1 -DNJ for use in the treatment of lymph node metastasis of gastric cancer

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to application of 1-DNJ in treating lymph node metastasis of gastric cancer. The present application firstly finds that 1-DNJ can play a role as a VCAM1 / VLA-4 interaction inhibitor, and further finds that 1-DNJ can be used for treating lymph node metastasis of gastric cancer, expands the application range of 1-DNJ in the field of antitumor, and provides a new technical scheme for prevention and treatment of lymph node metastasis of gastric cancer. 1-DNJ belongs to a small molecule compound, has the characteristics of convenient preparation, good potential tissue penetration ability and flexible drug administration mode, and has good prospects for drug development and clinical application.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of 1-DNJ in the treatment of lymph node metastasis in gastric cancer. Background Technology

[0002] Gastric cancer (GC) is one of the most common malignant tumors worldwide, with the highest incidence and mortality rates. Lymph node metastasis (LNM) is the most prevalent mode of metastasis in gastric cancer and a core factor influencing clinical stage, treatment strategy, and prognosis. The dense lymphatic network in the gastric submucosa and its extensive connections with perigastric lymph nodes endow gastric cancer with a strong tendency for lymphatic metastasis. Lymph nodes, as primary drainage hubs for interstitial fluid and tumor cells, occupy a central position in the tumor metastasis cascade and are key "gatekeepers" for systemic tumor spread. Traditionally, lymph node metastasis (LNM) was considered a result of tumor cells passively invading lymphatic vessels. However, increasing evidence suggests that LNM is an active, multi-step cascade process, precisely regulated by dynamic bidirectional interactions between tumor cells and the lymphatic microenvironment. In this process, tumor cells first acquire enhanced migration and invasion capabilities, breaching the basement membrane barrier and entering lymphatic vessels; subsequently, they establish specific adhesion interactions with lymphatic endothelial cells (LECs), a step crucial for transendothelial migration and eventual lymph node colonization. Within the tumor microenvironment (TME), tumor-derived signaling molecules actively remodel lymphatic vessels (LECs), promoting lymphangiogenesis and enhancing lymphatic permeability, thus jointly driving the development of lymphoblastic neoplasms (LNM). Therefore, LECs are not merely passive conduits in the process of tumor metastasis, but rather key effector cells that actively participate in and promote tumor spread.

[0003] Vascular cell adhesion molecule 1 (VCAM1) is a member of the immunoglobulin superfamily, a transmembrane glycoprotein that mediates cell adhesion through its binding to its ligand integrin α4β1 (VLA-4), playing a crucial role in inflammatory responses and immune cell migration. High expression of VCAM1 in gastric cancer tumors promotes lymph node metastasis. Mechanistic studies have shown that VCAM1... + Tumor cells activate the Ras / MAPK signaling pathway in LECs by interacting with the ligand-receptor of VLA-4 on the surface of LECs, driving lymphangiogenesis and thus promoting lymph node metastasis.

[0004] 1-Deoxynojirimycin (1-DNJ) is a natural polyhydroxy alkaloid extracted from mulberry leaves. Traditionally considered an α-glucosidase inhibitor, it possesses anti-diabetic and neuroprotective activities. Recent studies suggest it may also have some anti-tumor activity, but its potential to inhibit gastric cancer lymph node metastasis by blocking the VCAM1 / VLA-4 interaction has not yet been discovered or systematically studied.

[0005] Among existing drugs targeting the VCAM1 / VLA-4 axis, natalizumab, a humanized monoclonal antibody targeting the VLA-4 α4 subunit, has been approved for multiple sclerosis and Crohn's disease and is currently the standard clinical biologic for blocking VCAM1 / VLA-4 interaction. However, antibody drugs have limitations such as limited tissue penetration, inconvenient administration, and potential immunogenicity, and currently no small molecule inhibitors of VCAM1 / VLA-4 have entered clinical trials for gastric cancer.

[0006] Currently, there is a lack of effective targeted therapies specifically for lymph node metastasis in gastric cancer, and existing chemotherapy and immunotherapy regimens have limited specific inhibitory effects on LNM. Summary of the Invention

[0007] To address the lack of effective targeted therapies for gastric cancer lymph node metastasis in existing technologies, this invention provides the use of 1-deoxynojirimycin (1-DNJ) as a VCAM1 / VLA-4 interaction inhibitor, and its application in the preparation of drugs for treating gastric cancer lymph node metastasis.

[0008] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides the non-therapeutic use of 1-DNJ as an inhibitor of VCAM1 / VLA-4 interaction.

[0009] In a second aspect, the present invention provides the use of 1-DNJ in the preparation of VCAM1 / VLA-4 interaction inhibitors.

[0010] In a third aspect, the present invention provides the use of 1-DNJ in the preparation of a drug for treating lymph node metastasis of gastric cancer.

[0011] In some embodiments, the gastric cancer is VCAM1-positive gastric cancer.

[0012] In some embodiments, the drug is used to inhibit tumor-associated lymphangiogenesis.

[0013] In some embodiments, the 1-DNJ is combined with pharmaceutically acceptable excipients to form a pharmaceutical composition.

[0014] In some embodiments, the dosage form of the pharmaceutical composition is an oral formulation or an injectable formulation.

[0015] In some embodiments, the oral formulation is a tablet, capsule, granule, or oral liquid.

[0016] In some embodiments, the injectable formulation is an injection solution or a lyophilized powder for injection.

[0017] In some embodiments, the subject of the drug is a mammal, preferably a human.

[0018] The present invention has at least one of the following beneficial effects: (1) This invention is the first to discover that 1-DNJ can act as an inhibitor of VCAM1 / VLA-4 interaction, providing a new candidate compound for the study and drug development of VCAM1 / VLA-4 related diseases.

[0019] (2) This invention is the first to discover that 1-DNJ can be used to treat lymph node metastasis of gastric cancer, which expands the application scope of 1-DNJ in the field of anti-tumor treatment and provides a new technical solution for the prevention and treatment of lymph node metastasis of gastric cancer.

[0020] (3) This invention verified the inhibitory effect of 1-DNJ on gastric cancer cell migration, invasion, tumor cell-lymphoendothelial cell interaction and tumor-associated lymphangiogenesis through in vitro and in vivo experiments, and confirmed that it can significantly reduce the level of lymph node metastasis in gastric cancer orthotopic xenograft model.

[0021] (4) 1-DNJ is a small molecule compound with the characteristics of easy preparation, good potential tissue penetration ability and flexible administration method, and has good prospects for drug development and clinical application. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 Figure 1 shows the verification results of 1-DNJ inhibiting the interaction between VCAM1 and VLA-4. Figure A shows the molecular docking results of 1-DNJ with the VCAM1 / VLA-4 complex; Figure B shows the Co-IP experiment verifying the results of 1-DNJ inhibiting the binding of VCAM1 and VLA-4. The left figure shows the detection results after immunoprecipitation with ITGA4, and the right figure shows the detection results after immunoprecipitation with ITGB1.

[0024] Figure 2Figure 1 shows the experimental results of 1-DNJ inhibiting the proliferation, migration, and invasion of gastric cancer cells. Figure A shows the effect of 1-DNJ on the proliferation activity of gastric cancer cells HGC-27 and normal gastric mucosal epithelial cells GES-1 as detected by the CCK-8 assay. Figure B shows the effect of different concentrations of 1-DNJ on the migration ability of HGC-27 cells as detected by the scratch healing assay. The left image is a representative image of scratch healing, and the right image is the quantitative analysis result of scratch healing area. Figure C shows the effect of different concentrations of 1-DNJ on the migration and invasion ability of HGC-27 cells as detected by the Transwell assay. The left image is a representative staining image, the middle image is the statistical result of the number of migrating cells, and the right image is the statistical result of the number of invasive cells. This indicates that P < 0.01.

[0025] Figure 3 Figure 1 shows the experimental results of 1-DNJ inhibiting the interaction between tumor cells and lymphoendothelial cells and lymphangiogenesis. Figure A shows the results of the tumor cell-lymphoendothelial cell adhesion experiment, with the left image showing representative images of different treatment groups and the right image showing the statistical results of the number of adherent cells. Figure B shows the results of the transendothelial migration experiment, with the left image showing representative images of VE-cadherin immunofluorescence staining and tumor cell transendothelial migration, and the right image showing the statistical results of the number of migrating cells. Figure C shows the results of the lymphoendothelial cell tube formation and migration experiments, with the left image showing representative images of different treatment groups, the middle image showing the statistical results of relative branch length, and the right image showing the statistical results of the number of migrating cells. This indicates that P < 0.01.

[0026] Figure 4 Figure 1 shows the in vivo experimental results of 1-DNJ inhibiting lymph node metastasis in orthotopic gastric cancer xenografts. Figure A shows representative images of lymph node metastasis in different treatment groups and the statistical results of the number of metastatic lymph nodes detected by in vivo fluorescence imaging in mice. Figure B shows the HE staining results and immunohistochemical staining results of VCAM1, LYVE1, and VEGFC in the primary tumor tissue. Figure C shows the results of multiplex immunofluorescence staining of the primary tumor tissue, where Pan-CK is a gastric cancer cell marker, VCAM1 is vascular cell adhesion molecule 1, and LYVE1 is a lymphatic endothelial marker. Figure D shows the HE staining results and Pan-CK immunofluorescence staining results in the metastatic lymph node tissue. This indicates that P < 0.01. Detailed Implementation

[0027] The embodiments of the present invention will now be described in detail. The following examples are only used to explain the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. After reading this specification, those skilled in the art can make various modifications, substitutions or variations to the technical solutions of the present invention without departing from the concept and technical principles of the present invention, and all such modifications, substitutions or variations should fall within the scope of protection of the present invention.

[0028] Unless otherwise stated, the technical and scientific terms used herein shall have the meanings commonly understood by one of ordinary skill in the art. Unless otherwise specified, the experimental methods described herein shall be performed under standard experimental conditions, in accordance with relevant laboratory manuals, or as recommended by the manufacturers of reagents or instruments, or using other equivalent methods known in the art.

[0029] The terms "comprising," "including," and "containing" as used herein are open-ended expressions, and unless explicitly specified, they do not exclude the presence of other components, steps, or structures not explicitly listed. The terms "preferred," "optional," and "optional" as used herein are only for illustrating some embodiments and do not constitute a limitation on the scope of protection of this invention.

[0030] Unless otherwise specified, the numerical ranges mentioned in this article include any integers, fractions, decimals, and their upper and lower limits within that range.

[0031] Unless otherwise specified, the singular expressions such as "a," "a kind," and "the" used in this article also include the plural forms; "multiple" usually refers to two or more.

[0032] The term “and / or” as used in this document refers to any one or any combination of the relevant objects.

[0033] Where there is no contradiction, the technical features of the various embodiments and examples in this specification can be combined with each other. Any conventional adjustments, substitutions, or equivalent modifications made by those skilled in the art based on the content of this specification should be considered part of the disclosure herein.

[0034] The term "treatment" as used in this article includes prevention, delay of occurrence, reduction of risk of occurrence, inhibition, relief, improvement, control, stabilization or elimination of disease, symptom or abnormal state, as well as improvement of one or more symptoms or pathological changes caused therefrom.

[0035] The “1-DNJ” mentioned in this article refers to 1-deoxynojirimycin and includes its pharmaceutically acceptable salts, hydrates, solvates, and derivatives with the same pharmacological activity.

[0036] The “VCAM1” mentioned in this article refers to Vascular Cell Adhesion Molecule-1.

[0037] The “VLA-4” mentioned in this article refers to Very Late Antigen-4, a heterodimer receptor composed of integrin α4 subunit (ITGA4) and integrin β1 subunit (ITGB1).

[0038] The “VCAM1 / VLA-4 interaction inhibitor” mentioned in this article refers to substances that can reduce, block, or inhibit the binding between VCAM1 and VLA-4.

[0039] The term "gastric cancer lymph node metastasis" as used in this article refers to the process or state in which gastric cancer cells spread through the lymphatic system and form metastatic lesions in regional or distant lymph nodes.

[0040] The term "VCAM1-positive gastric cancer" as used in this article refers to gastric cancer in which the expression level of VCAM1 in the tumor tissue is higher than that in normal control tissue, or in which VCAM1 expression can be detected by immunohistochemistry, immunofluorescence, flow cytometry, PCR, Western blot, or other conventional detection methods in the field.

[0041] The term "pharmaceuticalally acceptable excipients" as used in this article refers to carriers, diluents, excipients, stabilizers, lubricants, disintegrants, binders, preservatives, buffers, or other commonly used excipients in the pharmaceutical field that are suitable for pharmaceutical formulations and will not cause unacceptable toxicity, irritation, or other adverse reactions in subjects.

[0042] The term "pharmaceutical composition" as used herein refers to a composition comprising an effective amount of 1-DNJ and one or more pharmaceutically acceptable excipients.

[0043] The term "subject" as used herein refers to an individual who requires prevention, mitigation, or treatment, including humans and non-human mammals. Preferably, the subject is a human.

[0044] The “effective dose” mentioned in this article refers to the dose of 1-DNJ that produces the expected biological, pharmacological, or therapeutic effects.

[0045] The first aspect of the invention relates to the non-therapeutic use of 1-DNJ as an inhibitor of VCAM1 / VLA-4 interaction.

[0046] This invention is the first to discover that 1-DNJ can effectively inhibit the interaction between VCAM1 and VLA-4. Molecular docking results show that 1-DNJ can stably bind to the VCAM1 / VLA-4 interaction interface; further verification through Co-IP experiments shows that 1-DNJ can significantly reduce the binding level between VCAM1 and VLA-4. These results indicate that 1-DNJ can function as an inhibitor of the VCAM1 / VLA-4 interaction.

[0047] The VCAM1 / VLA-4 signaling axis is widely involved in various biological processes, including cell adhesion, cell migration, inflammatory responses, immune regulation, angiogenesis, lymphangiogenesis, and tumor metastasis. Therefore, the 1-DNJ discovered in this invention, as a VCAM1 / VLA-4 interaction inhibitor, has broad research and application value.

[0048] In some implementations, 1-DNJ can be used as a laboratory research tool to study the biological functions and molecular regulatory mechanisms of VCAM1 / VLA-4 interactions.

[0049] In some implementations, 1-DNJ can be used to establish, validate, or optimize detection, evaluation, or screening systems related to VCAM1 / VLA-4 interactions.

[0050] In some implementations, 1-DNJ can be used as a positive control, reference material, or tool compound to evaluate the ability of candidate compounds, antibodies, peptides, nucleic acid drugs, or other active substances to regulate VCAM1 / VLA-4 interactions.

[0051] In some implementations, 1-DNJ can be used to study the mechanism of action of the VCAM1 / VLA-4 signaling axis in tumor cells, immune cells, vascular endothelial cells, or lymphoendothelial cells.

[0052] The above uses are all non-therapeutic and do not involve the administration of drugs to humans or animals for the purpose of disease prevention, diagnosis or treatment.

[0053] A second aspect of the invention relates to the use of 1-DNJ in the preparation of VCAM1 / VLA-4 interaction inhibitors.

[0054] The VCAM1 / VLA-4 signaling axis is widely involved in various pathophysiological processes, including inflammatory responses, immune cell recruitment, cell adhesion, angiogenesis, lymphangiogenesis, and tumor metastasis. Abnormally activated VCAM1 / VLA-4 signaling is closely related to the occurrence and development of many diseases. Therefore, drugs that can inhibit the VCAM1 / VLA-4 interaction have significant clinical application value.

[0055] In some embodiments, the VCAM1 / VLA-4 interaction inhibitor is used to prevent, alleviate, or treat diseases associated with abnormal VCAM1 / VLA-4 signaling.

[0056] In some embodiments, the diseases include inflammatory diseases, immune-related diseases, angiogenesis-related diseases, lymphangiogenesis-related diseases, and tumor metastasis-related diseases.

[0057] In some preferred embodiments, the disease is a tumor-related lymph node metastasis disease.

[0058] In a further preferred embodiment, the disease is gastric cancer with lymph node metastasis.

[0059] A third aspect of the invention relates to the use of 1-DNJ in the preparation of a medicament for treating lymph node metastasis in gastric cancer.

[0060] This invention has found that 1-DNJ can not only inhibit the interaction between VCAM1 and VLA-4, but also significantly inhibit the migration and invasion of gastric cancer cells, reduce the adhesion between tumor cells and lymphoendothelial cells, restore the integrity of the lymphoendothelial barrier, inhibit the transendothelial migration of tumor cells, and further inhibit the formation of tumor-associated lymphangiogenesis, thereby effectively reducing the level of lymph node metastasis in gastric cancer.

[0061] In some embodiments, the gastric cancer is VCAM1-positive gastric cancer.

[0062] In some implementations, when the expression level of VCAM1 protein or VCAM1 nucleic acid is detected in gastric cancer tissue at a higher level than that in the corresponding normal gastric tissue, it can be identified as VCAM1 positive gastric cancer.

[0063] In some implementations, the presence of VCAM1-positive tumor cells in gastric cancer tissue can be used to diagnose VCAM1-positive gastric cancer.

[0064] In some preferred embodiments, the VCAM1-positive gastric cancer has a high risk of lymph node metastasis and / or a strong tendency for lymph node metastasis.

[0065] In some embodiments, the 1-DNJ is used to prevent, delay, inhibit, or treat lymph node metastasis in VCAM1-positive gastric cancer.

[0066] In some embodiments, the drug is used to inhibit tumor-associated lymphangiogenesis. Tumor-associated lymphangiogenesis refers to lymphatic vessels located within or around tumor tissue, or formed or remodeled under the influence of the tumor microenvironment. These tumor-associated lymphangiogenesis can participate in processes such as tumor cell migration, invasion, transendothelial transport, lymphatic spread, and lymph node metastasis. Tumor-associated lymphangiogenesis refers to the process of new lymphatic vessel formation induced by the tumor microenvironment, as well as the expansion, elongation, increased branching, or enhanced function of existing lymphatic vessels. Tumor-associated lymphangiogenesis can be evaluated using lymphatic vessel density, microlymphatic vessel density, lymphatic vessel length, lymphatic vessel area, number of lymphatic vessel branches, LYVE1 expression level, VEGFC expression level, or other indicators known in the art.

[0067] In some embodiments, the 1-DNJ can reduce the density of microlymphatic vessels in tumor tissue, reduce the expression level of LYVE1, reduce the expression level of VEGFC, and / or reduce the level of lymphatic vessel invasion, thereby inhibiting tumor-associated lymphangiogenesis and reducing the incidence of lymph node metastasis in gastric cancer.

[0068] In some embodiments, the 1-DNJ is combined with pharmaceutically acceptable excipients to form a pharmaceutical composition. The pharmaceutical composition can be formulated using methods known to those skilled in the art. For example, it can be administered parenterally as an injection of a sterile solution or suspension of water or a pharmaceutically acceptable solution other than water. For example, a suitable combination of pharmacologically acceptable carriers or mediators can be used, specifically sterile water, physiological saline, vegetable oil, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, mediators, preservatives, binders, etc. The above pharmaceutical compositions can be prepared by mixing into unit dosage forms required for drug administration. The amount of active ingredient in these formulations is set to obtain an appropriate volume within the indicated range.

[0069] Sterile compositions for injection can be formulated using excipients such as distilled water for injection, following conventional formulation methods.

[0070] Examples of aqueous solutions for injection include isotonic solutions containing, for example, physiological saline, lactose, glucose, and other supplements (such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride). Appropriate dissolving aids can be used in combination, such as alcohols (ethanol, etc.), polyols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (polysorbate 80(TM), HCO-50, etc.).

[0071] As an oily liquid, sesame oil and soybean oil can be examples, and benzyl benzoate and / or benzyl alcohol can be used in combination as a solubilizing agent. The composition can be mixed with buffers (e.g., phosphate buffer solutions and sodium acetate buffer solutions), analgesics (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants. The prepared injection solution is typically packaged in appropriate ampoules.

[0072] In some embodiments, the dosage form of the pharmaceutical composition is an oral formulation or an injectable formulation.

[0073] Because 1-DNJ has good drug development potential, it can be formulated into drug dosage forms suitable for different routes of administration according to clinical needs.

[0074] In some embodiments, the pharmaceutical composition is an oral formulation.

[0075] The oral preparation may be a tablet, capsule, granule or oral liquid.

[0076] In some embodiments, the tablet may be a regular tablet, a film-coated tablet, a sugar-coated tablet, a chewable tablet, a dispersible tablet, or a sustained-release tablet.

[0077] In some embodiments, the capsule may be a hard capsule, a soft capsule, or a sustained-release capsule.

[0078] In some embodiments, the granules may be ordinary granules, instant granules, or sustained-release granules.

[0079] In some embodiments, the oral liquid may contain pharmaceutically acceptable solvents, solubilizers, flavoring agents, preservatives, or stabilizers.

[0080] In some embodiments, the pharmaceutical composition is an injectable formulation.

[0081] The injectable preparation may be an injection solution or a lyophilized powder for injection.

[0082] In some embodiments, the injection solution may be an aqueous solution, a suspension, or an emulsion.

[0083] In some embodiments, the lyophilized powder injection may be reconstituted with water for injection, physiological saline, glucose solution or other pharmaceutically acceptable solvents before administration.

[0084] Those skilled in the art can select appropriate dosage forms, excipients, and preparation processes according to actual needs to obtain pharmaceutical compositions suitable for treating lymph node metastasis of gastric cancer.

[0085] In some embodiments, the subject of the drug is a mammal. The mammal includes both laboratory animals used for pharmacodynamic, safety, and mechanistic studies, and non-laboratory animals used in clinical or veterinary settings for disease prevention or treatment. By applying 1-DNJ to different mammalian subjects, its pharmacological effects, administration methods, and therapeutic efficacy in vivo can be systematically evaluated, thereby validating its applicability and universality in the intervention of gastric cancer lymph node metastasis. Exemplary mammals include, but are not limited to, humans, as well as non-human mammals such as mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, pigs, sheep, cattle, pandas, elephants, horses, and non-human primates. More preferably, the subject is a human.

[0086] The present invention also provides a method for treating lymph node metastasis of gastric cancer, comprising administering an effective amount of 1-DNJ or a pharmaceutically acceptable salt thereof to a subject in need.

[0087] The present invention also provides a method for treating diseases associated with abnormal VCAM1 / VLA-4 interaction, comprising administering an effective amount of 1-DNJ or a pharmaceutically acceptable salt thereof to a subject in need.

[0088] In some implementations, the disease is a tumor metastasis-related disease.

[0089] In some preferred embodiments, the disease is gastric cancer with lymph node metastasis.

[0090] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these embodiments are only used to illustrate the technical content of the present invention and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the specific experimental conditions in the following embodiments are given priority reference to the guidelines provided in this specification, or may be carried out according to generally accepted experimental manuals or conventional experimental conditions, or other experimental methods known in the art, or according to the conditions recommended by the relevant reagent or instrument manufacturers. In specific embodiments, unless otherwise specified, minor deviations within the weighing accuracy range are allowed for the measurement parameters involving raw material components; reasonable deviations due to instrument detection accuracy or operational accuracy are also allowed for parameters such as temperature and time.

[0091] Example 1 (1) Using VCAM1 immunoglobulin domains 1 and 4 as targets, molecular docking was performed using Schrödinger software. The results were evaluated using ADME / T parameters and calculated using MM-GBSA binding energy. Figure 1 According to data from the Chinese database, 1-DNJ achieved the highest docking score of -8.649 and a binding energy of -66.53.

[0092] (2) Co-IP experiment confirmed the inhibition of VCAM1 and VLA-4 binding by 1-DNJ: Total protein was extracted, pre-incubated with 1-DNJ, followed by immunoprecipitation, and the bound proteins were detected by Western blotting after elution. The results showed that in the control group without 1-DNJ treatment, both ITGA4 and ITGB1 co-precipitated significantly with VCAM1, confirming a direct protein interaction between VCAM1 and VLA-4. After the addition of 1-DNJ, the signals of co-precipitated ITGA4 and ITGB1 were significantly reduced. The above results confirm that 1-DNJ can directly block the ligand-receptor binding of VCAM1 and VLA-4, providing direct biochemical evidence for the inhibition of VCAM1 / VLA-4 interaction by 1-DNJ. Figure 1 (B)

[0093] Example 2 (1) According to 5×10 3 Cells were seeded at a density of cells / well in 96-well plates and gently shaken to ensure even distribution. 10 μL of CCK-8 solution was added, and after incubation, the absorbance was measured at 450 nm using a microplate reader. The results were calculated and statistically analyzed. The results are as follows: Figure 2 As shown in Figure A, the IC50 inhibitory activity against the proliferation of gastric cancer cells HGC-27 is [value missing]. 50 = 15.10 μM, IC50 against normal gastric mucosal epithelial cells GES-1 50 = 250.10 μM, with a therapeutic window of approximately 16.6 times, and excellent safety.

[0094] (2) After the cells were seeded into 6-well plates and cultured to 100% confluence, a 200 μL sterile pipette tip was used to make a straight, uniform scratch. After washing away the detached cell debris with PBS, serum-free medium containing DMSO (10 μM) or 1-DNJ (5, 10, 15 μM) was added, respectively. Images were taken under an inverted microscope at 0 h, 24 h, and 48 h after scratching, and the scratch healing rate was calculated using ImageJ software. The results showed that compared with the DMSO control group, the scratch healing rate of the 1-DNJ treatment group was significantly reduced at 24 h and 48 h (p<0.01), suggesting that 1-DNJ has a sustained inhibitory effect on the migration ability of gastric cancer cells. Figure 2 (Middle B). Transwell assay was used to detect the inhibitory effect of 1-DNJ on tumor cell migration and invasion. The assay was performed at a rate of 3 × 10⁻⁶. 4 Cells were resuspended in 200 μL of serum-free cell culture medium per well, and DMSO (10 μM) and 1-DNJ (5, 10, 15 μM) were added respectively. Cells were seeded into the upper chamber of a Transwell chamber and cultured for 24 h. Crystal violet staining was performed, and the number of migrating and invading cells was quantified using ImageJ software. Figure 2(C). The results showed that compared with the DMSO control group, the number of transmembrane migrating and invading cells in the 5, 10, and 15 μM 1-DNJ treatment groups was significantly reduced (p<0.01), indicating that 1-DNJ can effectively inhibit the motility and matrix invasion of gastric cancer cells.

[0095] Example 3 (1) DiI fluorescently labeled VCAM1-overexpressing HGC-27 cells (5×10⁻⁶) 4 (2 × 10) added to the already formed HLEC monolayer (2 × 10) 5 In 24-well plates containing 100% confluence of cells, DMSO (10 μM), 1-DNJ (10 μM), IgG (68.5 nM), or nastatinumab (68.5 nM) were added, respectively. After incubation for 2 hours, the cells were washed with PBS and counted using a fluorescence microscope. Results showed that the number of adherent cells in the 1-DNJ group was significantly lower than that in the DMSO group (p<0.01), but there was no significant difference compared to the nastatinumab group. Figure 3 As shown in Figure A.

[0096] (2) VE-cadherin immunofluorescence staining showed that 1-DNJ restored the integrity of endothelial junctions disrupted by VCAM1. HLEC cells were digested and counted at a rate of 3 × 10⁻⁶ cells / cells. 4 Cells / well were seeded into the upper chamber of a pretreated Transwell chamber until a confluent monolayer was formed. DMSO (10 μM), 1-DNJ (10 μM), IgG (68.5 nM), or nastatinum antibody (68.5 nM) were added, followed by 1×10⁶ cells / well. 5 HGC-27 cells expressing GFP green fluorescence were seeded into the upper chamber of a Transwell cell. After culture, images were taken using an inverted fluorescence microscope, and the number of tumor cells penetrating the lymphoendothelial cells was counted using ImageJ software. The results showed that the number of adhering cells in the 1-DNJ group was significantly lower than that in the DMSO group, but not significantly different from that in the nastatinumab group. Figure 3 As shown in B.

[0097] (3) After co-culturing HGC-27 cells with HLECs for 48 h, HLECs were sorted by flow cytometry and divided into groups of 1×10⁻⁶ cells. 5 HLEC cells were resuspended at a density of 10 cells / well in 400 μL of serum-free endothelial cell culture medium, and DMSO (10 μM), 1-DNJ (10 μM), IgG (68.5 nM), or natamizumab (68.5 nM) were added, respectively. The cells were then seeded into pretreated 24-well plates and cultured for 5 h. After culture, the 24-well plates were removed, imaged using an inverted microscope, and the total length of the tubes was counted using ImageJ software. Cells were then cultured at a density of 3 × 10⁻⁶ cells / well. 4HLEC cells were resuspended in 200 μL of serum-free endothelial cell culture medium per well, and DMSO (10 μM), 1-DNJ (10 μM), IgG (68.5 nM), or nastatinum antibody (68.5 nM) were added, respectively. Cells were seeded into the upper chamber of a Transwell chamber and cultured for 24 h. Crystal violet staining was performed, and the number of migrating cells was quantified using ImageJ software. Figure 3 (C). HLEC formation and migration assays showed that 1-DNJ significantly inhibited the lymphangiogenic capacity of LECs, with no significant difference compared to the natezumab group. Figure 3 (C)

[0098] Example 4 5×10 5 HGC-27 cells (DiI fluorescently labeled) were injected into the stomach wall of 4-5 week old BALB / c nude mice to establish an orthotopic xenograft model. Two weeks later, mice were randomly divided into four groups (n=10 / group): control group (physiological saline, 40 mg / kg, daily intraperitoneal injection), 1-DNJ group (40 mg / kg, daily intraperitoneal injection), IgG group (3.125 mg / kg, weekly intraperitoneal injection), and nastatinumab group (3.125 mg / kg, weekly intraperitoneal injection), with continued administration for 4 weeks. The endpoint was determined by IVIS imaging to detect lymph node metastasis; primary tumor and metastatic lymph nodes were collected for mIHC analysis. Figure 4 The results showed that the incidence of lymph node metastasis, lymphatic vessel length and lymphatic vessel invasion in the 1-DNJ group were significantly lower than those in the control group (p<0.01), and the efficacy was comparable to that in the natetuzumab group.

[0099] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. The scope of protection of the present invention should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. Non-therapeutic use of 1-DNJ as an inhibitor of VCAM1 / VLA-4 interaction.

2. Use of 1-DNJ in the preparation of VCAM1 / VLA-4 interaction inhibitors.

3. Application of 1-DNJ in the preparation of drugs for treating lymph node metastasis of gastric cancer.

4. The application according to claim 3, wherein the gastric cancer is VCAM1-positive gastric cancer.

5. The application according to claim 3, wherein the drug is used to inhibit tumor-associated lymphangiogenesis.

6. The application according to any one of claims 3-5, wherein the 1-DNJ forms a pharmaceutical composition with pharmaceutically acceptable excipients.

7. The application according to claim 6, wherein the dosage form of the pharmaceutical composition is an oral formulation or an injectable formulation.

8. The application according to claim 7, wherein the oral preparation is a tablet, capsule, granule or oral liquid.

9. The application according to claim 7, wherein the injectable formulation is an injection solution or a lyophilized powder for injection.

10. The application according to any one of claims 3-5, 7-9, wherein the subject of the drug is a mammal; preferably a human.

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