLS) protein expression levels in tumor tissues of mice in the subcutaneous tumor model group and the amitinib group using Western blotting. Figure 10 This application aims to detect the protein expression level of glutaminase in PC-9 and H1975 lung cancer cells directly treated with amitinib in vitro using Western Blot. Figure 11 This application describes the detection of glutaminase protein expression levels in PC-9 and H1975 lung cancer cells under glutamine deficiency using Western blotting. Figure 12 The image shows the cell viability results of PC-9 lung cancer cells treated with different concentrations of glutamine for 24h, 48h and 72h, as determined by CCK8 assay in this application. Figure 13 The image shows the cell viability results of PC-9 lung cancer cells treated with different concentrations of glutamine for 24h, 48h and 72h, as determined by CCK8 assay in this application. Figure 14This image shows the results of Transwell assay of the migration and invasion ability of PC-9 lung cancer cells treated with specified concentrations (CTRL of 2 mM, 150 μM, and 0 μM) of glutamine for 48 h in this application. Figure 15 This image shows the results of Transwell assay of the migration and invasion ability of PC-9 lung cancer cells treated with specified concentrations (CTRL of 2 mM, 150 μM, and 0 μM) of glutamine for 48 h in this application. Figure 16 The image shows the cell cloning ability of PC-9 and H1975 lung cancer cells treated with glutamine at the specified concentrations (CTRL: 2 mM, 150 μM, 0 μM) for 48 h. Figure 17 The images show the tumor morphology of the PC-9 subcutaneous tumor model group, the group treated with a specified concentration of amitinib alone, and the group treated with a specified concentration of amitinib and glutamine. Figure 18 The figures show the tumor volume and weight statistics of the PC-9 subcutaneous tumor model group, the group treated with a specified concentration of amitinib alone, and the group treated with a specified concentration of amitinib and glutamine. Detailed Implementation
[0017] The following detailed descriptions are exemplary and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, components, and / or combinations thereof. Experimental methods in the following embodiments that do not specify specific conditions employ conventional techniques in this art or follow the conditions recommended by the manufacturer; reagents or instruments whose manufacturers are not specified are all commercially available conventional products. To enable those skilled in the art to more clearly understand the technical solutions of the present invention, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
[0018] Example 1: An application of amitinib in the preparation of a lung cancer treatment drug, wherein amitinib upregulates the abundance of species related to glutamine metabolism.
[0019] Amitinib inhibits lung cancer by upregulating the abundance of species related to amino acid metabolism, especially glutamine metabolism, in the gut; the EGFR mutant cell lines are PC-9 and / or H1975.
[0020] Fecal 16S rRNA and LC-MS sequencing analysis of PC-9 tumor-bearing mice: Experimental methods: To establish a PC-9 subcutaneous tumor model, PC-9 cells (5×10⁻⁶) were used. 6 Each tumor cell (one per mouse) was resuspended in PBS and injected subcutaneously into the right axilla of nude mice. The tumors were allowed to grow to 100 mm. 3 Mice were divided into three groups: a subcutaneous tumor model group (Model), a low-concentration amitinib treatment group (Almonrtinib-L), and a high-concentration amitinib treatment group (Almonertnib-H). The mice were treated with saline or amitinib (2 mg / kg or 5 mg / kg) by gavage, respectively. Approximately 200 mg of fresh fecal samples were collected upon sacrifice and flash-frozen in liquid nitrogen. Total bacterial DNA was extracted from the fecal samples. Microbial diversity sequencing was performed using the Illumina next-generation high-throughput sequencing platform to sequence the V1-V9 regions of 16S rRNA, identifying dominant, rare, and some unknown species in the samples to obtain the composition and relative abundance of the microbial community. Samples were analyzed using ultra-high performance liquid chromatography (UHPLC) coupled with high resolution mass spectrometry (LC-MS) for non-targeted metabolomics research. The experimental procedure mainly included: metabolite extraction from the samples, LC-MS / MS detection, and data analysis. First, the ratios corresponding to the quantitative values of differentially expressed metabolites were calculated and logarithmic transformations were performed to base 2. Then, the p-values of the T-test between groups of metabolites were calculated and the absolute values of the logarithmic transformations to base 10 were used to present the differential analysis results of all differentially expressed metabolites.
[0021] Experimental results: (1) Differential bacterial communities between the subcutaneous tumor model group and the amitinib treatment group were determined by 16S rRNA sequencing technology, such as Figure 3 As shown, the blue portion represents the bacterial flora enriched in the ametinib group, and the red portion represents the bacterial flora enriched in the subcutaneous tumor model group. LEfSe analysis results show... Anaerocolumna_sp0007029, Enterocloster_clostridioformis, Lacrimispora_ sp000526-575, Anaerosacchariphilus sp011959465, Anaerotruncus colihominis DSM 17241, Frankia sp902812705, Lacrimispora indolis, Longicatena caecimuris, Marvinbryantia_uncultured bacterium Species such as [list of species] were significantly enriched in the ametinib group.
[0022] (2) The upregulated and downregulated metabolites in the feces of mice in the subcutaneous tumor model group and the amitinib group were identified by LC-MS non-targeted metabolomics, such as Figure 1 As shown, this plot displays all differentially regulated metabolites (upregulated or downregulated) between the amitinib group and the subcutaneous tumor model group. The KEGG pathway enrichment map is shown below. Figure 2 As shown, the aminoacyl-tRNA biosynthesis pathway exhibited significant differences between the two groups, and was significantly downregulated in the ametinib group. According to KEGG annotations, as... Figure 4 As shown, the abundance heatmap of metabolites enriched in the aminoacyl-tRNA biosynthesis pathway between groups reveals that the six amino acids enriched in this pathway—glutamine, tryptophan, asparagine, phenylalanine, histidine, and methionine—were significantly downregulated in the ametinib group. This application hypothesizes that changes in the gut microbiota led to the significant downregulation of these amino acid metabolites in the ametinib group. Therefore, Spearman association analysis was used to explore the correlation between the differentially enriched gut microbiota in the ametinib group and the six amino acid metabolites. Figure 5 As shown, it was found Anaerosacchariphilus sp011959465, Anaerotruncus colihominis DSM 17241, Frankia sp902812705, Lacrimispora indolis, Longicatena caecimuris, Marvinbryantia_uncu- ltured bacterium These species showed a significant negative correlation with six amino acid metabolites, especially glutamine, suggesting they may be the main contributors to glutamine metabolism.
[0023] Example 2: Application of amitinib in the preparation of a lung cancer treatment drug, where amitinib regulates host glutamine metabolism: 1. Experimental Methods: PC-9 and H1975 lung cancer cells were treated in a single amino acid-deficient medium for 48 h. Cell absorbance at 450 nm was measured using a CCK8 assay, and cell viability was calculated. After anesthetizing mice, blood was collected from the orbital cavity, and the supernatant was frozen at -80℃. Tumor tissue samples were collected and frozen at -80℃. Glutamine levels in serum and tumors of mice in the subcutaneous tumor model group and the amitinib group were determined using a glutamine content detection kit. Western blotting was used to detect glutaminase expression in tumor tissues of mice in the subcutaneous tumor model group and the amitinib group. The main steps included sample preparation, gel electrophoresis, transfer to a membrane, blocking, and antibody incubation / detection.
[0024] 2. Experimental Results: (1) This application compared the effects of the deficiency of six single amino acids on the survival of lung cancer cells at the cellular level. The CCK8 experiment results showed that in PC-9 cells, compared with the control group, the cell survival rate was significantly reduced when glutamine and phenylalanine were deficient in the culture medium alone. However, the cell survival rate was lowest when glutamine was deficient alone, while the cell survival rate did not change significantly when other amino acids were deficient alone. In H1975 cells, compared with the control group, the cell survival rate was significantly reduced when glutamine was deficient in the culture medium alone, while the cell survival rate did not change significantly when other amino acids were deficient alone. Figure 6 This image shows the survival rate of PC-9 lung cancer cells detected by CCK8 in cases of single amino acid deficiency. Figure 7 The image shows the survival rate of H1975 lung cancer cells detected by CCK8 assay when a single amino acid is deficient. It is speculated that lung cancer cell survival is more sensitive to glutamine deficiency than to the deficiency of other amino acids, and some literature has reported that lung cancer cells are "addicted" to glutamine.
[0025] (2) This application used ELISA to measure the changes in glutamine levels in the blood and tumor tissues of mice in the subcutaneous tumor model group and the amitinib group. It was found that compared with the subcutaneous tumor model group, the glutamine levels in the blood and tumor tissues of mice in the amitinib group were significantly decreased, indicating that amitinib significantly downregulated host glutamine levels. Figure 8 As shown, the levels of glutamine (Gln) in tumor tissues and serum of mice in the subcutaneous tumor model group and the amitinib group were detected by ELISA.
[0026] (3) Glutamine deficiency played a synergistic anti-tumor role in tumor growth in the amitinib group. After amitinib treatment, the expression level of glutaminase protein in tumor tissues was significantly reduced, such as Figure 9 As shown, Western blotting was used to detect the protein expression level of glutaminase (GLS) in tumor tissues of mice in the subcutaneous tumor model group and the amitinib group. Furthermore, glutaminase protein expression was significantly reduced in PC-9 and H1975 mice treated with glutamine deficiency. When PC-9 and H1975 mice were directly treated with amitinib, there was no significant change in glutaminase protein expression compared to the control group, indicating that amitinib does not directly regulate glutaminase protein expression in lung cancer cells, but rather affects tumor glutaminase expression by downregulating glutamine levels in the host body. Figure 10 As shown, the protein expression level of glutaminase in PC-9 and H1975 lung cancer cells was detected by Western blotting. Figure 11 As shown, the protein expression level of glutaminase in PC-9 and H1975 lung cancer cells under glutamine deficiency was detected by Western blotting.
[0027] Example 3: An application of amitinib in the preparation of a lung cancer treatment drug, examining the effect of glutamine deficiency on lung cancer cell survival, proliferation and migration, and invasion: 1. Experimental Methods: CCK8 assay for cell viability: PC-9 and H1975 cells were seeded in 96-well plates and treated with amitinib at specified concentrations for 24h, 48h, and 72h, respectively. CCK8 reagent was then added, and the cells were incubated for 1–4 hours. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. Cell cloning assay: 1000 cells / well were seeded in 6-well plates and treated with amitinib at specified concentrations for 48h. After culturing for 10–14 days, visible clonal clusters were formed. Cells were fixed with paraformaldehyde and stained with crystal violet. The number of cell clones was counted to assess cell proliferation. Transwell migration / invasion assay: PC-9 and H1975 cells were resuspended in serum-free RPMI-1640 medium and seeded in the upper chamber of a Transwell plate. RPMI-1640 medium containing 10% serum was added to the lower chamber to induce cell migration or invasion. After treatment with amitinib at specified concentrations for 48 hours, the cells were fixed and stained. The number of migrating / invading cells was counted under a microscope.
[0028] 2. Experimental Results: The CCK8 assay results showed that glutamine deficiency significantly reduced the survival rate of PC-9 and H1975 cells, indicating that glutamine deficiency can significantly inhibit the survival and proliferation of lung cancer cells, and this inhibitory effect is dose- and time-dependent. Figure 12 and 13 The image shows the cell viability results of PC-9 and H1975 lung cancer cells treated with different concentrations of glutamine for 24h, 48h, and 72h, as detected by CCK8 assay. Cell cloning experiments showed that glutamine deficiency significantly reduced the number of cell clones compared to the control group, indicating that glutamine deficiency significantly inhibits the proliferation of lung cancer cells. Figure 16 The figure shows the cell clonal capacity of PC-9 and H1975 lung cancer cells after treatment with specified concentrations (CTRL: 2 mM, 150 μM, 0 μM) of glutamine for 48 h. Transwell results show that glutamine deficiency significantly reduced the number of migrating and invasive PC-9 and H1975 cells, significantly inhibiting the migration and invasion abilities of lung cancer cells. Figure 14 and Figure 15 The figure shows the results of Transwell assay to detect the migration and invasion abilities of PC-9 and H1975 lung cancer cells treated with specified concentrations (CTRL of 2mM, 150μM, and 0μM) of glutamine for 48 hours.
[0029] Example 4: An application of amitinib in the preparation of a lung cancer treatment drug, testing whether glutamine reuptake can reverse the antitumor effect of amitinib (Alm) in vivo: 1. Experimental Methods: PC-9 cell suspension was inoculated into the axilla of 6-week-old BALB / c-nu athymic nude mice. Tumors were allowed to grow to 100 mm. 3Mice were divided into three groups: a subcutaneous tumor model group (Model), an amitinib treatment group (Alm), and an amitinib + glutamine treatment group (Alm+Gln). The mice were administered saline, amitinib, and amitinib + glutamine via gavage / intraperitoneal injection, respectively, for 12 days. At the end of the experiment, all mice were euthanized, and tumor tissue was isolated, weighed, and frozen at -80°C or fixed in 4% paraformaldehyde. Tumor volume was measured as length × width. 2 / 2.
[0030] Ethical Statement: All studies were approved by the Institutional Animal Care and Therapy Committee of Jiangsu Normal University. Animal experimental procedures, including treatment, care, and endpoint selection, followed the guidelines for in vivo experiments in animal research reporting and were conducted on a randomized basis. Ethics Review Number: JSNU-IACUC-2025010.
[0031] 2. Experimental Results: From the tumor morphology observation images, it is clearly visible that compared to the subcutaneous tumor model group, the tumors in mice treated with amitinib were significantly smaller. The tumors in the amitinib + glutamine group were generally larger than those in the amitinib group. Figure 17 The image shows morphological observations of tumors in the PC-9 subcutaneous tumor model group, the group treated with a specified concentration of amitinib alone, and the group treated with a specified concentration of amitinib and glutamine together. From the overall trend of tumor volume statistics, the tumor volume in the amitinib + glutamine group was larger than that in the amitinib group, with a significant difference at day 12 of treatment. The tumor weight in the amitinib + glutamine group was also significantly greater than that in the amitinib group, indicating that glutamine reuptake significantly reversed the inhibitory effect of amitinib on mouse tumors, suggesting that amitinib has an anti-tumor effect through host glutamine metabolism. Figure 18 The figure shows the tumor volume and weight statistics of the PC-9 subcutaneous tumor model group, the group treated with a specified concentration of amitinib alone, and the group treated with a specified concentration of amitinib and glutamine.
[0032] The present invention has been described above by way of example. It should be noted that any simple modifications, alterations or other equivalent substitutions that can be made by those skilled in the art without creative effort without departing from the core of the present invention fall within the protection scope of the present invention.
Claims
1. An application of amitinib in the preparation of a lung cancer treatment drug, characterized in that, Ametinib inhibits lung cancer by regulating glutamine metabolism through gut microbiota.
2. The application of amitinib in the preparation of a lung cancer treatment drug according to claim 1, characterized in that: Ametinib inhibits lung cancer by regulating the abundance of gut microbiota related to amino acid metabolism, thereby affecting glutamine metabolism.
3. The application of amitinib in the preparation of a lung cancer treatment drug according to claim 2, characterized in that: Ametinib acts on non-small cell lung cancer carrying EGFR mutations, causing an increase in gut microbiota related to amino acid metabolism, thereby reducing gut glutamine levels and restricting the growth of glutamine-dependent lung cancer cells.
4. The application of amitinib according to claim 3 in the preparation of a lung cancer treatment drug, characterized in that: The EGFR mutant cell lines are PC-9 and / or H1975.
5. The application of amitinib according to claim 1 in the preparation of a lung cancer treatment drug, characterized in that: The lung cancer mentioned is non-small cell lung cancer.
6. The application of amitinib according to claim 1 in the preparation of a lung cancer treatment drug, characterized in that: The lung cancer mentioned is lung adenocarcinoma.
7. A drug for treating lung cancer, characterized in that: It contains amitinib and pharmaceutically acceptable excipients.
8. The drug for treating lung cancer according to claim 7, characterized in that: The medication for treating lung cancer is available in oral or non-oral formulations.
9. The drug for treating lung cancer according to claim 7, characterized in that: The drugs for treating lung cancer are pharmaceutically permissible injections, powders, ointments, transdermal patches, tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, suppositories, inhalers, or sprays.
10. The medicament for treating lung cancer according to claim 7, characterized in that: The excipients include one or more of the following: fillers, stabilizers, diluents, adjuvants, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorbent carriers, and lubricants.