Application of an integrin αv inhibitor combined with a γ-secretase inhibitor in the preparation of drugs for treating tumors

Abstract

This invention discloses the application of integrin αv inhibitors combined with γ-secretase inhibitors in the preparation of drugs for treating tumors. The study found that the combined use of integrin αv inhibitors and γ-secretase inhibitors in tumor treatment exhibits a synergistic effect, not only inhibiting in situ tumor growth but also significantly suppressing tumor invasion, distant metastasis, and other malignant progression, thus helping to slow tumor progression, improve treatment efficacy, and enhance patient prognosis. Furthermore, the study found that the combined use of integrin αv inhibitors and γ-secretase inhibitors can reduce the production of the intracellular free domain of integrin αv and inhibit the activation of the classical downstream pathway of integrin αv. The combined use of integrin αv inhibitors and γ-secretase inhibitors shows broad application prospects in tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of an integrin αv inhibitor combined with a γ-secretase inhibitor in the preparation of a drug for treating tumors. Background Technology

[0002] Cancer has become a major threat to public health and life. According to a recent study in 2022 by Academician He Jie's team at the Chinese Academy of Medical Sciences, the top five cancers in terms of incidence—lung cancer, colorectal cancer, stomach cancer, liver cancer, and breast cancer in women—account for 57.4% of all new cancer cases. Because early-stage cancers often have an insidious onset, by the time symptoms appear and a definitive diagnosis is made, distant metastasis and regional lymph node metastasis have often already occurred, making local surgical resection no longer feasible and severely limiting patient prognosis. Therefore, in-depth analysis of the molecular mechanisms of tumor development, local invasion, and metastasis is crucial for screening clinical therapeutic targets and improving the prognosis of cancer patients.

[0003] In recent years, numerous studies have reported the crucial role of the tumor microenvironment (TME) in the malignant progression of tumors. The tumor microenvironment refers to the stromal cellular components within a tumor, excluding tumor cells, including fibroblasts, immune cells, vascular endothelial cells, and the extracellular matrix. Tumor cells communicate with non-tumor components through direct contact and paracrine cytokine secretion, forming a complex and interwoven communication network that ultimately leads to tumor progression.

[0004] The integrin family, a class of transmembrane dimer proteins, is a key molecule in tumor cells that sense extracellular signals. Integrins are activated primarily by binding to and recognizing extracellular matrix proteins, and then transducing signals into the cell. As key molecules directly mediating extracellular-intracellular signal transduction, numerous studies have reported the crucial roles of integrins in various processes such as tumor survival, proliferation, invasion, and metastasis. In 2015, Alanko's team discovered that integrins and their downstream signaling pathways can promote tumor cells' resistance to anointing apoptosis, thereby promoting their metastasis. In addition to being highly expressed on the surface of tumor cells, integrins are also highly expressed in various stromal cells, such as fibroblasts and vascular endothelial cells, and play a crucial role in processes such as microenvironmental fibrosis, angiogenesis, and tumor extubation. Handa's team found that tumor cells can bind to integrin αvβ1 on the surface of vascular endothelial cells, leading to cell adhesion and vascular leakage. Based on the extensive reports on the key roles of the integrin family and its related signaling pathways in various stages of tumor progression, numerous clinical trials targeting integrins are currently underway. More than 70 clinical trials using integrin inhibitors to treat tumors have been registered on ClinicalTrials.gov.

[0005] However, clinical trials of these integrin inhibitors for cancer treatment have not been successful to date. Among them, silengilide, a small molecule inhibitor targeting integrin αv (CD51), was considered a promising candidate for clinical treatment; however, its Phase III clinical trial also failed. Silengilide is a small cyclic polypeptide molecule containing an RGD sequence, designed by German chemist Kessler, which can competitively antagonize integrin αvβ3 and αvβ5 receptors. The molecular structure of silengilide is shown below:

[0006]

[0007] Cilengiline first entered Phase I clinical trials in 2000. This trial recruited 51 patients with malignant gliomas to investigate its safety. In this study, the tolerated dose of cilengiline reached 2400 mg / m². 2 Of the four patients, two achieved complete symptom remission, three achieved partial symptom remission, and four had stable conditions. Subsequently, silengiptide successfully completed a Phase II clinical trial and in 2008 entered its first multicenter, randomized, controlled Phase III clinical trial (CENTRIC EORTC26071-22072, NCT00689221) for the treatment of glioblastoma patients. However, this clinical trial failed in 2013: silengiptide failed to significantly improve overall survival. To date, no other integrin-targeting drugs have entered Phase III clinical trials in cancer treatment research.

[0008] However, the specific reasons for the failure of integrin inhibitors such as silengiptide in clinical applications remain unclear. Therefore, finding effective methods to improve the clinical efficacy of cancer treatment is of considerable necessity. Summary of the Invention

[0009] This invention addresses the shortcomings and deficiencies of existing technologies regarding the poor clinical efficacy of integrin αv inhibitors in tumor treatment. It aims to provide an application of integrin αv inhibitors combined with γ-secretase inhibitors in the preparation of drugs for treating tumors. By using integrin αv inhibitors in combination with γ-secretase inhibitors for tumor treatment, the two substances work synergistically to achieve significant therapeutic effects against tumors. Drugs containing both integrin αv inhibitors and γ-secretase inhibitors can be used for tumor treatment.

[0010] The primary objective of this invention is to provide the application of integrin αv inhibitors in combination with γ-secretase inhibitors in the preparation of medicaments for treating tumors.

[0011] Another object of the present invention is to provide the use of γ-secretase inhibitors in the preparation of products that inhibit transmembrane cleavage of integrin αv to produce functional intracellular free fragment proteins.

[0012] Another object of the present invention is to provide the use of γ-secretase inhibitors in the preparation of integrin αv inhibitor tumor therapeutic enhancers.

[0013] Another object of the present invention is to provide a drug for treating cancer.

[0014] The present invention achieves the above-mentioned objectives through the following technical means:

[0015] Through extensive research, this invention has demonstrated that combined therapy targeting integrin αv and γ-secretase, particularly using cilengitide and nigasstat (LY3039478), can significantly enhance the tumor treatment efficacy of integrin αv inhibitors. The combination of these two drugs can exert a synergistic effect, showing remarkable efficacy against the malignant progression of various solid tumors, including tumor proliferation, tumor invasion, and distant metastasis.

[0016] This invention first demonstrated through in vitro drug sensitivity testing that combined therapy targeting integrin αv and γ-secretase can significantly inhibit various solid tumors. Among them, compared with the monotherapy group, the combined use of cilengitide and nigastricostat (LY3039478) can significantly inhibit tumor cell lines. The combined use of cilengitide and nigastricostat (LY3039478) showed the most significant therapeutic effect, and the two drugs have a synergistic effect.

[0017] Further Western blot analysis revealed that the combination therapy significantly reduced the production level of free integrin αv intracellular fragments. Furthermore, while silengiptide could relatively completely inhibit downstream signaling pathways of integrin αv (including FAK phosphorylation activation and F-actin polymerization), it could not completely block the epithelial-mesenchymal transition (EMT) of tumor cells. Building on this, the addition of the γ-secretase inhibitor nigastric acid significantly inhibited EMT, thus suppressing tumor invasion and other related malignant phenotypes.

[0018] The applicant further discovered through nude mouse in situ hepatocellular carcinoma tumorigenesis experiments that the combined treatment can significantly reduce the epithelial-mesenchymal transition process of tumor cells in in situ liver tumors; in addition, the combined treatment significantly reduced the level of lung metastasis of in situ tumors.

[0019] Furthermore, by constructing subcutaneous tumor-bearing models of solid tumors in nude mice from various sources and conducting combination drug therapy, the applicant found that the combination of integrin αv inhibitor silengilide and γ-secretase inhibitor nigastric acid significantly reduced the tumor volume and weight of liver cancer, pancreatic cancer, colorectal cancer, and glioma. The combination of silengilide and nigastric acid not only inhibited tumor invasion and metastasis but also significantly suppressed the growth of in situ tumors. In addition, the combination of silengilide and nigastric acid showed similar therapeutic effects in various solid tumors, demonstrating its universality.

[0020] In conclusion, combination therapy targeting integrin αv and γ-secretase has shown significant efficacy in the treatment of solid tumors. Combination therapy using integrin αv and γ-secretase inhibitors can not only inhibit the growth of tumors in situ but also significantly suppress tumor invasion, distant metastasis, and other malignant progressions, helping to slow tumor progression, improve treatment effectiveness, and enhance patient prognosis.

[0021] Therefore, the following technical solutions should all fall within the protection scope of this invention:

[0022] This invention provides the application of integrin αv inhibitors in combination with γ-secretase inhibitors in the preparation of drugs for treating tumors.

[0023] The combination of silengiptide and nigastric acid in this application has similar therapeutic effects in a variety of tumors and is universally applicable. Obviously, tumors from other sources not mentioned in this application are also included in the scope of tumors described in this application.

[0024] Preferably, the tumor expresses integrin α. Ⅴ The tumor.

[0025] More preferably, the expression of integrin α Ⅴ The tumors are those that express integrin αvβ3 or integrin αvβ5.

[0026] Preferably, the tumor includes, but is not limited to, liver cancer, pancreatic cancer, glioma, and colorectal cancer.

[0027] Preferably, the integrin αv inhibitor is an integrin αvβ3 inhibitor or an integrin αvβ5 inhibitor.

[0028] More preferably, the integrin αvβ3 inhibitor or integrin αvβ5 inhibitor is silengitide.

[0029] Preferably, the γ-secretase inhibitor is nigasstat.

[0030] Preferably, the treatment of tumors involves inhibiting tumor growth, inhibiting local tumor invasion, or inhibiting distant metastasis.

[0031] This invention also provides the use of γ-secretase inhibitors in the preparation of products that inhibit transmembrane cleavage of integrin αv to produce functional intracellular free fragment proteins.

[0032] This invention also provides the application of γ-secretase inhibitors in the preparation of integrin αv inhibitor tumor therapeutic enhancers.

[0033] The present invention also provides a medicament for treating tumors, comprising an integrin αv inhibitor and a γ-secretase inhibitor.

[0034] Preferably, the integrin α Ⅴ The inhibitors are integrin αvβ3 inhibitors or integrin αvβ5 inhibitors.

[0035] More preferably, the integrin αvβ3 inhibitor or integrin αvβ5 inhibitor is silengitide.

[0036] Preferably, the γ-secretase inhibitor is nigasstat.

[0037] Preferably, the tumor expresses integrin α. Ⅴ The tumor.

[0038] More preferably, the expression of integrin α Ⅴ The tumors are those that express integrin αvβ3 or integrin αvβ5.

[0039] Preferably, the treatment of tumors involves inhibiting tumor growth, inhibiting local tumor invasion, or inhibiting distant metastasis.

[0040] Preferably, the drug further comprises pharmaceutically acceptable excipients and carriers.

[0041] The tumor treatment drug provided by this invention can be administered via any physiologically acceptable route, such as oral administration or injection. In the embodiments provided by this invention, silengiptide is preferably administered as an injection, and nigasstat is preferably administered as an oral solution.

[0042] Therefore, the medicament for treating tumors provided by this invention can be formulated into any pharmaceutically acceptable dosage form, suitable for different routes of administration, such as suspensions, syrups, oral solutions, injections, or any combination thereof. Generally, the active ingredient constitutes 1–95% of the total therapeutic dose.

[0043] The term "pharmaceutically acceptable" as used in this invention means that the excipient or carrier is compatible with the active pharmaceutical ingredient, preferably capable of stabilizing the active pharmaceutical ingredient and being harmless to the individual being treated.

[0044] When used to treat solid tumors, the dosage and frequency of the drug of this invention must be determined by the attending physician within the scope of reliable medical judgment. For any specific patient, the specific effective therapeutic dose level must be determined based on a number of factors, including the disorder being treated and its severity; the patient's age, weight, general health condition, sex, and diet, etc.

[0045] In actual use, the following dosages can be used as a reference: the dosage of silengiptide is 1-500 mg / kg, administered 5-7 times per week; the dosage of nigasstat is 1-100 mg / kg, administered 2-3 times per week.

[0046] More preferably, the dose of silengiptide is 75 mg / kg, five times a week; and the dose of nigasstat is 8 mg / kg, three times a week.

[0047] The final results of a Phase III clinical trial investigating the therapeutic effect of silengilide on glioblastoma patients showed that silengilide failed to achieve the expected efficacy, did not improve overall survival, and the specific reasons for the failure remain unclear. This application study concludes that the combined use of silengilide and a γ-secretase inhibitor, such as nigastric acid (LY3039478), can significantly inhibit the growth, invasion, and distant metastasis of various solid tumors, significantly improving the therapeutic effect of silengilide in various solid tumors and providing a new approach to solid tumor treatment. Furthermore, a series of in vitro and in vivo experiments verified that the main reason for the enhanced efficacy of the combination therapy is that the γ-secretase inhibitor inhibits γ-secretase-mediated transmembrane cleavage of integrin αv and the production of its free intracellular fragment; however, the integrin αv inhibitor does not inhibit this process. Therefore, the combination with a γ-secretase inhibitor mechanistically explains the failure of silengilide in the Phase III clinical trial. This combination therapy can effectively inhibit tumor progression, improve the prognosis of cancer patients, and provide a new approach to solid tumor treatment.

[0048] The present invention has the following beneficial effects:

[0049] This invention provides the application of integrin αv inhibitors in combination with γ-secretase inhibitors in the preparation of drugs for treating tumors. The combined use of integrin αv inhibitors and γ-secretase inhibitors in tumor treatment exhibits a synergistic effect, not only inhibiting in situ tumor growth but also significantly suppressing tumor invasion, distant metastasis, and other malignant progressions, thus helping to slow tumor progression, improve treatment efficacy, and enhance patient prognosis. Furthermore, this invention demonstrates that the combined use of integrin αv inhibitors and γ-secretase inhibitors can reduce the production of the intracellular free domain of integrin αv and inhibit the activation of the classical downstream pathway of integrin αv. The combined use of integrin αv inhibitors and γ-secretase inhibitors shows broad application prospects in tumor treatment. Attached Figure Description

[0050] Figure 1 Example 1 involved treatment with different combinations of integrin αv inhibitors (Cilengitide, Cyclo(-RGDfK)) and γ-secretase inhibitors (Avagacestat, Semagacestat, LY3039478) on the hepatocellular carcinoma cell line MHCC-97H. Figure 1 A) Pancreatic cancer cell line Panc-1 ( Figure 1 B) Colorectal cancer cell line SW480 ( Figure 1 C) and glioma cell line U87MG ( Figure 1 D) Experimental results on the effect of survival ability.

[0051] Figure 2 Example 2: Viability of the hepatocellular carcinoma cell line MHCC-97H after treatment with different doses of silengiptide and nigastric acid. Figure 2 A) and the results of predicting the synergistic effect of combination therapy using the SynergyFinder website ( Figure 2 B).

[0052] Figure 3 Example 3 describes the level of free intracellular fragments generated by integrin αv cleavage in the hepatocellular carcinoma cell line MHCC-97H after treatment with a combination of silengitide and nigasstat.

[0053] Figure 4 Example 4 illustrates the activation level of the integrin αv downstream signaling pathway in the hepatocellular carcinoma cell line MHCC-97H after treatment with a combination of silengiptide and nigastric acid; wherein, Figure 4 A represents the activation level of the FAK signaling pathway; Figure 4 B and Figure 4 C represents the F-actin polymerization level and statistics.

[0054] Figure 5 Example 5 shows the expression of epithelial-mesenchymal transition-related proteins in the hepatocellular carcinoma cell line MHCC-97H after treatment with a combination of silengilide and nigastric acid.

[0055] Figure 6 In Example 6, after injecting hepatocellular carcinoma cell lines into the livers of nude mice, they were treated with a combination of silengiptase and nigastric acid. Lung tissue was then obtained from the mice, and HE staining was performed on the nude mouse lungs to detect lung metastasis. Figure 6 A) and statistics Figure 6 B).

[0056] Figure 7 Example 7: Subcutaneous inoculation of nude mice with liver cancer ( Figure 7 A) Pancreatic cancer ( Figure 7 B) Colorectal cancer ( Figure 7 C) Glioma ( Figure 7 D) Statistical results of tumor volume after treatment with silengiptide and nigasstat following cell line therapy.

[0057] Figure 8 Example 7: Subcutaneous inoculation of nude mice with liver cancer ( Figure 8 A) Pancreatic cancer ( Figure 8 B) Colorectal cancer ( Figure 8 C) Glioma ( Figure 8 D) Statistical results of tumor weight after treatment with silengiptide and nigastric acid following cell line therapy. Detailed Implementation

[0058] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0059] Unless otherwise specified, all reagents used in the examples are conventional reagents available in the art and can be purchased commercially. Experimental procedures not specifically described in the examples are conventional procedures in the art or can be understood or known by those skilled in the art based on their prior knowledge or common general knowledge.

[0060] The sources of some of the drugs used in this invention are as follows:

[0061] Cilengiptide was purchased from Selleck, drug code S6387.

[0062] Nigastric was purchased from Selleck, drug code S7169.

[0063] Cyclo(-RGDfK) was purchased from Selleck, drug code S7844.

[0064] Avagacestat was purchased from Selleck, drug code S1262.

[0065] Semagacestat was purchased from Selleck, drug code S1594.

[0066] Example 1: Screening of different combinations of integrin αv inhibitors and γ-secretase inhibitors

[0067] This embodiment uses integrin αv and γ-secretase as targets for combined therapy to explore the inhibitory effects of different combinations of integrin αv inhibitors and γ-secretase inhibitors on tumors.

[0068] I. Experimental Methods

[0069] The cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C, 5% CO2, and 90% relative humidity. The cells adhered to the culture vessel and were discarded after they reached the bottom of the vessel. The cells were then digested with 0.25% trypsin and passaged in separate flasks.

[0070] The control group consisted of no drug administration and administration of an integrin αv inhibitor (Cilengitide or Cyclo(-RGDfK)) alone.

[0071] The following experimental groups were set up according to different drug combinations: Cilengitide + Avagacestat group, Cilengitide + Semagacestat group, Cilengitide + LY3039478 group, Cyclo(-RGDfK) + Avagacestat, Cyclo(-RGDfK) + Semagacestat, and Cyclo(-RGDfK) + LY3039478; a total of 6 experimental groups with different drug combinations.

[0072] Hepatocellular carcinoma cell line MHCC-97H, pancreatic cancer cell line Panc-1, colorectal cancer cell line SW480, and glioma cell line U87MG were treated with different combinations of integrin αv inhibitors (Cilengitide or Cyclo(-RGDfK)) and γ-secretase inhibitors (Avagacestat or Semagacestat or LY3039478).

[0073] The specific procedure was as follows: Logarithmic growth phase cells were used for the experiment. Before the experiment, the medium was changed using DMEM containing 10% fetal bovine serum. The corresponding drug combinations were mixed and added to the cell culture medium to reach the final concentration. The final concentrations of each drug were: Cilengitide (1 μM), Cyclo(-RGDfK) (50 nM), Avagacestat (50 nM), Semagacestat (50 μM), and LY3039478 (1 μM). After adding the drugs, the cells were placed back into an incubator at 37°C, 5% CO2, and 90% relative humidity. After 48 hours, cell viability was detected using the CellTiter-Glo kit (Promega).

[0074] II. Experimental Results

[0075] Experimental results are as follows Figure 1As shown, combination therapy with integrin αv inhibitors and γ-secretase inhibitors exhibits stronger inhibitory effects on tumor cells in various solid tumor cell lines compared to monotherapy. Among these, the combination of cilengitide and nigastricostat (LY3039478) showed the most significant therapeutic effect. Therefore, the following examples use a combination of cilengitide and nigastricostat (LY3039478).

[0076] Example 2: Culture and in vitro drug inhibition assay of human-derived hepatocellular carcinoma cell line MHCC-97H

[0077] I. Experimental Methods

[0078] 1. Culture of human-derived hepatocellular carcinoma cell line MHCC-97H:

[0079] The cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C, 5% CO2, and 90% relative humidity. The cells adhered to the culture medium and were cultured in a flask after they reached the bottom of the flask. The cells were then digested with 0.25% trypsin and passaged in separate flasks. Cells in the logarithmic growth phase were used for experiments.

[0080] 2. In vitro drug inhibition test:

[0081] The hepatocellular carcinoma cell line MHCC-97H was treated with cilengitide at different final concentrations (0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, or 30 μM), and simultaneously treated with nigasstat (LY3039478) or dimethyl sulfoxide (DMSO) at different final concentrations (0.01 μM, 0.1 μM, 1 μM, 10 μM, or 30 μM). Cell viability was assessed using the CellTiter-Glo kit (Promega) after 48 hours.

[0082] The specific method for the combined treatment of the above-mentioned hepatocellular carcinoma cell line MHCC-97H is as follows:

[0083] Logarithmic growth phase cells were used for experiments. Before drug stimulation, the medium was changed using DMEM medium containing 10% fetal bovine serum. The drugs were added to the corresponding cell culture medium according to the final concentration of DMSO, silengiptide, or nigastric acid used in each group. The cells were then placed back into an incubator at 37°C, 5% CO2, and 90% relative humidity. After 48 hours, cell viability was assessed using the CellTiter-Glo kit (Promega).

[0084] 3. Synergistic effect prediction:

[0085] Cell survival levels after different concentration combinations of drug inhibition assays were analyzed using the HSA model on the SynergyFinder website.

[0086] II. Experimental Results

[0087] The experimental results on the effect of silengiptide combined with nigastric acid at different concentration levels on the survival of hepatocellular carcinoma cell line MHCC-97H are as follows: Figure 2 As shown in Figure A, it can be seen that, compared with the inhibition by cilengitide alone, the combined use of cilengitide and nigasstat (LY3039478) can significantly inhibit the survival level of tumor cells.

[0088] Based on the HSA model prediction from the SynergyFinder website (results are as follows) Figure 2 As shown in Figure B), the synergistic index of cilengitide and nigastricostat (LY3039478) was 17.011, indicating a synergistic effect when cilengitide and nigastricostat are used in combination. Furthermore, at doses of cilengitide (1 μM) or higher and nigastricostat (LY3039478) ( ), Figure 2 The dosage combination shown in the white box in section B) shows that silengitide and nigasstat have a strong synergistic effect.

[0089] Example 3: Effect of in vitro drug inhibition on the production level of the intracellular free domain of integrin αv in the hepatocellular carcinoma cell line MHCC-97H

[0090] Based on the results of Example 2, a combination of drug concentrations with strong synergistic effects was selected: Cilengitide at a dose of 1 μM and Nigastrol (LY3039478) at a dose of 1 μM for subsequent experiments.

[0091] I. Experimental Grouping

[0092] a. Control group: Add an equal volume of solvent DMSO;

[0093] b. Cilengitide monotherapy group: Cilengitide was added to a final concentration of 1 μM;

[0094] c. Nigastrol (LY3039478) monotherapy group: Nigastrol was added to a final concentration of 1 μM;

[0095] d. Combined use of cilengitide and nigalstatin (LY3039478) experimental group: cilengitide and nigalstatin were added, with a final concentration of 1 μM for both cilengitide and nigalstatin.

[0096] II. Experimental Methods

[0097] (1) Drug inhibition experiment

[0098] According to the above experimental grouping, the liver cancer cell line MHCC-97H was treated with the corresponding dose of drug, and the cells were placed back into an incubator at 37°C, 5% CO2 and 90% relative humidity for 48 hours before detection.

[0099] (2) Extraction of total cellular protein and Western blot experiment:

[0100] After drug treatment in each experimental group, the culture medium was discarded, the cells were washed with PBS, and digested with 0.25% trypsin. Cells were lysed using RIPA lysis buffer (with added phosphatase and protease inhibitors), centrifuged at 14000g for 15 minutes at 4°C, and the supernatant was collected to obtain total cellular protein.

[0101] The total cellular protein obtained above was subjected to Western blot experiments. GAPDH levels were detected using integrin αv carboxyl-terminal specific primary antibody (Millipore ab1930) and GAPDH antibody as internal control proteins. After incubation with horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody, the images were developed using a Bio-Rad gel imaging system.

[0102] III. Experimental Results

[0103] Experimental results are as follows Figure 3 As shown, compared with the cilengitide monotherapy group, the combination of cilengitide and nigasstat (LY3039478) significantly reduced the level of integrin αv free intracellular domain (CD51-ICD).

[0104] Example 4: Effect of in vitro drug inhibition on the activation level of the classical downstream pathway of integrin αv in the hepatocellular carcinoma cell line MHCC-97H

[0105] FAK is a classic downstream pathway of integrin αv, and its main activation mechanism is phosphorylation at Tyr397.

[0106] I. Experimental Grouping

[0107] a. Control group: Add an equal volume of solvent DMSO;

[0108] b. Cilengitide monotherapy group: Cilengitide was added to a final concentration of 1 μM;

[0109] c. Nigastrol (LY3039478) monotherapy group: Nigastrol was added to a final concentration of 1 μM;

[0110] d. Combined use of cilengitide and nigalstatin (LY3039478) experimental group: cilengitide and nigalstatin were added, with a final concentration of 1 μM for both cilengitide and nigalstatin.

[0111] II. Experimental Method

[0112] 1. Drug inhibition experiment

[0113] According to the above experimental grouping, the liver cancer cell line MHCC-97H was treated with the corresponding dose of drug, and the cells were placed back into an incubator at 37°C, 5% CO2 and 90% relative humidity for 48 hours before detection.

[0114] 2. Western blot was used to detect the phosphorylation activation level of FAK:

[0115] After treatment with silengiptide (10 μM) and nigasstat (1 μM), total cell protein was extracted, labeled with FAK antibody and FAK (Tyr397) antibody, and GAPDH level was detected using GAPDH antibody as an internal control protein. After further incubation with horseradish peroxidase (HRP)-conjugated secondary antibody, the cells were developed.

[0116] 3. Detection of F-actin polymerization level using immunofluorescence staining:

[0117] After treatment with silengilide (10 μM) and nigasstat (1 μM), the cells were fixed, penetrated, labeled with phalloidin, stained with DAPI, and photographed using a confocal microscope.

[0118] III. Experimental Results

[0119] Experimental results are as follows Figure 4 As shown, compared with the cilengitide monotherapy group, the combination of cilengitide and nigasstat (LY3039478) can further inhibit the activation of the classical downstream pathway of integrin αv, including FAK phosphorylation activation. Figure 4 A) and the level of F-actin polymerization ( Figure 4 B. Figure 4 C).

[0120] Example 5: Effect of in vitro drug inhibition on the expression level of epithelial-mesenchymal transition specific marker protein in the hepatocellular carcinoma cell line MHCC-97H

[0121] Epithelial-mesenchymal transition (EMT) is a manifestation of enhanced invasive and metastatic abilities of tumor cells. Therefore, detecting the expression levels of EMT-specific marker proteins can be used to assess tumor invasiveness and metastasis. Specifically, the expression levels of N-cadherin and Vimentin are positively correlated with the level of EMT in tumor cells, while the expression level of E-cadherin is negatively correlated with the level of EMT in tumor cells.

[0122] I. Experimental Grouping

[0123] a. Control group: Add an equal volume of solvent DMSO;

[0124] b. Cilengitide monotherapy group: Cilengitide was added to a final concentration of 1 μM;

[0125] c. Nigastrol (LY3039478) monotherapy group: Nigastrol was added to a final concentration of 1 μM;

[0126] d. Combined use of cilengitide and nigalstatin (LY3039478) experimental group: cilengitide and nigalstatin were added, with a final concentration of 1 μM for both cilengitide and nigalstatin.

[0127] II. Experimental Methods

[0128] (1) Drug inhibition experiment

[0129] According to the above experimental grouping, the liver cancer cell line MHCC-97H was treated with the corresponding dose of drug, and the cells were placed back into an incubator at 37°C, 5% CO2 and 90% relative humidity for 48 hours before detection.

[0130] (2) Effect of Western blot analysis on the expression levels of epithelial-mesenchymal transition-specific proteins:

[0131] After treatment with silengidide (1 μM) and nigasstat (1 μM), total cell protein was extracted and labeled with E-cadherin (E-cad), N-cadherin (N-cad), and Vimentin antibodies, respectively. GAPDH level was detected using GAPDH antibody as an internal control protein. After incubation with secondary antibody, the cells were developed.

[0132] III. Experimental Results

[0133] Experimental results are as follows Figure 5As shown, compared with the cilengitide monotherapy group, the combination of cilengitide and nigasstat (LY3039478) can significantly inhibit the level of epithelial-mesenchymal transition in tumor cells, that is, inhibit the invasion and metastasis phenotype of tumors.

[0134] Example 6: Constructing a mouse liver orthotopic tumor-bearing model and investigating the effect of combined therapy on tumor cell metastasis.

[0135] I. Construction of an orthotopic tumor-bearing model of the mouse liver:

[0136] Log-phase hepatocellular carcinoma cell line MHCC-97H was selected, digested, and counted. MHCC-97H cells were resuspended in DMEM medium and subcutaneously implanted in nude mice. To further promote tumorigenicity, human-derived hepatic stellate cells (LX-2) were co-injected. Each nude mouse was injected with 5 × 10⁶ cells. 6 (MHCC-97H)+5×10 6 (LX-2) / 100μL. The specific injection procedure was as follows: after anesthetizing mice with sodium pentobarbital via intraperitoneal injection, the liver of the mice was exposed by laparotomy, and a subcapsular injection was performed. The mice were observed after model establishment, and drug treatment experiments were conducted 3 weeks later.

[0137] II. Experimental Grouping

[0138] a. Control group: Add an equal volume of solvent DMSO;

[0139] b. Cilengitide monotherapy group: Cilengitide was added;

[0140] c. Nigastrol (LY3039478) monotherapy group: Nigastrol was added;

[0141] d. Combined use of Cilengitide and Nigalstatin (LY3039478) experimental group: Cilengitide and Nigalstatin were added.

[0142] III. Experimental Procedure

[0143] (1) Drug Experiment

[0144] According to the experimental groups described above, the mice were administered the corresponding drugs. Cilengitide was administered at a dose of 75 mg / kg, five times a week, via intraperitoneal injection; nigastric acid (LY3039478) was administered at a dose of 8 mg / kg, three times a week, via gavage. After 3 weeks, the mice were sacrificed and tissues were obtained by perfusion.

[0145] (2) HE staining

[0146] Mouse lungs were obtained, fixed, dehydrated, embedded in paraffin, and sectioned. After staining with hematoxylin and eosin, slides were mounted, and the number of metastatic lesions in the mouse lungs was counted.

[0147] IV. Experimental Results

[0148] Experimental results are as follows Figure 6 As shown, compared with the cilengitide monotherapy group or the nigastric acid monotherapy group, the combined use of cilengitide and nigastric acid (LY3039478) can significantly reduce the level of lung metastasis of tumors, and has a significant inhibitory effect on tumor invasion and distant metastasis malignant phenotype, and has a significant therapeutic effect.

[0149] Example 7 investigates the effects of combined silengiptide and nigarstat on tumor volume and weight in various solid tumors.

[0150] I. Constructing a subcutaneous tumor-bearing model of solid tumors and detecting their volume and weight:

[0151] Four tumor cell lines from different tissue sources were selected for experiments: human hepatocellular carcinoma cell line MHCC-97H, human pancreatic cancer cell line Panc-1, human colorectal cancer cell line SW480, and human glioma cell line U87MG. Tumor cells in the logarithmic growth phase were selected, digested, and counted. The tumor cells were resuspended in DMEM medium and then subcutaneously implanted in nude mice. Each nude mouse was injected with 5 × 10⁵ cells / mL. 6 / 100μL. Mice were observed after injection, and tumor volume was measured using calipers. The calculation formula was: V=π / 6×L(length)×W(width)×H(height). Tumors were considered complete when they reached a volume of 30mm². 3 Drug treatment experiments were conducted on both sides.

[0152] II. Experimental Methods

[0153] The experiment was divided into four groups:

[0154] a. Control group: Add an equal volume of solvent DMSO;

[0155] b. Cilengitide monotherapy group: Cilengitide was added;

[0156] c. Nigastrol (LY3039478) monotherapy group: Nigastrol was added;

[0157] d. Combined use of Cilengitide and Nigalstatin (LY3039478) experimental group: Cilengitide and Nigalstatin were added.

[0158] According to the above experimental groups, the corresponding drugs were administered. Cilengitide was administered at a dose of 75 mg / kg, 5 times a week, via intraperitoneal injection; Nigastamine (LY3039478) was administered at a dose of 8 mg / kg, 3 times a week, via gavage.

[0159] The tumor size was measured every 3 days. After 3 weeks, the mice were sacrificed, and the subcutaneous tumors were removed for weighing and statistical analysis.

[0160] III. Experimental Results

[0161] Experimental results are as follows Figure 7 and Figure 8 As shown; where, Figure 7 The results of experiments on the effect of silengiptide and nigasstat combination therapy on tumor volume in various solid tumors; Figure 8 The results of an experiment on the effect of silengiptide and nigasstat combination therapy on tumor weight in various solid tumors; Figure 7 A, Figure 8 A is the experimental result of the human-derived liver cancer cell line MHCC-97H. Figure 7 B. Figure 8 B represents the experimental results of the human pancreatic cancer cell line Panc-1. Figure 7 C Figure 8 C represents the experimental results of the human colorectal cancer cell line SW480. Figure 7 D、 Figure 8 D shows the experimental results of the human-derived glioma cell line U87MG.

[0162] It is evident that, compared with the cilengitide monotherapy group or the nigasstat monotherapy group, the combined use of cilengitide and nigasstat (LY3039478) can significantly inhibit the volume and weight of tumors in subcutaneous tumor-bearing models of liver cancer, pancreatic cancer, colorectal cancer, and glioma, and has a significant inhibitory effect on the growth of various solid tumors.

[0163] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of integrin αv inhibitors combined with γ-secretase inhibitors in the preparation of drugs for treating tumors. The integrin αv inhibitor is Cilengitide. The γ-secretase inhibitor is LY3039478. The tumor is liver cancer.

2. A drug for treating tumors, characterized in that, It includes an integrin αv inhibitor and a γ-secretase inhibitor, wherein the integrin αv inhibitor is Cilengitide. The γ-secretase inhibitor is LY3039478. The tumor is liver cancer.

3. The drug according to claim 2, characterized in that, The drug also contains pharmaceutically acceptable excipients.

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