Use of ampakine compounds for the preparation of a medicament for the treatment of gastric cancer peritoneal metastasis

Abstract

This invention relates to the application of AMPA receptor antagonists in the preparation of drugs for treating peritoneal metastases of gastric cancer, belonging to the field of biomedical technology. This invention discloses the application of GRIA2 as a drug target in the preparation of drugs for the prevention or treatment of peritoneal metastases of gastric cancer. It discloses the use of AMPA receptor antagonists NBQX and / or Selurampanel in the preparation of drugs for the prevention or treatment of peritoneal metastases of gastric cancer. NBQX and Selurampanel can significantly inhibit the migration, invasion, spheroidization ability, and adhesion to mesothelial cells of gastric cancer cells; in vivo experiments have confirmed that AMPA receptor antagonists can significantly reduce tumor burden and the number of metastatic nodules in a mouse model of peritoneal metastases of gastric cancer. Patient-derived organoid xenograft models further demonstrate that AMPA receptor antagonists have significant efficacy against peritoneal metastases of gastric cancer with high GRIA2 expression. This invention provides a new treatment strategy for peritoneal metastases of gastric cancer.

Description

Technical Field

[0001] This invention relates to the application of an AMPA receptor antagonist in the preparation of a drug for treating peritoneal metastasis of gastric cancer, belonging to the field of biomedical technology. Background Technology

[0002] Gastric cancer is the fourth leading cause of cancer death worldwide. The peritoneum is the most common site of metastasis for gastric cancer, with approximately 10%-21% of patients presenting with peritoneal metastases at initial diagnosis. The median survival for patients with peritoneal metastases from gastric cancer is only 3-6 months, indicating a very poor prognosis. Although modern multimodal treatments, including systemic chemotherapy, intraperitoneal chemotherapy, and hyperthermic intraperitoneal chemotherapy (HIPEC), have shown benefits in some studies, large-scale evidence-based trials have not yet demonstrated significant improvements in long-term survival. Therefore, current treatment strategies remain insufficient, and a deeper understanding of the molecular mechanisms of peritoneal metastasis in gastric cancer and the discovery of feasible therapeutic targets remain crucial. Dysregulation of the Wnt / β-catenin signaling pathway is closely associated with tumor stem cell characteristics, malignant proliferation, epithelial-mesenchymal transition (EMT)-driven invasion, and treatment resistance. In this signaling pathway, glycogen synthase kinase-3β (GSK3β) is a major negative regulator: GSK3β constitutively phosphorylates β-catenin, causing it to be labeled for proteasome degradation. Inhibition of GSK3β kinase activity stabilizes β-catenin and releases a TCF / LEF-dependent transcriptional program, promoting a proliferative and invasive phenotype. However, the mechanism by which peritoneal microenvironment signaling directly inhibits GSK3β to promote peritoneal dissemination of gastric cancer remains unclear. GRIA2, also known as the AMPA-like subunit 2 of the glutamate ionotropic receptor, encodes the GluR-2 protein, a key subunit of the tetrameric AMPA receptor mediating rapid excitatory synaptic transmission in the central nervous system. Notably, GluR-2 critically determines the calcium permeability of the AMPA receptor: RNA-edited GluR-2 prevents calcium ion penetration, while unedited GluR-2 allows calcium influx. In recent years, increasing evidence suggests that ionotropic glutamate receptors, including the AMPA receptor subunit, are expressed and functionally relevant in various epithelial malignancies, including thyroid cancer, lung cancer, breast cancer, colon cancer, gastric cancer, as well as melanoma and hepatocellular carcinoma. Functional studies have shown that AMPA receptors promote tumor cell proliferation in a calcium influx-dependent manner. However, the role of GRIA2 in peritoneal metastasis of gastric cancer remains unexplored. NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzoquinoxaloline-7-sulfonamide) is a selective competitive AMPA receptor antagonist originally designed for the treatment of epilepsy. Selurampanel (BGG492) is another AMPA receptor competitive antagonist that has been tested in small clinical trials and shown reasonably acceptable safety. However, there are currently no studies on the use of AMPA receptor antagonists for the treatment of peritoneal metastasis of gastric cancer. Summary of the Invention

[0003] The purpose of this invention is to solve the technical problem of how to obtain a drug that inhibits peritoneal metastasis of gastric cancer.

[0004] To address the aforementioned problems, this invention provides the application of GRIA2 as a drug target in the preparation of drugs for the prevention or treatment of peritoneal metastasis of gastric cancer.

[0005] This invention provides the application of an AMPA receptor antagonist in the preparation of a drug for the prevention or treatment of peritoneal metastasis of gastric cancer.

[0006] Preferably, the AMPA receptor antagonist works by inhibiting GRIA2-mediated calcium ion influx.

[0007] Preferably, the AMPA receptor antagonist works by inhibiting GSK3β-Ser9 phosphorylation and reducing the stability of β-catenin protein.

[0008] Preferably, the AMPA receptor antagonist inhibits the interaction between GRIA2 and GSK3β by blocking GRIA2-mediated calcium ion influx, thereby relieving the inhibition of GSK3β kinase activity, promoting β-catenin phosphorylation and degradation, inhibiting the activation of the Wnt / β-catenin signaling pathway, and ultimately inhibiting peritoneal metastasis of gastric cancer.

[0009] Preferably, the AMPA receptor antagonist includes NBQX and / or Selurampanel.

[0010] Preferably, the drug is used in patients with peritoneal metastases from gastric cancer who have high GRIA2 expression.

[0011] This invention provides the use of an AMPA receptor antagonist in the preparation of a drug that inhibits the migration, invasion, stem cell-like characteristics, and / or adhesion of gastric cancer cells to peritoneal mesothelial cells.

[0012] Preferably, the AMPA receptor antagonist includes NBQX and / or Selurampanel.

[0013] Preferably, the dosage form of the drug includes tablets, powders, granules, capsules, oral liquids, injections, or sustained-release formulations.

[0014] The present invention provides a gastric cancer treatment system, the treatment system comprising: a drug delivery system; the drug delivery system comprising an AMPA receptor antagonist.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This invention, through extensive experiments, has demonstrated that AMPA receptor antagonists can effectively inhibit peritoneal metastasis of gastric cancer. In in vitro cell studies, NBQX and Selurampanel dose-dependently inhibited the migration and invasion of gastric cancer cells; suppressed their spheroidization ability; reduced tumor stem cell characteristics; and decreased adhesion between gastric cancer cells and peritoneal mesothelial cells. Mechanistic studies show that AMPA receptor antagonists inhibit the activation of the Wnt / β-catenin signaling pathway by blocking GRIA2-mediated calcium ion influx, inhibiting GSK3β-Ser9 phosphorylation, and reducing β-catenin protein stability. In vivo animal studies, in a mouse peritoneal metastasis model, treatment with NBQX and Selurampanel significantly reduced the intensity of intraperitoneal bioluminescent signals and the number of metastatic nodules. Patient-derived organoid xenograft models further demonstrate that AMPA receptor antagonists have selective efficacy against peritoneal metastases of gastric cancer with high GRIA2 expression. This invention provides a novel treatment strategy for peritoneal metastasis of gastric cancer, and GRIA2 can serve as a biomarker for precision treatment. Attached Figure Description

[0017] Figure 1 Figure showing the in vivo validation results of GRIA2 promoting peritoneal metastasis of gastric cancer;

[0018] Figure 2 Figure showing the experimental results of GRIA2 promoting gastric cancer cell migration, invasion, stemness, and mesothelial adhesion in a glutamate-dependent manner;

[0019] Figure 3 Figure showing the experimental results of a study on the mechanism by which GRIA2 promotes peritoneal metastasis by activating the Wnt / β-catenin signaling pathway;

[0020] Figure 4 The figure shows the experimental results of GRIA2 directly interacting with GSK3β kinase in a glutamate- and calcium-dependent manner and inhibiting its activity.

[0021] Figure 5 Figure 1 shows the results of in vitro and in vivo experiments on the inhibition of peritoneal metastasis of gastric cancer by AMPA receptor antagonists;

[0022] Figure 6 Figure showing the results of a study on the activation of GRIA2 signaling by glutamate secretion from tumor-associated fibroblasts. Detailed Implementation

[0023] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings:

[0024] Unless otherwise specified, all reagents and materials used in this invention are commercially available. All data processing in this invention was performed using the GraphPad Prism software package, with p < 0.05 considered statistically significant.

[0025] Example 1

[0026] Genome-wide CRISPR screening identified GRIA2 as a key driver of peritoneal metastasis in gastric cancer.

[0027] 1. Genome-scale CRISPR library screening:

[0028] Lentiviral transduction of MKN74 cells was performed using a human whole-genome CRISPR knockout (GeCKO) v2 library (containing 123,411 sgRNAs) at an MOI of 0.3. After selection and amplification with puromycin, 1×10⁶ cells were collected. 8 One cell was used as a pre-injection control sample. Thirty-six nude mice were used for in vivo screening and randomly divided into three replicate groups (n=12 per group). Each mouse received an intraperitoneal injection of 1×10⁻⁶ cells. 7 Transduced cells were collected. At the experimental endpoint, all visible peritoneal metastases were collected for genomic DNA extraction and sgRNA sequencing analysis.

[0029] 2. Results Analysis

[0030] The MAGeCK-RRA algorithm was used to analyze sgRNA enrichment and depletion. 201 significantly depleted genes were identified (P < 0.01, log2 fold change ≤ -2, and ≥ 3 congruent sgRNAs). Depleted genes represent candidate metastasis drivers. Among the candidate genes, GRIA2 showed a consistent association with poor prognosis in Kaplan-Meier analysis (probe 236538_at: HR = 1.6, P = 3.6e-05; probe 205358_at: HR = 1.34, P = 0.00084). GRIA2 encodes the core subunit of the AMPA-type glutamate receptor, and its function in peritoneal metastasis of gastric cancer has not been previously explored.

[0031] Example 2

[0032] In vivo validation of GRIA2 promoting peritoneal metastasis of gastric cancer

[0033] 1. Cell line construction

[0034] MKN74 and MKN45 cells, which highly express endogenous GRIA2, were selected for knockout studies, while YTN16, a mouse gastric cancer cell line with low GRIA2 expression, was selected for overexpression studies. Stable GRIA2 knockout (sgMKN74, sgMKN45) or overexpression (oeYTN16) cell lines were constructed using CRISPR-Cas9 technology and labeled with luciferase.

[0035] 2. Animal models

[0036] Human gastric cancer cells (sgMKN74 and sgMKN45) were intraperitoneally injected into nude mice to establish a xenograft model. Mouse gastric cancer cells (oeYTN16) were implanted into homologous C57BL / 6J mice to assess tumor behavior in the immune-intact peritoneal microenvironment. The bioluminescence signal intensity of intraperitoneal tumors was monitored periodically using IVIS imaging.

[0037] 3. Results

[0038] GRIA2 knockout significantly reduced intraperitoneal bioluminescence signal intensity and the number of disseminated nodules (P<0.01). Conversely, mice injected with GRIA2-overexpressing tumor cells showed significantly increased intraperitoneal bioluminescence signal intensity and tumor burden (P<0.001). These data validated the CRISPR knockout screening results and confirmed that increased GRIA2 expression promotes peritoneal metastasis of gastric cancer.

[0039] Example 3

[0040] AMPA receptor antagonists inhibit malignant phenotypes of gastric cancer cells.

[0041] 1. Experimental Design

[0042] MKN74 and MKN45 cells were treated with two selective competitive AMPA receptor antagonists, NBQX and Selurampanel. NBQX and Selurampanel were dissolved in DMSO to prepare stock solutions, which were then diluted in culture medium to specified final concentrations (0, 5, 10, 20, 50 μM), with a final DMSO concentration of 0.1% (v / v).

[0043] 2. Migration and Invasion Experiments

[0044] Cell migration and invasion were assessed using Transwell chambers (8 μm pore size). In the invasion assay, the upper chamber membrane was pre-coated with Matrigel. Cells were resuspended in serum-free medium and seeded in the upper chamber, while the lower chamber was supplemented with medium containing 10% FBS as a chemokine. After culturing for 24 hours (migration) or 48 hours (invasion), cells were fixed, stained, and counted.

[0045] 3. Pelletization Experiment

[0046] Cells (1×10) 4 (Numbers / well) were inoculated into ultra-low adsorption plates, and the culture medium was DMEM / F12 supplemented with 20 ng / mL recombinant EGF, 10 ng / mL recombinant bFGF and 1×B27. After 14 days of culture, the number and size of the spheres were analyzed.

[0047] 4. Tumor-mesothelial cell adhesion assay

[0048] Human peritoneal mesothelial cells (HMrSV5) were seeded in 96-well plates and cultured overnight until confluence. Gastric cancer cells were labeled with 15 μM calcein AM for 30 minutes (5 × 10⁻⁶ cells / well). 4 Mesothelial cell monolayers were added to cells / well and co-cultured for 3 hours. After washing, the cells were observed, photographed, and the number of adhered tumor cells was counted.

[0049] 5. Results

[0050] NBQX and Selurampanel significantly inhibited the migration and invasion abilities of MKN74 and MKN45 cells in a dose-dependent manner (P<0.05 to P<0.001). NBQX showed a stronger inhibitory effect compared to Selurampanel. Similarly, NBQX and Selurampanel significantly reduced the spheroidization ability of gastric cancer cells compared to the solvent control (P<0.01). Furthermore, NBQX and Selurampanel treatment reduced the adhesion of gastric cancer cells to HMrSV5 cells (P<0.01).

[0051] Example 4

[0052] Efficacy evaluation of AMPA receptor antagonists in patient-derived organoid models

[0053] 1. Patient-derived organoid culture

[0054] Tumor specimens were obtained from patients with peritoneal metastases from gastric cancer who expressed high or low GRIA2. After digestion and lysis of red blood cells, the tumor tissue was resuspended in matrix gel and seeded into 24-well plates, and maintained in culture with complete gastric cancer culture medium.

[0055] 2. Drug treatment

[0056] Organoids were treated with NBQX (50 μM) or Selurampanel (50 μM) to observe morphological changes. The expression of GluR2 and β-catenin proteins was detected by immunohistochemistry.

[0057] 3. Results

[0058] Morphological examination revealed that GRIA2-high expression organoids developed invasive structures with neurite-like branching, while GRIA2-low expression organoids formed smoother, epithelial-like spherical structures. Notably, treatment with NBQX or Selurampanel resulted in significant shrinkage of GRIA2-high expression organoids, accompanied by a substantial reduction in branching. In contrast, NBQX or Selurampanel had minimal effect on the volume or morphology of GRIA2-low expression organoids. Immunohistochemical analysis showed that NBQX or Selurampanel treatment reduced β-catenin expression in GRIA2-high expression PDOs, while having negligible effect on β-catenin levels in GRIA2-low expression PDOs.

[0059] Example 5

[0060] AMPA receptor antagonists inhibit peritoneal metastasis of gastric cancer in vivo.

[0061] 1. Mouse peritoneal transfer model

[0062] Male nude mice and C57BL / 6J mice (5-6 weeks old) were used to establish a peritoneal dissemination model. Luciferase-labeled MKN74 or MKN45 cells (1×10⁻⁶) were used. 6 Or 1×10 7 Cells / animal were intraperitoneally inoculated on day 0.

[0063] 2. Drug treatment plan

[0064] Mice were randomly assigned to a control group, an NBQX group (30 mg / kg / day, intraperitoneal injection), and a Selurampanel group (10 mg / kg / day, intraperitoneal injection), with six mice in each group. Administration began on day 3 post-inoculation and continued for 14 days. Tumor progression was monitored periodically using IVIS imaging.

[0065] 3. Patient-derived organ xenograft (PDOX) model

[0066] Sex-matched NKG mice (6 weeks old) were used for PDOX establishment. PDO (1×10⁻⁶) 6 Cells were suspended in 50 μL of L Atrigel and injected intraperitoneally. The drug treatment regimen was the same as above.

[0067] 4. Results

[0068] In a mouse peritoneal metastasis model, IVIS imaging showed that the intraperitoneal bioluminescence intensity and the number of metastatic tumor nodules in the NBQX or Selurampanel treatment groups were significantly lower than those in the control group (P<0.01 to P<0.001). In the PDOX model, GRIA2-overexpressing PDO generated extensive intraperitoneal metastases. NBQX or Selurampanel treatment further inhibited the peritoneal metastasis of GRIA2-overexpressing PDO and reduced the number of metastatic nodules. In contrast, these drugs had no significant therapeutic effect on intraperitoneal dissemination of GRIA2-low-expressing PDO. These data indicate that AMPA receptor antagonists have selective efficacy against GRIA2-overexpressing gastric cancer peritoneal metastases, suggesting that GRIA2 expression level can serve as a predictive biomarker for precision treatment.

[0069] Example 6

[0070] Mechanism of action of AMPA receptor antagonists

[0071] 1. Interaction between GRIA2 and GSK3β

[0072] Interacting proteins of GRIA2 were identified by immunoprecipitation-mass spectrometry. Co-IP experiments confirmed the direct interaction between GRIA2 and GSK3β, which was enhanced by glutamate stimulation. Molecular dynamics simulations revealed that glutamate binding effectively constrained the structural dynamics of the complex, conferring higher structural stability.

[0073] 2. Regulation of GSK3β activity by GRIA2

[0074] Western blot analysis showed that GRIA2 knockout led to decreased GSK3β-Ser9 phosphorylation, while overexpression led to increased Ser9 phosphorylation. In vitro kinase activity assays confirmed that GRIA2 knockout increased GSK3β enzyme activity, while overexpression decreased it. Treatment with the GSK3β inhibitor LiCl (10 mM) reversed the decrease in β-catenin expression caused by GRIA2 knockout.

[0075] 3. The role of calcium ions in the regulation of GSK3β by GRIA2

[0076] In gastric cancer cells, GRIA2 contains unedited glutamine (Q) at position 607, enabling it to be permeable to calcium ions. Calcium influx assays showed that GRIA2 knockout decreased calcium influx, while overexpression increased it. In calcium-containing media, wild-type GRIA2 overexpression inhibited GSK3β activity and upregulated TCF / LEF transcriptional activity, while the GRIA2 Q607R mutant (a calcium channel inactivation mutant) did not induce these changes. Under calcium-free conditions, GRIA2 knockout, wild-type overexpression, or Q607R mutant overexpression did not significantly affect GSK3β activity or TCF / LEF transcriptional activity.

[0077] 4. Conclusion

[0078] AMPA receptor antagonists inhibit the interaction between GRIA2 and GSK3β by blocking GRIA2-mediated calcium ion influx, thereby relieving the inhibition of GSK3β kinase activity, promoting β-catenin phosphorylation and degradation, inhibiting the activation of the Wnt / β-catenin signaling pathway, and ultimately suppressing the malignant phenotype associated with peritoneal metastasis of gastric cancer.

[0079] Detailed description of the attached diagram:

[0080] in, Figure 1 Figure 1 shows the in vivo validation results of GRIA2 promoting peritoneal metastasis of gastric cancer. Figure A is a schematic diagram of the experimental workflow of genome-wide CRISPR screening; Figure B shows the sequencing results of candidate genes; Figures C and D are Kaplan-Meier survival analyses of GRIA2 expression and overall survival in gastric cancer patients; Figures E, F, G, H, I, and J show the effects of GRIA2 knockout or overexpression on tumor burden in a mouse peritoneal metastasis model.

[0081] Figure 2 This is a graph showing the experimental results of GRIA2 promoting gastric cancer cell migration, invasion, stemness, and mesothelial adhesion in a glutamate-dependent manner. Figure A shows the effect of GRIA2 on gastric cancer cell migration and invasion; Figure B shows the effect of GRIA2 on spheroidization ability; Figure C shows the effect of GRIA2 on tumor cell-mesothelial cell adhesion; Figures D, E, and F show the effects of glutamate stimulation on the above phenotypes.

[0082] Figure 3 Figure 1 shows the experimental results of a study on the mechanism by which GRIA2 promotes peritoneal metastasis by activating the Wnt / β-catenin signaling pathway. Figure A shows the differentially expressed gene analysis by RNA sequencing; Figure B shows the GSEA enrichment analysis; Figures C and D show the expression and stability of β-catenin protein; Figures E, F, G, H, and I show the β-catenin mutant reversion experiment.

[0083] Figure 4This diagram shows the experimental results of GRIA2 directly interacting with GSK3β kinase in a glutamate- and calcium-dependent manner and inhibiting its activity. Figure A shows the identification of GRIA2-interacting proteins by co-precipitation-mass spectrometry; Figure B shows the Co-IP verification of the interaction between GRIA2 and GSK3β; Figures C, D, and E show the effects of GRIA2 on GSK3β phosphorylation and activity; Figures F, G, H, and I show molecular dynamics simulations and identification of key binding sites; Figures J, K, L, and M show the role of calcium ions in the regulation of GSK3β activity by GRIA2.

[0084] Figure 5 Figures show the results of in vitro and in vivo experiments on the inhibition of peritoneal metastasis of gastric cancer by AMPA receptor antagonists; Figures A, B, C, and D show the effects of NBQX and Selurampanel on the migration, invasion, spheroidization, and adhesion of MKN74 cells; Figures E and F show the effects of AMPA receptor antagonists on the morphology and protein expression of patient-derived organoids; Figure G shows the experimental design of animal experiments and PDOX models; Figures H and I show the effects of drug treatment in mouse peritoneal metastasis models; Figure J shows the effects of drug treatment in PDOX models.

[0085] Figure 6 Figure 1 shows the results of a study on the activation of GRIA2 signaling by glutamate secretion in tumor-associated fibroblasts. Figure A shows cell type clustering from single-cell RNA sequencing; Figures B and C show the expression of glutamate transporters in different cell types; Figure D shows the glutamate concentration in the culture supernatant of different cell types; Figures E, F, G, H, I, J, K, and L show the effect of fibroblast-specific knockout of Slc17a7 on tumor metastasis.

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. Application of GRIA2 as a drug target in the preparation of drugs for the prevention or treatment of peritoneal metastasis of gastric cancer.

2. Application of AMPA receptor antagonists in the preparation of drugs for the prevention or treatment of peritoneal metastasis of gastric cancer.

3. The application according to claim 1, characterized in that, The AMPA receptor antagonist inhibits the interaction between GRIA2 and GSK3β by blocking GRIA2-mediated calcium ion influx, thereby relieving the inhibition of GSK3β kinase activity, promoting β-catenin phosphorylation and degradation, inhibiting the activation of the Wnt / β-catenin signaling pathway, and ultimately inhibiting peritoneal metastasis of gastric cancer.

4. The application according to claim 1, characterized in that, The AMPA receptor antagonist works by inhibiting GRIA2-mediated calcium ion influx.

5. The application according to claim 1, characterized in that, The AMPA receptor antagonist works by inhibiting GSK3β-Ser9 phosphorylation and reducing the stability of β-catenin protein.

6. The application according to claim 1, characterized in that, The AMPA receptor antagonists include NBQX and / or Selurampanel.

7. The application according to claim 1, characterized in that, The drug is used for patients with peritoneal metastases from gastric cancer who have high GRIA2 expression.

8. Application of AMPA receptor antagonists in the preparation of drugs that inhibit the migration, invasion, stem cell-like characteristics and / or adhesion of gastric cancer cells to peritoneal mesothelial cells.

9. The application according to claim 8, characterized in that, The AMPA receptor antagonists include NBQX and / or Selurampanel.

10. A gastric cancer treatment system, characterized in that, The treatment system includes a medication delivery system; the medication delivery system includes an AMPA receptor antagonist.

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