Application of EMD638683 and compound of EMD638683 as therapeutic target in preparation of medicine for treating gastric cancer

Abstract

The invention provides application of EMD638683 and a compound thereof as a therapeutic target to preparation of a medicine for treating gastric cancer, relates to the technical field of biomedicine, and is technically characterized by providing application of EMD638683 (R-Form) as a therapeutic target to preparation of a medicine for treating gastric cancer. According to the application, a specific verification test proves that EMD638683 (R-Form) is targeted to an N40 glycosylation site of LT beta R, a TRIM28 / PCBP2 / SARM1 pathway is specifically blocked, and the off-target toxicity is low. After the cRGD modified liposome is delivered, the tumor specific enrichment can be realized, the bioavailability is improved, the damage to normal organs is reduced, and the safety is good. The medicine can effectively reverse gastric cancer radiotherapy resistance, is adaptive to radiotherapy non-responders with high LT betaR expression, assists in precise stratified treatment, and improves the survival rate of patients. A traditional DNA repair related mechanism is broken through, the sensitization effect is achieved by regulating and controlling a pyroptosis pathway, and a brand new direction is provided for research and development of gastric cancer radiotherapy sensitization drugs.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to the application of EMD638683 (R-Form) and its compounds as therapeutic targets in the preparation of drugs for treating gastric cancer. Background Technology

[0002] Gastric cancer (GC) is the fifth most common malignant tumor worldwide and the fourth leading cause of cancer-related death. Despite significant advances in treatment, overall survival rates for patients with advanced gastric cancer remain unsatisfactory.

[0003] Radiotherapy, as an important treatment for gastric cancer, exerts its anti-tumor effect by inducing DNA damage and triggering regulatory cell death (such as pyroptosis). Among these, pyroptosis mediated by the NLRP3 / Caspase1 / GSDMD pathway is a key step in regulating tumor radiosensitivity. However, in clinical practice, most gastric cancer patients gradually develop resistance during radiotherapy, leading to an increased risk of tumor recurrence and metastasis. The core reason for this is the lack of effective radiosensitivity biomarkers, and existing research mainly focuses on radiotherapy resistance interventions related to DNA repair mechanisms. The development of radiosensitizing targeted drugs targeting other pathways is significantly insufficient, failing to meet the needs of precision clinical treatment.

[0004] Lymphotoxin β receptor (LTβR), a member of the tumor necrosis factor receptor superfamily, has traditionally been considered involved in immune regulation and lymphoid organogenesis. Recent studies have confirmed its high expression in various solid tumors, including gastric cancer, and its close association with tumor proliferation, metastasis, and poor prognosis. Further research has revealed that radiotherapy can induce N-glycosylation modification and nuclear localization of LTβR, promoting PCBP2 SUMOylation and nuclear export through TRIM28 recruitment, enhancing SARM1 translation efficiency, and subsequently inhibiting the pyroptosis pathway, ultimately mediating radioresistance in gastric cancer. This mechanism provides important evidence for the screening of radiosensitization targets. Currently, there are no clinically available radiosensitizing drugs targeting LTβR for gastric cancer. Summary of the Invention

[0005] The purpose of this application is to address the technical shortcomings in the development of radiosensitizing targeted drugs in the existing technology.

[0006] The application of EMD638683 (R-Form) as a therapeutic target in the preparation of drugs for treating gastric cancer.

[0007] Preferably, the EMD638683 (R-Form) is used to reverse radiotherapy resistance in gastric cancer.

[0008] This application also provides a compound, cRGD-Lipo@EMD, for preparing a drug for treating gastric cancer, wherein cRGD-Lipo@EMD is prepared by a rapid mixing method to encapsulate EMD in cRGD-modified liposomes.

[0009] Preferably, the preparation method of the cRGD-Lipo@EMD is as follows:

[0010] A lipid mixture consisting of phospholipids, sterol lipids, DSPE-PEG, and DSPE-PEG-cRGD was dissolved in ethanol; the organic phase was then rapidly mixed with an aqueous solution containing EMD under continuous stirring to induce self-assembly.

[0011] Preferably, the specific preparation method of the cRGD-Lipo@EMD is as follows:

[0012] Weigh out soybean phosphatidylcholine, dioleoylphosphatidylethanolamine-polyethylene glycol-cyclic peptide arginine-glycine-aspartic acid and cholesterol, dissolve them in chloroform, transfer them to a round-bottom flask and evaporate under reduced pressure to form a uniform film of lipids on the flask wall.

[0013] An EMD aqueous solution was added to the lipid membrane, and after thorough vortex hydration, it was subjected to ultrasonic treatment.

[0014] The obtained liposome suspension was repeatedly extruded through a polycarbonate membrane using a liposome extruder to obtain liposomes with uniform particle size.

[0015] The liposome suspension was purified using a nanodialysis device, and the volume was adjusted with deionized water after purification.

[0016] Add an appropriate amount of freeze-drying protectant to the liposome solution, freeze-dry, and store away from light.

[0017] This application also provides the application of cRGD-Lipo@EMD in the preparation of drugs to reverse radiotherapy resistance in gastric cancer.

[0018] A radiosensitizing targeted drug, wherein the drug contains EMD638683 (R-Form).

[0019] Preferably, the drug also includes other medically acceptable adjuvants.

[0020] Compared with the prior art, this application has at least the following beneficial effects:

[0021] 1. This application clarifies that EMD638683 targets the N40 glycosylation site of LTβR, specifically blocking radiotherapy-induced LTβR nuclear localization and subsequent signaling pathways (TRIM28 / PCBP2 / SARM1), avoiding interference with normal cellular DNA repair mechanisms, reducing off-target toxicity, and exhibiting higher therapeutic safety compared to traditional non-specific radiosensitizers.

[0022] 2. cRGD-modified liposomes (cRGD-Lipo@EMD) achieve specific enrichment of EMD in gastric cancer tissue through tumor-targeting design, solving the problems of low bioavailability and uneven distribution of small molecule drugs. While improving the radiosensitization effect, it reduces damage to normal organs such as heart, liver, spleen, lungs and kidneys, and has good in vivo safety.

[0023] 3. The drug provided in this application can reverse radiotherapy resistance in gastric cancer and is suitable for patients with advanced gastric cancer who have poor radiotherapy response, especially for radiotherapy non-responders with high LTβR expression, providing a solution to the current situation of lacking effective radiosensitization targets in clinical practice; at the same time, LTβR can serve as a radiosensitivity biomarker to help patients receive precise stratified treatment, improve the overall efficacy of radiotherapy for gastric cancer and the survival rate of patients. Attached Figure Description

[0024] Figure 1 This document presents the stability and N-glycosylation level detection of LTβR in one embodiment of this application, including the results of Western blot experiments on AGS / HGC-27 cells treated with tunicamycin (A, B) or N40Q mutant (C, D) after radiation treatment (IR).

[0025] Figure 2 The experimental steps and results of the screening are shown below. A shows the screening process; B shows the chemical structure of EMD638683; and C shows the molecular docking results of EMD638683 (R-Form) with the LTβR N40 pocket.

[0026] Figure 3 The results are from surface plasmon resonance (SPR) experiments. A and B represent the binding affinity (dissociation constant KD) and binding kinetics between EMD and LTβR, respectively, as analyzed by surface plasmon resonance (SPR).

[0027] Figure 4 This demonstrates the dose-dependent inhibition of LTβR glycosylation by EMD.

[0028] Figure 5The images show the results of in vitro sensitization experiments in one embodiment of this application, specifically the EdU incorporation assay (A, B), colony formation assay (C, D), and apoptosis detection (E, F) in AGS cells treated with 12.5 μM EMD with or without radiotherapy (IR). Scale bar: 100 μm.

[0029] Figure 6 This invention describes the effect of organoids derived from radioresistant patients (PDO, Patient 1) in one embodiment. Specifically, it includes: bright-field image (A), diameter quantification analysis (B), LTβR expression level (C), EdU incorporation level (D), and viability detection (E). Scale bars: bright-field (BF) 200 μm, immunofluorescence (IF) 50 μm. Data are expressed as mean ± standard deviation (n=3). Statistical methods: one-way or two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, #P > 0.05.

[0030] Figure 7 This is a schematic diagram of the preparation process of cRGD-Lipo@EMD;

[0031] Figure 8 Characterization of the prepared cRGD-Lipo@EMD nanoparticles: specifically, particle size (A), zeta potential (B), polydispersity index (PDI) (C), and morphology under transmission electron microscopy (TEM) (D). Scale bar: 1 μm.

[0032] Figure 9 For cellular uptake experiments, the cellular uptake efficiency of Cy5.5-labeled liposomes at 12 / 24 hours was observed by microscopy (A, B) and detected by flow cytometry (C). Scale bar: 50 μm.

[0033] Figure 10 This is an in vivo distribution experiment. The results of in vivo imaging system (IVIS) at 24 hours (A, B) and quantitative analysis of the ex vivo organs (C, D).

[0034] Figure 11 For in vivo therapeutic experiments. Mice treated with solvent, EMD, cRGD-Lipo, or cRGD-Lipo@EMD with or without radiation (IR) were shown in tumor images (A), tumor weight (B), and tumor growth curves (C).

[0035] Figure 12Immunohistochemical (IHC) staining results of LTβR and Ki-67 in tumor tissue. A represents LTβR, B represents Ki-67, scale bar: 50 μm. Data are expressed as mean ± standard deviation (n=5). Statistical methods: unpaired t-test, one-way or two-way ANOVA. *P<0.05, **P<0.01, ***P<0.001.

[0036] Figure 13 To present the results of the ubiquitination experiment, the ubiquitination levels of wild-type (WT) and N40Q mutant LTβR were detected in AGS cells (A) and HGC-27 cells (B) treated with MG132 and radiation (IR).

[0037] Figure 14 Characterization of UNBS5162: chemical structure (A), molecular docking results with the LTβR N40 pocket (B), and binding kinetics (C) and affinity curves detected by SPR (D).

[0038] Figure 15 This study focuses on gene ontology (GO) enrichment analysis of EMD-related biological processes based on the Comparative Toxicogenomics Database (CTD database).

[0039] Figure 16 Hematoxylin-eosin (H&E) staining results of major organs in mice treated with cRGD-Lipo@EMD. Scale bar: 20 μm. Detailed Implementation

[0040] The following are some English definitions:

[0041] English Explanation EMD This application specifically refers to EMD638683 DSPE-PEG Distearate phosphatidylethanolamine-polyethylene glycol DSPE-PEG-cRGD Distearate phosphatidylethanolamine-polyethylene glycol-cyclic arginine-glycine-aspartic peptide

[0042] This application provides the use of EMD638683 as a therapeutic target in the preparation of drugs for treating gastric cancer, wherein EMD638683 (R-Form) is used to reverse radiotherapy resistance in gastric cancer.

[0043] Based on the above applications, this application also provides a compound cRGD-Lipo@EMD, wherein cRGD-Lipo@EMD is a cRGD-modified liposome encapsulating EMD prepared by a rapid mixing method.

[0044] In one embodiment, the preparation method of the cRGD-Lipo@EMD is as follows:

[0045] A lipid mixture consisting of phospholipids, sterol lipids, DSPE-PEG, and DSPE-PEG-cRGD was dissolved in ethanol; the organic phase was then rapidly mixed with an aqueous solution containing EMD under continuous stirring to induce self-assembly.

[0046] This application also provides the application of the aforementioned cRGD-Lipo@EMD in the preparation of drugs to reverse radiotherapy resistance in gastric cancer.

[0047] The above content will be explained in conjunction with specific verification experiments:

[0048] I. Experimental Materials and Their Sources:

[0049] Serial Number Experimental materials source 1 EMD638683 (R-Form) MedChemExpress 2 Soybean phosphatidylcholine RUIXIBIO 3 Cholesterol RUIXIBIO 4 DOPE–PEG–cRGD RUIXIBIO

[0050] II. Experimental Procedure

[0051] 1. Construction of AGS tumor-bearing mouse model

[0052] AGS cells and HGC-27 cells were resuspended in PBS:Matrix gel (1:1), and 100 μL (containing 5 × 10⁻⁶ cells) was subcutaneously injected into the flank of each mouse. 6 (Each cell). Tumor size was measured with calipers every 3 days, and tumor volume was calculated using the formula (length × width²) / 2. Once the tumor became palpable (day 6 post-inoculation), mice were randomly assigned to different groups for treatment.

[0053] 2. Virtual Filtering:

[0054] This application uses the Schrödinger software package for virtual screening. Human LTβR structures were prepared using the OPLS4 force field, and a receptor grid (20×20×20 Å box) was generated centered on Asn40, with specific hydrogen bond constraints set. A bioactive compound library Plus (MCE, 27,524 compounds) was prepared using LigPrep, and screening was performed sequentially using high-throughput virtual screening (HTVS), standard precision (SP), and ultra-high precision (XP) docking modes, retaining the top 10% of compounds at each stage. Binding free energy was optimized using Prime MM-GBSA. The top 100 candidate compounds satisfying MM-GBSA ΔGbind < -40 kcal / mol and XP GScore < -5 were selected, and then verified using CS-DOCK2 (Vina score < -6 as the threshold). Finally, two lead compounds were selected: EMD638683 and UNBS5162.

[0055] 3. Surface Plasmon Resonance (SPR) Experiment

[0056] SPR experiments were performed using a Biacore X100 instrument (Cytiva) equipped with a CM5 sensor chip. Recombinant human LTβR protein (C328, Novizan Suzhou, China) was immobilized on the activated chip surface using the standard amine coupling method. Small molecule analytes EMD (HY-15193A, MCE) and UNBS5162 (HY-16509, MCE) were diluted to different concentrations with running buffer and injected into the protein-immobilized chip surface. The binding and dissociation phases were monitored in real time, and the equilibrium dissociation constant (KD) was calculated using Biacore X100 Evaluation software.

[0057] 4. Preparation and characterization of cRGD-Lipo@EMD

[0058] cRGD-modified liposomes encapsulating EMD (cRGD-Lipo@EMD) were prepared using a rapid mixing method. Brief steps: A lipid mixture consisting of phospholipids, sterol lipids, DSPE-PEG, and DSPE-PEG-cRGD (molar ratio...) was prepared. Figure 7 The organic phase (as shown) was dissolved in ethanol; under continuous stirring, the organic phase was rapidly mixed with an aqueous solution containing EMD (or Cy5.5 for fluorescence tracking) to induce self-assembly; the resulting liposome suspension was dialyzed against PBS to remove unencapsulated drug and organic solvent; particle size distribution, polydispersity index (PDI) and zeta potential were measured using a ZetaView nanoparticle tracking analyzer (PMX120-Z, Partrex, Germany); the microstructure of the synthesized nanoparticles was characterized by TEM.

[0059] Specifically, the preparation method of the cRGD-Lipo@EMD is as follows:

[0060] Please see Figure 7 Accurately weigh 18 mg of soybean phosphatidylcholine, 1 mg of dioleoylphosphatidylethanolamine-polyethylene glycol-cyclic peptide arginine-glycine-aspartic acid (DOPE-PEG-cRGD), and 1 mg of cholesterol (all purchased from Ruixi Biotechnology, Xi'an), dissolve them in 1 mL of chloroform, transfer to a round-bottom flask, and evaporate under reduced pressure to form a uniform lipid film on the flask wall. Add 1.5 mL of Cy5.5-EMD aqueous solution with a concentration of 1 mg·mL⁻¹ to the lipid film, vortex hydration thoroughly, and then sonicate. Extrude the resulting liposome suspension through a liposome extruder repeatedly through a polycarbonate membrane with a pore size of 100 nm to obtain liposomes with uniform particle size. Purify the liposome suspension using a nanodialysis device equipped with a 30 nm polycarbonate membrane, and adjust the volume to 2 mL with deionized water. Add an appropriate amount of lyophilization protectant to the liposome solution, freeze-dry, and store in the dark.

[0061] The characterization was performed using the following methods:

[0062] The particle size and polydispersity index (PDI) of liposomes were determined using dynamic light scattering (DLS). The zeta potential of liposomes was detected by electrophoretic light scattering. The encapsulation efficiency (EE%) of protein in liposomes was determined using a dioctoctanic acid (BCA) protein quantification kit, calculated as: Encapsulation efficiency = (Total protein amount − Free protein amount) / Total protein amount × 100%. The successful co-loading of EMD and Cy5.5 fluorescent dye into liposomes was verified by measuring UV-Vis absorption and fluorescence emission spectra.

[0063] 5. In vitro cell uptake experiment

[0064] Human gastric cancer cell line AGS cells were co-incubated with cRGD-modified liposomes loaded with Cy5.5-EMD (cRGD-Lipo@Cy5.5-EMD) for 12 / 24 h (incubation conditions: 37°C). After thoroughly washing the cells with PBS to remove untaken nanoparticles, the uptake efficiency of the cells was quantitatively detected by flow cytometry based on the fluorescence intensity of Cy5.5.

[0065] The Cy5.5-EMD preparation method described in the above experiment is as follows:

[0066] The protein was fluorescently labeled using sulfonic acid-Cy5.5 succinimide ester (DuoFluor, catalog number D10020): EMD at a concentration ≥1 mg·mL⁻¹ was dialyzed into coupling buffer at pH 9.0. Fluorescent dye was added at a molar ratio of EMD (EMD638683) to Cy5.5 at 1:5, and the mixture was incubated overnight at 4°C in the dark. Ammonium chloride was added to the reaction system to a final concentration of 50 mmol·L⁻¹, and the reaction was terminated by incubation at 4°C for 2 h. The reaction product was then thoroughly dialyzed against PBS to remove free dye, yielding Cy5.5-EMD. The fluorescently labeled Cy5.5-EMD was finally stored in 1×PBS (pH 7.4) containing 0.1% sodium azide (NaN3) and 1% bovine serum albumin (BSA) at 4°C in the dark.

[0067] 6. In vivo assessment: biodistribution, efficacy and safety

[0068] In vivo distribution experiment:

[0069] To assess the in vivo distribution characteristics of the drug, free Cy5.5-labeled cRGD-Lipo@EMD (administered at a Cy5.5 equivalent dose) was injected into the tail vein of nude mice bearing subcutaneous tumors; whole-body fluorescence images of mice were acquired using an in vivo imaging system at 24 and 18 hours after administration.

[0070] At the end of the experiment, the mice were sacrificed, and major organs such as the heart, liver, spleen, lungs, and kidneys, as well as tumor tissues, were harvested for in vitro fluorescence imaging and quantitative analysis of the fluorescence intensity of each tissue.

[0071] Treatment efficacy evaluation experiment:

[0072] Mice were randomly divided into 6 groups (n=5 in each group): control group, EMD group, radiotherapy alone group, EMD combined with radiotherapy group, cRGD-Lipo combined with radiotherapy group, and cRGD-Lipo@EMD combined with radiotherapy group.

[0073] Dosage regimen: On days 6, 9, 12, and 15 after subcutaneous tumor formation, fractionated local radiotherapy was administered to the tumor area, with a total dose of 6 Gy in 4 fractions; cRGD-Lipo@EMD was administered via tail vein injection at the equivalent EMD dose of 5 mg·kg⁻¹ twice weekly. Throughout the treatment period, tumor volume and body weight changes in mice were monitored regularly.

[0074] Finally, the tumor tissue was dissected, separated, and weighed. The expression levels of LTβR and Ki-67 in the tumor tissue were detected by immunohistochemistry. At the same time, the major organs were collected to prepare pathological sections, and H&E staining was performed to assess the systemic toxicity of the drug.

[0075] 7. Bioinformatics Analysis

[0076] Based on TCGA-STAD data, gene set enrichment analysis (GSEA) was used to identify relevant biological processes. To further elucidate the specific associations of LTβR, TRIM28, PCBP2, and SARM1 with radiotherapy response phenotypes, single-sample GSEA (ssGSEA) was employed, followed by Pearson correlation analysis to assess their linear association with target gene expression levels. Bioinformatics analysis was performed using R software (version 4.3.2). Furthermore, interacting genes of EMD were screened from the Comparative Toxicology Genomics Database (CTD), and gene ontology (GO) enrichment analysis was used to infer the potential biological functions of EMD.

[0077] 8. Statistical Analysis

[0078] Statistical analysis was performed using GraphPad Prism software (version 10.4.2) and R software (version 4.3.1). Quantitative data are expressed as mean ± standard deviation (unless otherwise specified); for comparisons of two independent samples, unpaired t-tests or Mann-Whitney U tests were selected based on the data distribution characteristics; one-way ANOVA was used to analyze differences among multiple groups, followed by Tukey's post-hoc test; two-way ANOVA was used to compare tumor growth curves; a two-sided p-value < 0.05 was defined as statistically significant.

[0079] The routine cell culture, transfection procedures, routine qRT-PCR procedures, standard detection steps of Western blotting, routine flow cytometry analysis, and routine animal model construction (such as basic seeding methods for subcutaneous xenograft tumors) involved in this application are all basic experimental techniques known in the field. The relevant operating procedures have been widely reported and are well known to those skilled in the art, so they are not described in detail in this application.

[0080] III. Experimental Results

[0081] Previous studies, using immunoblotting analysis, revealed that irradiation not only increased LTβR expression but also significantly increased its molecular weight. Since LTβR is a glycoprotein, this study explored whether this change in migration rate reflected alterations in N-glycosylation: tunicamycin treatment eliminated high-molecular-weight LTβR species and prevented their accumulation after irradiation, indicating that irradiation promotes the formation of glycosylated LTβR (G-LTβR). Figure 1 A, B).

[0082] To further clarify the glycosylation sites involved, this application focuses on asparagine 40 (N40), which has been reported to regulate the stability of LTβR. An N40Q mutant was constructed in this application. Unlike wild-type LTβR, the N40Q mutant did not show an increased migration rate after irradiation, and protein accumulation was significantly reduced. Figure 1 C, D); Ubiquitination experiments showed that this reduction was associated with increased polyubiquitination in the N40Q mutant, suggesting enhanced proteasome degradation (C, D). Figure 13 ).

[0083] Given the necessity of N40 glycosylation for LTβR stability, structure-based virtual screening was performed to identify small molecules targeting the N40 region; two candidate molecules (EMD and UNBS5162) were screened from 27,524 compounds in the MCE database. Figure 2 AC; Figure 1 C, D). Surface plasmon resonance analysis showed that EMD had a higher binding affinity to LTβR than UNBS5162, exhibiting dose-dependent binding kinetics, with an equilibrium dissociation constant (KD) of 0.92 μM (C, D). Figure 3 A, B; Figure 14 C, D).

[0084] Notably, GO analysis based on CTD showed that the targets of EMD were enriched in radiation response and cell death pathways. Figure 15 Consistently, EMD treatment reduced the accumulation of G-LTβR in gastric cancer cells after irradiation in a dose-dependent manner. Figure 4 ).

[0085] To assess the functional consequences of LTβR glycosylation inhibition, cellular responses to irradiation were examined in the presence of EMD: EMD treatment significantly reduced colony formation, survival, and proliferation of irradiated gastric cancer cells. Figure 5 AD); Flow cytometry analysis further showed that irradiation alone induced limited cell death, while EMD combined with irradiation significantly increased the apoptotic cell population (AD); Figure 5 E, F).

[0086] The translational relevance of these findings was further evaluated using patient-derived organoids established from a treatment non-responder (Patient 1) with high LTβR expression; immunohistochemical analysis confirmed that LTβR expression decreased after EMD treatment. Figure 6 A, C); Combined EMD irradiation resulted in significant structural damage, decreased proliferative activity, and increased cell death in organoids. Figure 6 AE).

[0087] Secondly, this application employs a rapid mixing technique to construct cRGD-modified liposomes (cRGD-Lipo@EMD) encapsulating EMD, in order to improve targeted delivery efficiency and radiosensitization effect. Figure 7 Dynamic light scattering (DLS) analysis revealed that these liposomes exhibited a uniform particle size distribution, low polydispersity (PDI < 0.3), and a stable negative zeta potential. Figure 8 AC); consistently, transmission electron microscopy (TEM) images showed well-dispersed spherical nanoparticles, consistent with DLS measurement data ( Figure 8 D). Fluorescence microscopy and flow cytometry confirmed that AGS cells could efficiently and time-dependently internalize the liposomes, as evidenced by a significant increase in intracellular fluorescence intensity within 12-24 hours and a marked rightward shift in the flow cytometry histogram. Figure 9 AC).

[0088] Next, the in vivo tumor targeting was assessed by intravenously injecting cRGD-Lipo@Cy5.5-EMD into AGS tumor-bearing mice; real-time IVIS imaging showed that fluorescence signals specifically accumulated in the tumor region within 24 hours. Figure 10 A, B); In vitro imaging of the excised tissue confirmed this trend, showing that the enrichment level in the tumor was significantly higher than in major organs (heart, liver, spleen, lung, kidney) ( Figure 10 C, D); Furthermore, H&E staining of these organs showed no pathological abnormalities, indicating that the liposome preparation has good biocompatibility and no significant systemic toxicity. Figure 16 ).

[0089] In the treatment evaluation, mice bearing xenograft tumors were treated with free EMD, cRGD-Lipo, or cRGD-Lipo@EMD, with or without combined irradiation; although single therapy (EMD alone or irradiation alone) had moderate inhibitory effects, cRGD-Lipo@EMD combined with irradiation produced the strongest antitumor response, significantly reducing tumor volume and weight compared to all other groups. Figure 11 AC); IHC analysis of the resected tumor showed that this combination therapy resulted in the most significant downregulation of LTβR and Ki-67 expression ( Figure 12 A, B).

[0090] In summary, these results indicate that cRGD-Lipo@EMD is an effective targeted radiosensitizer for gastric cancer.

[0091] Through the above verification experiments, this application demonstrates that EMD638683 specifically blocks radiotherapy-induced LTβR nuclear localization and subsequent TRIM28 / PCBP2 / SARM1 signaling pathways by targeting the N40 glycosylation site of LTβR, avoiding interference with normal cellular DNA repair mechanisms and reducing off-target toxicity. Compared with traditional non-specific radiosensitizers, it has higher therapeutic safety. After targeted delivery via cRGD-modified liposomes (cRGD-Lipo@EMD), it can achieve specific enrichment of EMD in gastric cancer tissue, solving the problems of low bioavailability and uneven systemic distribution of small molecule drugs. While improving the radiosensitization effect, it reduces the impact on the heart, liver, spleen, lungs, and kidneys. This drug exhibits good in vivo safety and can effectively reverse radiotherapy resistance in gastric cancer. It is suitable for patients with advanced gastric cancer who have poor radiotherapy response, especially radiotherapy non-responders with high LTβR expression. It provides a solution to the current situation of lacking effective radiosensitizing targets in clinical practice. At the same time, LTβR can serve as a radiosensitivity biomarker to help patients receive precise stratified treatment, thereby improving the overall efficacy of radiotherapy for gastric cancer and the survival rate of patients. In addition, this application breaks through the traditional DNA repair-related radiotherapy resistance mechanism by regulating the NLRP3 / Caspase1 / GSDMD pyroptosis pathway to exert a sensitizing effect, providing a new direction for the development of radiosensitizing drugs for gastric cancer and enriching the intervention strategies for tumor radiotherapy resistance.

Claims

1. Application of EMD638683 (R-Form) as a therapeutic target in the preparation of drugs for treating gastric cancer.

2. The application of EMD638683 as a therapeutic target in the preparation of drugs for treating gastric cancer according to claim 1, characterized in that: The EMD638683 (R-Form) is used to reverse radiotherapy resistance in gastric cancer.

3. A compound, characterized in that: The compound is cRGD-Lipo@EMD, used to prepare drugs for treating gastric cancer. The cRGD-Lipo@EMD is prepared by a rapid mixing method to encapsulate EMD in cRGD-modified liposomes.

4. A compound according to claim 3, characterized in that: The preparation method of the cRGD-Lipo@EMD is as follows: A lipid mixture consisting of phospholipids, sterol lipids, DSPE-PEG, and DSPE-PEG-cRGD was dissolved in ethanol; the organic phase was then rapidly mixed with an aqueous solution containing EMD under continuous stirring to induce self-assembly.

5. A compound according to claim 4, characterized in that: The specific preparation method of the cRGD-Lipo@EMD is as follows: Weigh out soybean phosphatidylcholine, dioleoylphosphatidylethanolamine-polyethylene glycol-cyclic peptide arginine-glycine-aspartic acid and cholesterol, dissolve them in chloroform, transfer them to a round-bottom flask and evaporate under reduced pressure to form a uniform film of lipids on the flask wall. An EMD aqueous solution was added to the lipid membrane, and after thorough vortex hydration, it was subjected to ultrasonic treatment. The obtained liposome suspension was repeatedly extruded through a polycarbonate membrane using a liposome extruder to obtain liposomes with uniform particle size. The liposome suspension was purified using a nanodialysis device, and the volume was adjusted with deionized water after purification. Add an appropriate amount of freeze-drying protectant to the liposome solution, freeze-dry, and store away from light.

6. Application of cRGD-Lipo@EMD in the preparation of drugs to reverse radiotherapy resistance in gastric cancer.

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