Use of tegaserod in the treatment of renal cancer

Abstract

The application discloses application of tegaserod in treatment of renal cancer. It is found for the first time that tegaserod maleate and sunitinib are combined to use, and the sensitivity of sunitinib-resistant renal cancer cells to sunitinib can be recovered, and it is found that the combination of the two has a significant synergistic effect on the treatment of renal cancer. The application provides a brand-new treatment scheme for the technical field of renal cancer treatment, and has important scientific significance and broad clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of tegaserod in the treatment of renal cell carcinoma. Background Technology

[0002] Renal cell carcinoma (RCC) is a malignant tumor originating from renal tubular epithelial cells and is one of the most common malignant tumors that seriously threaten human health. Despite improvements in early diagnosis and treatment, approximately 40%–50% of patients are already at an advanced stage at initial diagnosis or progress to an advanced stage during follow-up, resulting in a poor prognosis for advanced RCC. Receptor tyrosine kinase inhibitors, such as sunitinib, are first-line targeted therapies for advanced RCC, significantly improving efficacy compared to previous cytokine therapies. Although treatment options for advanced RCC are constantly evolving, targeted therapies remain the cornerstone of its treatment.

[0003] Sunitinib is a receptor tyrosine kinase inhibitor that inhibits the activity of multiple receptor tyrosine kinases, including epidermal growth factor receptor, platelet-derived growth factor receptor, and vascular endothelial growth factor receptor, thereby suppressing tumor angiogenesis and cell proliferation and ultimately effectively controlling tumor progression. It plays a crucial role in the treatment of renal cell carcinoma (RCC). Sunitinib is a first-line targeted therapy for renal cell carcinoma. However, a significant number of RCC patients develop resistance to sunitinib within 1-2 years of treatment, leading to a substantial decrease in its efficacy and posing a new challenge to RCC treatment. Therefore, increasing sunitinib sensitivity and improving its efficacy are of significant clinical importance for prolonging the survival of renal cell carcinoma patients. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the purpose of this invention is to provide a drug that can increase the sensitivity of sunitinib and improve the efficacy of sunitinib.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides any one of the following applications, the application comprising: 1) The use of tegaserod or a pharmaceutically acceptable salt thereof in the preparation of drugs that enhance the sensitivity of patients with renal cell carcinoma to sunitinib; 2) Use of tegaserod or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving the therapeutic effect of sunitinib on renal cell carcinoma; 3) The use of tegaserod or its pharmaceutically acceptable salt in combination with sunitinib in the preparation of a drug for the treatment of renal cell carcinoma; 4) Use of sunitinib in the preparation of medicaments for improving the therapeutic effect of tegaserod or its pharmaceutically acceptable salts on renal cell carcinoma; 5) Use of tegaserod or its pharmaceutically acceptable salts in the preparation of medicaments for the treatment of renal cell carcinoma.

[0006] Specific pharmaceutically acceptable salts include those salts that, within the bounds of reliable medical judgment, are suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and in proportion to a reasonable benefit or risk. Pharmaceutically acceptable salts (pharmaceutically acceptable salts) are well known in the art. Pharmaceutically acceptable salts of the compounds described in this invention include salts derived from suitable inorganic and organic acids and inorganic and organic bases.

[0007] Furthermore, the pharmaceutically acceptable salts include maleate, hydrochloride, methanesulfonate, benzenesulfonate, fumarate, succinate, malate, acetate, citrate, tartrate, lactate, sulfate, or phosphate.

[0008] In a specific embodiment of the present invention, the pharmaceutically acceptable salt is maleate.

[0009] In this invention, tegaserod maleate, TM, and tegaserod maleate are synonymous. Its trade name is Zemarco, and its molecular formula is C. 20 H 27 N5O5 has a molecular weight of 417.459.

[0010] Furthermore, the dosage forms of the drug include non-gastrointestinal dosage forms and / or gastrointestinal dosage forms.

[0011] Furthermore, the non-gastrointestinal dosage forms include injectable dosage forms, cavity dosage forms, mucosal dosage forms, and / or skin dosage forms.

[0012] Furthermore, the injectable dosage forms include, but are not limited to, various injectable preparations such as intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections.

[0013] Furthermore, the cavity drug delivery dosage forms include, but are not limited to: suppositories, aerosols, effervescent tablets, drops, and pills.

[0014] Furthermore, the mucosal drug delivery dosage forms include, but are not limited to: eye drops, nasal drops, ophthalmic ointments, mouthwashes, sublingual tablets, adhesive tablets, and films.

[0015] Furthermore, the skin delivery dosage forms include, but are not limited to: topical solutions, lotions, liniments, ointments, plasters, pastes, patches, etc.

[0016] Furthermore, the gastrointestinal dosage forms include tablets, granules, capsules, solutions, powders, sustained-release preparations, emulsions, suspensions, syrups, and / or drops.

[0017] A second aspect of the present invention provides a pharmaceutical composition for treating kidney cancer.

[0018] Furthermore, the pharmaceutical composition comprises tegaserod or a pharmaceutically acceptable salt thereof and sunitinib.

[0019] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or excipients.

[0020] Furthermore, the pharmaceutically acceptable salt is maleate.

[0021] Furthermore, the dosage ratio of tegaserod or its pharmaceutically acceptable salt to sunitinib is 1:1 to 1:8.

[0022] In this invention, the pharmaceutically acceptable carriers and / or excipients include, but are not limited to: diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

[0023] In some embodiments, the diluent includes, but is not limited to, lactose, sodium chloride, glucose, urea, starch, and water. The binder includes, but is not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, alginate and alginates, xanthan gum, and hydroxypropyl cellulose. The surfactant includes, but is not limited to, sodium lauryl sulfate, glyceryl monostearate, and hexadecyl alcohol. The humectant includes, but is not limited to, glycerol and starch. The adsorbent carrier includes, but is not limited to, starch, lactose, bentonite, and soap clay. The lubricant includes, but is not limited to, zinc stearate, talc, calcium and magnesium stearate, polyethylene glycol, polyoxyethylene monostearate, monolauric sucrose, and magnesium lauryl sulfate. The filler includes, but is not limited to, mannitol, xylitol, sorbitol, maltose, glucose, lactose, sucrose, dextrin, and starch. The disintegrants include, but are not limited to: crosylvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, crosylcarboxymethyl cellulose sodium, soybean polysaccharides, etc.

[0024] Furthermore, the pharmaceutical composition is a single compound preparation or a combination of two separate single preparations.

[0025] Furthermore, the compound preparation is a compound preparation comprising tegaserod or a pharmaceutically acceptable salt thereof and sunitinib.

[0026] Furthermore, the combination of the single-agent formulations is a combination of a single-agent formulation containing tegaserod or a pharmaceutically acceptable salt thereof and a single-agent formulation containing sunitinib.

[0027] Furthermore, in the combination of the single-component preparations, the two single-component preparations are applied simultaneously or sequentially.

[0028] In some embodiments, tegaserod or its pharmaceutically acceptable salts and sunitinib in the pharmaceutical composition may be administered simultaneously, separately, or sequentially. Simultaneous administration means that the two drugs are administered concurrently. If not simultaneous, they are administered sequentially within a time frame so that both are therapeutically effective within the same time frame. Therefore, sequential administration allows for administration of one drug 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, or several hours after administering one drug, provided that the circulating half-life of the first administered drug allows for the simultaneous presence of therapeutically effective amounts of both. The time delay between administrations of the components will vary depending on the exact nature of the components, their interactions, and their respective half-lives. This differs from simultaneous or sequential administration, which refers to a significant interval between administration of one drug and another, i.e., when the second drug is administered, the first administered drug may no longer be present in the bloodstream at a therapeutically effective amount.

[0029] In some embodiments, the pharmaceutical composition or pharmaceutical preparation may also contain other drugs or reagents that can be used to treat and prevent kidney cancer. There are no particular limitations on the other drugs or reagents, as long as they can produce the expected therapeutic and / or preventive, or adjuvant therapeutic and / or preventive effects on kidney cancer, they are all within the scope of protection of this invention.

[0030] In some embodiments, the other drugs or agents that can be used to treat renal cell carcinoma include, but are not limited to: chemotherapy drugs, specifically, the chemotherapy drugs include, but are not limited to: taxanes (paclitaxel, docetaxel, cabazitaxel), antibiotics (doxorubicin, epirubicin, daunorubicin, pirarubicin, etoposide, irinotecan, mitoxantrone), and antimetabolites (cisplatin, carboplatin, lobaplatin, nedaplatin, oxaliplatin, gemcitabine, 5-hydroxychloroquine). Fluorouracil and alkylating agents (cyclophosphamide, ifosfamide).

[0031] In some embodiments, the appropriate dosage of the pharmaceutical composition or pharmaceutical preparation described in this invention can be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, condition, diet, administration time, route of administration, excretion rate, and responsiveness. Skilled physicians can usually easily determine the prescription and the desired dosage that is effective for treatment, as long as it can produce the expected therapeutic and / or preventive effect on renal cell carcinoma. Such dosage is within the protection scope of this invention.

[0032] The third aspect of the present invention provides a method for inhibiting the proliferation, clonogenic ability, migration and invasion ability of renal cancer cells and / or promoting apoptosis of renal cancer cells in vitro for non-therapeutic purposes.

[0033] Furthermore, the method includes treating renal cell carcinoma cells with the pharmaceutical composition described in the second aspect of the present invention.

[0034] This invention demonstrates through experiments that the pharmaceutical composition has a significant synergistic effect in inhibiting the growth and proliferation of renal cell carcinoma cell lines. Therefore, the pharmaceutical composition can be used as an inhibitor for non-therapeutic purposes to suppress the growth and proliferation of renal cell carcinoma cells in the field of scientific research, such as to further study the growth and metabolic mechanisms or behavior of renal cell carcinoma cell lines and to screen potential drugs that can be used to treat renal cell carcinoma.

[0035] The fourth aspect of this invention provides a method for inhibiting the proliferation, migration, invasion, and angiogenesis of sunitinib-resistant renal cell carcinomas in vitro for non-therapeutic purposes.

[0036] Furthermore, the method includes treating renal cell carcinoma cells with tegaserod or a pharmaceutically acceptable salt thereof.

[0037] Furthermore, the pharmaceutically acceptable salt is maleate.

[0038] The fourth aspect of this invention provides the use of tegaserod or a pharmaceutically acceptable salt thereof in combination with sunitinib in the preparation of an in vitro non-therapeutic agent for inhibiting the proliferation, colony formation, migration and invasion of renal cell carcinoma cells and / or promoting apoptosis of renal cell carcinoma cells.

[0039] Furthermore, the pharmaceutically acceptable salt is maleate.

[0040] The fifth aspect of this invention provides the use of tegaserod or a pharmaceutically acceptable salt thereof in the preparation of an in vitro, non-therapeutic agent for inhibiting the proliferation, migration, invasion, and angiogenesis of sunitinib-resistant renal cell carcinomas.

[0041] Furthermore, the pharmaceutically acceptable salt is maleate.

[0042] The present invention also provides a method for treating renal cell carcinoma, the method comprising: administering tegaserod or a pharmaceutically acceptable salt thereof or the pharmaceutical composition described in the second aspect of the present invention to a subject in need.

[0043] Furthermore, the subjects include mammals and non-mammals, preferably humans.

[0044] Advantages and beneficial effects of the present invention: This invention is the first to discover that the combined use of tegaserod maleate and sunitinib can restore the sensitivity of sunitinib-resistant renal cell carcinoma cells to sunitinib, and also reveals a significant synergistic effect of the two in the treatment of renal cell carcinoma. This invention provides a novel treatment option for the field of renal cell carcinoma treatment and has broad clinical application prospects. Attached Figure Description

[0045] Figure 1 This study demonstrated the therapeutic efficacy of tegaserod maleate (TM) in combination with sunitinib (Su) against renal cell carcinoma xenografts in mice; among which, Figure 1 A represents the tumor growth status. Figure 1 Image B is a picture of tumor tissue. Figure 1 C represents the tumor weight result. Figure 1 D represents the expression levels of proliferation and apoptosis markers.

[0046] Figure 2 This study demonstrates the effects of tegaserod maleate (TM) on the in vitro migration, invasion, and angiogenesis of sunitinib-resistant renal cell carcinomas; among which... Figure 2 A represents the results of the scratch test. Figure 2 B represents the results of the Transwell experiment. Figure 2 C represents the results of the angiogenesis experiment.

[0047] Figure 3 This study demonstrates the effects of tegaserod maleate (TM) combined with sunitinib (Su) on the proliferation and apoptosis of renal cell carcinoma cells; among which, Figure 3 A represents the result of the cell proliferation capacity assay. Figure 3 B represents the result of the clone-forming ability test. Figure 3 C represents the result of apoptosis detection. Figure 3 D represents the synergistic effect calculation result. Detailed Implementation

[0048] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents.

[0049] The drugs, reagents and raw materials used in this invention are readily available to those skilled in the art and can be obtained commercially unless otherwise specified. Experimental methods not specified in this invention are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0050] The materials used in the following embodiments of the present invention are sourced from the following sources: Renal cell carcinoma cells CAKI-1 and A498 were provided by ATCC. Sunitinib-resistant cell lines CAKI-1-R and A498-R were induced using a half-maximal concentration (WMC) escalation method. MyCoys 5A and RPMI 1640 basal media were purchased from Gibco. Fetal bovine serum was purchased from Zetalife. Cell Counting Kit-8 reagents were purchased from DOJINDO, and apoptosis kits and cell cycle reagents were purchased from eBioscience. Tegaserod maleate was purchased from MCE (HY-14153A); other reagents were domestically produced analytical grade. Nude mice were purchased from Jiangsu Jicui Pharmaceutical Co., Ltd.

[0051] The experimental methods of the following embodiments of the present invention are as follows: 1. Cell culture: A498-R was grown in RPMI 1640 medium containing 10% fetal bovine serum, and CAKI-1-R was grown in RPMI 1640 medium containing 10% fetal bovine serum. Both were cultured at 37°C in a 5% CO2 humidified incubator and passaged every other day.

[0052] 2. CCK-8 cell proliferation experiment: Cell proliferation assays were performed using the DOJINDO Cell Proliferation and Cytotoxicity Assay Kit (CCK-8): (1) Take cells in the logarithmic growth phase and seed them in 96-well plates at 3000 cells / well. Add culture medium containing different drugs to each well to a final volume of 100 μL. Incubate at 37℃ and 5% CO2. Set up 3 replicates and 3 control wells (blank control with no cells and only culture medium).

[0053] (2) After cell seeding, start timing at 0h. At 0h, 24h, 48h and 72h, add 10μL of CCK-8 solution to the experimental wells and incubate at 37℃ for 4h.

[0054] (3) The absorbance at a wavelength of 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) monitor. The control was zeroed, and the cell growth curve was plotted with time as the x-axis and absorbance as the y-axis.

[0055] 3. Cloning experiment: (1) Take cells in the logarithmic growth phase and seed them at 1000 cells / well in a 6-well plate. Add culture medium containing different drugs to each well to a final volume of 2 mL. Incubate at 37°C with 5% CO2. Set up 3 replicates.

[0056] (2) Continue culturing for 7-14 days, changing the medium regularly during this period.

[0057] (3) When obvious clonal formation occurs, discard the culture medium, rinse with PBS, and fix the cells in 4% paraformaldehyde at room temperature for 20 minutes.

[0058] (4) Discard the paraformaldehyde, rinse with PBS, add crystal violet staining solution, and stain at room temperature for 30 minutes. Recover the crystal violet staining solution from each well, rinse the 6-well plate with distilled water to remove residual staining solution.

[0059] (5) After the 6-well plate is dried, observe the cells on the surface of the 6-well plate and take pictures to record the data. Count the number of cell clones in each group.

[0060] 4. Apoptosis experiment: (1) Take cells in the logarithmic growth phase and seed them in a 6-well plate. Add culture medium containing different drugs to each well to a final volume of 3 ml. Incubate at 37°C and 5% CO2. Set up 3 replicate wells and 3 control wells (DMSO).

[0061] (2) Collect cells, take 100 μL of cell suspension and resuspend in a flow cytometer, add Annexin V-APC and 7-AAD flow cytometry antibodies respectively, and incubate at room temperature in the dark for 15 min.

[0062] (3) Flow cytometry was used to detect cell apoptosis, with the DMSO group as the control, and the proportion of apoptotic cells was compared.

[0063] 5. Cell scratch assay: (1) Pretreatment of drug-resistant renal cell carcinoma cells with complete culture medium containing the corresponding concentration of TM for 48 hours.

[0064] (2) After rinsing with PBS, adherent cells were digested with TE, and digestion was terminated with complete culture medium. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in basal culture medium to prepare a cell suspension. The cells were quantitatively seeded into a six-well plate. When the cells adhered to the wall and the cell confluence was close to 90%, a 200 μL pipette tip was attached to make a scratch. The cell migration was observed regularly.

[0065] 6. Cell migration experiment: (1) Pretreatment of drug-resistant renal cell carcinoma cells with complete culture medium containing the corresponding concentration of TM for 48 hours.

[0066] (2) After rinsing with PBS, adherent cells were digested with TE buffer, and digestion was terminated with complete culture medium. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in basal culture medium to prepare a cell suspension. After cell counting, the cell density was adjusted to 1×10⁶ cells / mL. 5 Cells were evenly seeded into 8.0 μm wells of a 24-well Transwell plate, with 200 μL of cell suspension added to each well, resulting in a cell count of 2 × 10⁶ cells per well. 4 indivual.

[0067] (3) Add 600 μL of complete culture medium to the lower chamber of the Transwell chamber of the 24-well plate to avoid air bubbles between the Transwell chamber and the liquid at the bottom. Then place the 24-well plate in a cell culture incubator for culture.

[0068] (4) After culturing for 15 hours, gently remove the cells that have not yet migrated from the upper surface of the Transwell chamber using a cotton swab. Discard the supernatant from each well of the 24-well plate, add 1 ml of PBS and gently rinse the cells on the lower surface of the Transwell chamber. Repeat the PBS rinsing twice.

[0069] (5) Immerse the Transwell chamber in 4% paraformaldehyde and fix the cells at room temperature for 20 minutes. Then transfer the Transwell chamber to PBS and rinse gently. Repeat the PBS rinsing twice.

[0070] (6) Immerse the Transwell chamber in crystal violet staining solution and stain at room temperature for 30 minutes. Recover the crystal violet staining solution from each well and rinse the Transwell chamber with distilled water to remove any residual staining solution.

[0071] (7) After the Transwell chamber is dried, observe the cells on the lower surface of the Transwell chamber under an inverted optical microscope and take pictures to record the data. Count the number of cells that migrated in each group.

[0072] 7. Cell invasion experiment: (1) Pretreatment of drug-resistant renal cell carcinoma cells with complete culture medium containing the corresponding concentration of TM for 48 hours.

[0073] (2) Take the matrix gel out of the -20℃ freezer in advance and place it on ice to thaw. All pipette tips, 1.5 ml EP tubes and basal culture medium used in the operation related to the matrix gel need to be pre-cooled in the 4℃ freezer in advance and taken out when needed.

[0074] (3) Perform the operation on ice. Dilute the matrix gel with pre-cooled basal medium at a ratio of 1:8. After mixing evenly, add 50 μL of diluted matrix gel to each 8.0 μm Transwell chamber and make it evenly cover the upper surface of the bottom of the chamber. Then place the 24-well plate containing the Transwell chamber in a cell culture incubator and incubate for 45 minutes.

[0075] (4) After rinsing with PBS, adherent cells were digested with TE, and digestion was terminated with complete culture medium. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in basal culture medium to prepare a cell suspension. The cells were counted and the cell density was adjusted to 2 × 10⁶ cells / mL. 5Cells were then evenly seeded into 8.0 μm wells of a 24-well Transwell plate, with 200 μL of cell suspension added to each well, resulting in a cell count of 4 × 10⁶ cells per well. 4 indivual.

[0076] (5) Add 600 μL of complete culture medium to the lower chamber of the Transwell compartment of the 24-well plate, taking care to avoid air bubbles forming between the Transwell compartment and the liquid at the bottom. Place the 24-well plate in a cell culture incubator for incubation.

[0077] (6) After culturing for 20 hours, carefully remove the uninvaded cells and residual matrix gel from the upper surface of the Transwell chamber using a cotton swab. Discard the supernatant from each well of the 24-well plate, add 1 ml of PBS and gently rinse the cells on the lower surface of the Transwell chamber. Repeat the PBS rinse twice.

[0078] (7) Immerse the Transwell chamber in 4% paraformaldehyde and fix the cells at room temperature for 20 minutes. Then transfer the Transwell chamber to PBS and rinse gently. Repeat the PBS rinsing twice.

[0079] (8) Immerse the Transwell chamber in crystal violet staining solution and stain at room temperature for 30 minutes. Recover the crystal violet staining solution from each well and rinse the Transwell chamber with distilled water to remove any residual staining solution from the surface of the chamber.

[0080] (9) After the Transwell chambers are dried, observe the cells on the lower surface of the Transwell chambers under an inverted optical microscope and take pictures to record the data. Count the number of cells that invaded the lower surface of the chambers in each group.

[0081] 8. Angiogenesis experiment: (1) Melt the matrix gel at 4 degrees and apply 10 μL of matrix gel per well evenly to the 48-well plate on ice. Place it in a 4°C refrigerator for 30 minutes, then transfer it from the refrigerator to an incubator for 30 minutes until the gel dries.

[0082] (2) Digest the vascular endothelial cells (HUVECs) treated with the appropriate concentration of TM, centrifuge and discard the liquid, and seed 10,000 / 500 uL / well into 48-well plates. Observe the angiogenesis regularly.

[0083] 9. Chicken embryo intracellular blood vessel detection experiment: (1) Incubate the fertilized chicken eggs at 37°C and 60%–70% relative humidity. On the 7th day of embryonic development, disinfect the eggs with ethanol and make a small hole with a diameter of about 10 mm in the air cell.

[0084] (2) Subsequently, 200 μL of the corresponding concentration of TM was gently injected into each chorioallantoic membrane. The entire procedure was performed under sterile conditions. On day 5 of incubation, the CAM was imaged, and the number of blood vessels around the drug administration area in each CAM was counted and analyzed.

[0085] 10. Subcutaneous tumor formation in nude mice: Collect 1×10 6 Cells were resuspended in 200 μL of PBS and aseptically injected into the armpits of 6-8 week old male nude mice. One week later, tumors appeared in the armpits of the nude mice, confirming successful tumor formation. The mice were given the drug and weighed every other day to observe the size of the tumors and their vital status.

[0086] 11. Immunohistochemical detection: (1) Place the paraffin sections of tissue into a glass slide staining rack and bake in a 60°C oven for 120 minutes.

[0087] (2) Soak the slices in a dyeing vat containing xylene for 10 minutes, then transfer them to the next xylene dyeing vat and soak for 10 minutes, then transfer them to the next xylene dyeing vat and soak for another 10 minutes.

[0088] (3) Transfer the slices to a dyeing vat containing anhydrous ethanol and soak for 3 minutes, transfer them to another anhydrous ethanol dyeing vat and soak for 3 minutes, transfer them to a 95% ethanol dyeing vat and soak for 3 minutes, transfer them to an 85% ethanol dyeing vat and soak for 3 minutes, and then transfer them to a 75% ethanol dyeing vat and soak for 3 minutes.

[0089] (4) Immerse the slides in a staining jar containing distilled water for 5 minutes, and repeat the immersion in distilled water twice. Heat the staining jar containing sodium citrate buffer in a microwave oven for 3 minutes beforehand, then immerse the slides in sodium citrate buffer and microwave intermittently for 10 minutes to perform antigen retrieval.

[0090] (5) Carefully remove the sodium citrate buffer staining jar from the microwave oven and allow it to cool naturally at room temperature for 45 minutes. Then, immerse the slides in a staining jar containing distilled water for 5 minutes, and repeat the immersion in distilled water.

[0091] (6) Mix Tween-20 and PBS at a ratio of 1:1000 to prepare PBST washing solution. Immerse the slides in a staining jar containing PBST washing solution for 5 minutes, and repeat the PBST washing solution immersion of the slides.

[0092] (7) Place the sections in a staining jar containing 3% H2O2-methanol solution and incubate at room temperature for 15 minutes to block peroxidase in the tissue. Then, immerse the sections in a staining jar containing PBST washing solution for 5 minutes, and repeat the PBST washing solution immersion twice.

[0093] (8) Place the slides in a humidified chamber, aspirate any remaining liquid from the slides, use a histochemical pen to circle the tissue on the slides, add 50 μL of 3% BSA-PBST to each slide, and block at room temperature for 30 minutes. Then aspirate the blocking solution, add 100 μL of primary antibody solution to each slide, and incubate the humidified chamber overnight at 4°C.

[0094] (9) The next day, remove the wet box from the refrigerator and allow it to warm to room temperature for 40 minutes. Then, place the slides in a staining jar containing PBST washing solution and wash for 5 minutes. Repeat the washing with PBST washing solution twice. Place the slides in the wet box, aspirate any remaining liquid from the slides, add 50 μL of secondary antibody solution to each slide, and incubate at room temperature for 60 minutes.

[0095] (10) Immerse the slides in a staining jar containing PBST washing solution for 5 minutes, and repeat the PBST washing solution immersion twice. Prepare DAB colorimetric solution, place the slides in a humidified chamber, remove the residual liquid on the slides, add 50 μL DAB colorimetric solution to each slide, develop the color at room temperature for 1 minute, and stop the colorimetric reaction by rinsing with distilled water.

[0096] (11) Place the slides in a humidified chamber, remove any residual liquid from the slides, add 50 μL of hematoxylin staining solution to each slide, stain at room temperature for 1 minute, and then rinse thoroughly with distilled water. Place the slides in a staining jar containing 1% HCl-ethanol solution for decolorization, remove them quickly after 5 seconds, and rinse them in distilled water to terminate the decolorization reaction. Then, immerse the slides in PBST washing solution for inversion blue.

[0097] (12) Soak the slices in a dyeing vat containing 75% ethanol for 3 minutes, then transfer them to a dyeing vat containing 85% ethanol for 3 minutes, then transfer them to a dyeing vat containing 95% ethanol for 3 minutes, then transfer them to a dyeing vat containing anhydrous ethanol for 3 minutes, and finally transfer them to the next dyeing vat containing anhydrous ethanol for 3 minutes.

[0098] (13) Transfer the slice to a dyeing vat containing xylene and soak for 10 minutes, then transfer it to the next xylene dyeing vat and soak for 10 minutes, then transfer it to the next xylene dyeing vat and soak for another 10 minutes.

[0099] (14) Remove the slide from the xylene staining vat, drop a small amount of neutral resin near the tissue on the slide, and cover with a coverslip. After the slide has dried in a fume hood, observe the staining of each tissue under an optical microscope and take photos for record.

[0100] Example 1: Sequencing analysis of clinical renal cell carcinoma samples The inventors collected 115 renal cell carcinoma samples treated with sunitinib (Su) and performed multi-omics sequencing analysis including Western blotting (WES), transcriptomics, proteomics, and phosphoproteomics. Proteomic analysis revealed that the N6-adenosine monophosphate (m6A) recognition protein YTHDF1 (Gene ID: 54915) was significantly upregulated in sunitinib-resistant renal cell carcinoma patients, with YTHDF1 protein expression increasing 8.63-fold in the resistant group samples. P =0.003).

[0101] Example 2: In vivo experiments demonstrating the application of tegaserod maleate in the treatment of renal cell carcinoma. Based on the clinical findings of Example 1, the inventors further investigated the role of tegaserod maleate, a small molecule inhibitor targeting YTHDF1, in renal cell carcinoma.

[0102] Tegaserod maleate (TM) was purchased from MCE. Six- to eight-week-old nude mice were randomly divided into three groups of five mice each. CAKI-1-R cells were injected subcutaneously to induce tumor formation. Tumors were induced when the mice reached a size of 300 mm. 3 Mice in three groups were treated with saline, sunitinib, TM, and sunitinib combined with TM, respectively. The TM dose was 5 mg / kg, and the sunitinib dose was 40 mg / kg. Tumor volume and mouse weight were recorded every 2-3 days. On day 18, the xenografts were removed and their size was recorded. The tumor tissue was then fixed, embedded, sectioned, stained with H&E, and immunohistochemically analyzed to detect Ki-67 and Caspase-3 expression levels.

[0103] In vivo mouse xenograft models confirmed that the combined use of sunitinib (Su) and tegaserod maleate (TM) can restore the sensitivity of sunitinib-resistant renal cell carcinoma cells to sunitinib. Figure 1 Compared with the control group and the single-drug group, the combination therapy group showed a significant slowdown in tumor growth. Figure 1 A) The tumor volume and weight were significantly reduced ( Figure 1 B- Figure 1 C). Moreover, the combination therapy group showed lower expression of the tumor tissue proliferation marker Ki-67 and higher expression of the apoptosis marker Cleaved-Caspase3. Figure 1 D).

[0104] Example 3: In vitro experimental evaluation of the antitumor effect of TM in sunitinib-resistant renal cell carcinoma cells Sunitinib (Su) long-term treatment of CAKI-1 and A-498 cells yielded sunitinib-resistant cell lines CAKI-1-R and A498-R. After treating sunitinib-resistant CAKI-1 (6 μM) and A-498 (4 μM) renal cell carcinoma cells with TM for 48 hours, scratch assays were performed to assess cell migration, and Transwell assays were performed to assess cell migration and invasion. Cell proliferation was assessed using CCK-8 assays and colony formation assays after treatment with Su (CAKI-1-R 5 μM, A498-R 3 μM) and TM (CAKI-1 6 μM, A-498 4 μM) as monotherapy or in combination. Synergy Finder was used to calculate the synergistic effect of the two drugs to confirm their efficacy. The effect of TM on angiogenesis was assessed after treatment of human umbilical vein endothelial cells (HUVECs) with TM.

[0105] Scratch assays showed that TM significantly inhibited the migration ability of sunitinib-resistant renal cell carcinomas at the same time interval. Figure 2 A). Similarly, Transwell assays showed that TM significantly inhibited the migration and invasion of sunitinib-resistant renal cell carcinomas (A). Figure 2 B). Angiogenesis experiments have shown that TM can also inhibit angiogenesis ( Figure 2 C). In vitro cell experiments also confirmed that TM combined with Su can significantly reduce cell proliferation capacity (C). Figure 3 A) Cloning ability ( Figure 3 B) and the proportion of apoptosis ( Figure 3 C). Furthermore, the synergistic index of TM combined with Su was calculated using the Synergy Finder model. In both cell types, the synergistic index was greater than 10, indicating that the two cells produced a synergistic effect. Figure 3 D).

[0106] In summary, the results above suggest that tegaserod maleate, a drug clinically used for irritable bowel syndrome, may enhance the efficacy of sunitinib in patients with renal cell carcinoma and reduce sunitinib resistance.

[0107] It should be understood that after reading the above description of the present invention, those skilled in the art can make various alterations or modifications to the relevant conditions of the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The following application, characterized in that, The applications include: 1) The use of tegaserod or a pharmaceutically acceptable salt thereof in the preparation of drugs that enhance the sensitivity of patients with renal cell carcinoma to sunitinib; 2) Use of tegaserod or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving the therapeutic effect of sunitinib on renal cell carcinoma; 3) The use of tegaserod or its pharmaceutically acceptable salt in combination with sunitinib in the preparation of a drug for the treatment of renal cell carcinoma; 4) Use of sunitinib in the preparation of medicaments for improving the therapeutic effect of tegaserod or its pharmaceutically acceptable salts on renal cell carcinoma; 5) Use of tegaserod or its pharmaceutically acceptable salts in the preparation of medicaments for the treatment of renal cell carcinoma.

2. The application according to claim 1, characterized in that, The pharmaceutically acceptable salts include maleate, hydrochloride, methanesulfonate, benzenesulfonate, fumarate, succinate, malate, acetate, citrate, tartrate, lactate, sulfate, or phosphate. Preferably, the pharmaceutically acceptable salt is a maleate.

3. The application according to claim 1, characterized in that, The dosage forms of the drug include non-gastrointestinal dosage forms and / or gastrointestinal dosage forms; Preferably, the non-gastrointestinal dosage form includes injectable dosage form, cavity dosage form, mucosal dosage form and / or skin dosage form; Preferably, the gastrointestinal dosage form includes tablets, granules, capsules, solutions, powders, sustained-release preparations, emulsions, suspensions, syrups, and / or drops.

4. A pharmaceutical composition for treating renal cell carcinoma, characterized in that, The pharmaceutical composition comprises tegaserod or a pharmaceutically acceptable salt thereof and sunitinib.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipients; Preferably, the pharmaceutically acceptable salt is a maleate.

6. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition is a single compound preparation or a combination of two separate single preparations; Preferably, the combination formulation is a combination formulation comprising tegaserod or a pharmaceutically acceptable salt thereof and sunitinib; Preferably, the combination of the single-agent formulations is a combination of a single-agent formulation containing tegaserod or a pharmaceutically acceptable salt thereof and a single-agent formulation containing sunitinib; Preferably, the two single-component formulations in the combination are applied simultaneously or sequentially.

7. A method for inhibiting the proliferation, colony formation, migration and invasion of renal cell carcinoma cells, and / or promoting apoptosis of renal cell carcinoma cells in vitro for non-therapeutic purposes, characterized in that, The method includes treating renal cell carcinoma cells with the pharmaceutical composition according to any one of claims 4-6.

8. A method for inhibiting the proliferation, migration, invasion, and angiogenesis of sunitinib-resistant renal cell carcinomas in vitro for non-therapeutic purposes, characterized in that, The method includes treating renal cell carcinoma cells with tegaserod or a pharmaceutically acceptable salt thereof; Preferably, the pharmaceutically acceptable salt is a maleate.

9. The use of tegaserod or a pharmaceutically acceptable salt thereof in combination with sunitinib in the preparation of an in vitro non-therapeutic agent for inhibiting the proliferation, colony formation, migration and invasion of renal cell carcinoma cells and / or promoting apoptosis of renal cell carcinoma cells; Preferably, the pharmaceutically acceptable salt is a maleate.

10. The use of tegaserod or a pharmaceutically acceptable salt thereof in the preparation of reagents for the in vitro non-therapeutic purposes of inhibiting the proliferation, migration, invasion, and angiogenesis of sunitinib-resistant renal cell carcinomas; Preferably, the pharmaceutically acceptable salt is a maleate.

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