Application of paliprevir in the preparation of drugs for the treatment of osteosarcoma

Abstract

This invention belongs to the field of targeted drug technology, and provides the application of paliprevir in the preparation of drugs for treating osteosarcoma. Paliprevir treats osteosarcoma by inhibiting the proliferation and migration of osteosarcoma cells and promoting osteosarcoma cell apoptosis. This invention also provides the application of paliprevir in the preparation of drugs that enhance the sensitivity of osteosarcoma cells to chemotherapeutic drugs. Paliprevir, when used in combination with chemotherapeutic drugs, enhances the sensitivity of osteosarcoma cells to chemotherapeutic drugs and can be used to treat patients with chemotherapy-resistant osteosarcoma.

Description

Technical Field

[0001] This invention belongs to the field of targeted drug technology, and in particular relates to the application of paliprevir in the preparation of drugs for the treatment of osteosarcoma. Background Technology

[0002] Osteosarcoma is the most common primary malignant bone tumor, frequently occurring in children and adolescents. Current standard treatment includes surgical resection and preoperative and postoperative adjuvant chemotherapy, with commonly used chemotherapy drugs including methotrexate, doxorubicin, and cisplatin. However, some patients develop metastases (primarily lung metastases). After metastasis, the disease progresses rapidly, and existing surgical and chemotherapy regimens are often insufficient to effectively control metastatic lesions. Furthermore, chemotherapy resistance is a frequent problem. All of these factors contribute to a sharp decline in the five-year survival rate for patients with metastatic or recurrent osteosarcoma. Therefore, there is an urgent need to find a treatment for osteosarcoma and a chemotherapy sensitizer. Summary of the Invention

[0003] In view of the shortcomings of the prior art, the present invention provides the application of paliprevir in the preparation of a drug for treating osteosarcoma, the purpose of which is to solve the problems mentioned in the background art.

[0004] In a first aspect, the present invention provides the use of paliprevir in the preparation of a medicament for the treatment of osteosarcoma.

[0005] Furthermore, the drug treats osteosarcoma by inhibiting the proliferation and migration of osteosarcoma cells and promoting osteosarcoma cell apoptosis.

[0006] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0007] Furthermore, the drug is formulated into a clinically acceptable dosage form.

[0008] Furthermore, the dosage form includes tablets, pills, capsules, patches, or injections.

[0009] Secondly, the present invention provides the use of paliprevir in the preparation of a medicament that enhances the sensitivity of osteosarcoma cells to chemotherapeutic drugs.

[0010] Furthermore, the chemotherapy drug is cisplatin.

[0011] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0012] Furthermore, the drug is formulated into a clinically acceptable dosage form.

[0013] Thirdly, the present invention provides a pharmaceutical composition for the combined treatment of patients with chemotherapy-resistant osteosarcoma, the pharmaceutical composition comprising cisplatin and parizurevir.

[0014] The present invention has the following technical effects: (1) Paliprevir treats osteosarcoma by inhibiting the proliferation and migration of osteosarcoma cells and promoting osteosarcoma cell apoptosis.

[0015] (2) When used in combination with chemotherapy drugs, parizurevir can effectively enhance the sensitivity of osteosarcoma cells to chemotherapy drugs and can be used to treat patients with chemotherapy-resistant osteosarcoma. Attached Figure Description

[0016] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures: Figure 1 This is a graph showing the experimental results of the effect of paliprevir on the proliferation ability of osteosarcoma cells in Example 1 of this invention, wherein: Figure 1 In the figure, A represents the experimental results of CCK-8 assay for detecting the IC50 of a drug; Figure 1 Figures B and C in the diagram show the results of the CCK-8 assay for detecting osteosarcoma cell activity. Figure 1 B in the equation changes over time. Figure 1 In the figure, C represents the change with parizurevir concentration, ** indicates P < 0.01, and *** indicates P < 0.001; Figure 1 Figures D and E in the diagram represent the results of a clonogenic assay for osteosarcoma cell proliferation. Figure 1 In the diagram, D represents the results of a clonogenic experiment. Figure 1 E in the graph represents the clonal formation rate. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0017] Figure 2 This is a diagram showing the experimental results of the effect of paliprevir on osteosarcoma cell apoptosis in Example 2 of the present invention, wherein: Figure 2 Figures A and B in the diagram show the results of an immunofluorescence assay for osteosarcoma cell apoptosis. Figure 2 In the image, A represents the result of immunofluorescence staining. Scale bar: 20μm. Figure 2 In the graph, B represents the statistical analysis of BAX protein expression intensity. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. Figure 2 The figure shown in Figure C is the result of Western blotting assay for detecting osteosarcoma cell apoptosis.

[0018] Figure 3 This is a graph showing the results of the Transwell assay for detecting osteosarcoma cell migration ability in Example 3, where: Figure 3 In the image, A represents the staining result from the Transwell experiment; Figure 3In the graph, B represents the statistical results of the relative cell proportion (migrating cells). * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0019] Figure 4 The results are from the CCK-8 assay for cell viability in Example 4. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. Detailed Implementation

[0020] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0022] This invention provides the use of paliprevir in the preparation of a medicament for the treatment of osteosarcoma.

[0023] In some embodiments, the drug treats osteosarcoma by inhibiting the proliferation and migration of osteosarcoma cells and promoting osteosarcoma cell apoptosis.

[0024] In some embodiments, the medicament may also include pharmaceutically acceptable excipients.

[0025] In some embodiments, the drug is formulated into a clinically acceptable dosage form.

[0026] In some embodiments, the dosage form includes tablets, pills, capsules, patches, or injections.

[0027] In some embodiments, the present invention provides the use of paliprevir in the preparation of a medicament that enhances the sensitivity of osteosarcoma cells to chemotherapeutic drugs.

[0028] In some embodiments, the chemotherapy drug is cisplatin.

[0029] In some embodiments, the medicament may also include pharmaceutically acceptable excipients.

[0030] In some embodiments, the drug is formulated into a clinically acceptable dosage form.

[0031] In some embodiments, the present invention provides a pharmaceutical composition for the combined treatment of patients with chemotherapy-resistant osteosarcoma, the pharmaceutical composition comprising cisplatin and pariravir.

[0032] Experimental methods: 1. Cell viability assay (CCK-8 assay): Cell preparation: Osteosarcoma cells (143B and U2OS) in logarithmic growth phase were digested with trypsin, resuspended, counted, and the cell density was adjusted to an appropriate range.

[0033] Cell seeding: Add 100 μL of cell-containing culture medium to each well of a 96-well plate. For typical cell proliferation experiments, the number of cells per well is 2000. Incubate at 37°C. Pre-culture in an incubator for 24 hours to ensure full cell adhesion and recovery.

[0034] Drug treatment: Remove the old culture medium and add fresh culture medium containing different concentrations of drugs according to the experimental design, and continue to culture for 24 hours, 48 ​​hours or 72 hours.

[0035] CCK-8 assay: Add 10 μL of CCK-8 solution to each well and incubate in the dark for 2 hours. After incubation, measure the absorbance (OD value) at 450 nm using a microplate reader.

[0036] 2. Cloning experiment: Day 1: Cell Seeding and Drug Treatment Preparation of single-cell suspensions: Osteosarcoma cells in the logarithmic growth phase (143B and U2OS) were digested with trypsin, resuspended, and counted. Ensure the cell suspension was in good condition and free of cell clumps.

[0037] Cell seeding: Seed 1000 cells per well in a 6-well plate (final culture medium volume added to each well is 2 ml).

[0038] Cell adhesion: Gently shake to distribute cells evenly, then place in a container at 37°C. Incubate the cells in an incubator for 24 hours until they adhere completely.

[0039] Drug addition process: Remove the old culture medium and add 2 ml of fresh culture medium containing different concentrations of drug. Set up 3 replicates for each drug concentration to ensure data reliability.

[0040] Control group: Culture medium containing only an equal amount of solvent (DMSO).

[0041] Days 7-14: Cloning and Interpretation of Results Continuous culture: Return the cells to the incubator and continue culturing, replacing the medium with fresh drug-containing medium every 3-4 days. The culture time is typically 7-14 days, or until clearly visible cell clonal clusters are formed. Culture can be terminated when the control group cell clonal cluster reaches 50 cells.

[0042] A cell cluster of ≥50 cells can be counted as one clone.

[0043] Cell fixation: Gentle washing: Aspirate the culture medium and gently wash the cells 1-2 times with PBS.

[0044] Fixation: Add 1 ml of paraformaldehyde and fix at room temperature for 15-30 minutes. Then remove the fixative and allow the well plate to air dry naturally.

[0045] Cell staining: Staining: Add 1 ml of 0.1% crystal violet staining solution to each well and stain at room temperature for 15-30 minutes.

[0046] Washing: After recovering the staining solution, slowly rinse the well plate with running PBS or tap water until the background is clean and the clones are clearly visible.

[0047] Photography and counting: Invert the plate, let it dry, and take photos with a camera or scanner for record-keeping. Count the number of clones in each well under a microscope.

[0048] 3. Immunofluorescence experiment: Day 1: Cell Seeding and Treatment Cell preparation: Osteosarcoma cells in the logarithmic growth phase are digested, resuspended, counted, and seeded onto cell spreaders placed in confocal culture dishes or 24-well plates. The density is usually [missing information - likely a specific density per well (24-well plate)]. Each cell was used to ensure that the cell confluence was approximately 70-80% at the end of the experiment.

[0049] Treatment: If drug treatment is required, replace the drug-containing culture medium and treat for the appropriate time (e.g., 24 hours).

[0050] Wall-mounted: 37℃ Incubate overnight in an incubator to allow the cells to adhere fully to the incubator wall.

[0051] Day 2: Fixation, permeabilization, blocking, and immunostaining Fixation: Aspirate the culture medium and gently wash once with PBS. Add 4% paraformaldehyde (fix at room temperature for 15-20 minutes). After fixation, wash three times with PBS, 5 minutes each time.

[0052] Permeability testing: Add 0.1-0.5% Triton X-100 (diluted with PBS) and incubate at room temperature for 10-15 minutes. Wash three times with PBS, 5 minutes each time.

[0053] Blocking: Add blocking solution (e.g., 5% BSA) and block at room temperature for 1 hour.

[0054] Primary antibody incubation: Dilute the primary antibody with blocking buffer (1:100-1:500). Incubate overnight in a humidified chamber at 4°C (or 2 hours at room temperature).

[0055] Secondary antibody incubation: Recover the primary antibody, wash three times with PBST (PBS + 0.1% Tween-20), 10 minutes each time. Dilute the fluorescent secondary antibody with blocking buffer (protect from light, 1:200-1:500), and incubate at room temperature for 1 hour. All subsequent steps should be performed in the dark.

[0056] Nuclear counterstaining: Wash three times with PBST, 5 minutes each time. Add DAPI staining solution (1:1000 dilution) and incubate at room temperature for 5-10 minutes. Wash three times with PBST, 5 minutes each time.

[0057] Mounting and observation: Add anti-fluorescence quenching mounting medium and cover with a coverslip. Observe and photograph using a fluorescence microscope or confocal microscope.

[0058] 4. Transwell experiment: Day 1: Preparation of cell suspension and Transwell setup Preparation of single-cell suspension: Digest cells, resuspend in serum-free medium, and count. Adjust density to... cells / mL.

[0059] Transwell preparation: Place the Transwell chamber (24-well plate size, 8μm pore diameter) into the matching well plate.

[0060] Add cells to the upper chamber: Add 200 μL of cell suspension (i.e., ...) to the upper chamber. (cells).

[0061] Add chemotactic agent to the lower chamber: Add 700 μL of complete culture medium containing 20% ​​FBS to the lower chamber.

[0062] Incubation: Place the chamber in an incubator and incubate for 24 hours.

[0063] Day 2: Fixation, staining, and counting Terminate culture: Remove the chamber and gently wipe away any cells that have not penetrated the upper chamber with a cotton swab.

[0064] Fixation: Immerse the chamber in pre-cooled methanol for 10 minutes to fix.

[0065] Staining: Immerse the chamber in 0.1% crystal violet staining solution and stain at room temperature for 15-30 minutes.

[0066] Cleaning and observation: Gently rinse the chamber with PBS and wipe the inner surface of the upper chamber with a cotton swab. Turn the chamber over and randomly select 5-10 fields of view under a microscope (100-200x) to count the number of cells on the lower surface of the membrane.

[0067] Data analysis: Calculate the average number of cells per field of view.

[0068] Example 1: (1) Set up control group: osteosarcoma cells 143B and U2OS were cultured in complete medium without drugs (0 μM parizurevir); parizurevir treatment group: osteosarcoma cells 143B and U2OS were treated in complete medium containing different concentrations of parizurevir.

[0069] To evaluate the effect of paliprevir on the short-term proliferative capacity of osteosarcoma cells, osteosarcoma cells 143B and U2OS from the control group and the paliprevir-treated group were subjected to cell viability assays using the CCK-8 assay.

[0070] The results of the CCK-8 assay for detecting the IC50 (half-maximal inhibitory concentration) of a drug are as follows: Figure 1 As shown in Figure A, the results indicate that parizurevir has a significant inhibitory effect on the proliferation of osteosarcoma cells.

[0071] The results of the CCK-8 assay for cell viability are as follows: Figure 1 As shown in B and C, the results indicate that parizurevir can significantly inhibit the activity of osteosarcoma cells in a concentration- and time-dependent manner.

[0072] (2) Set up control group: osteosarcoma cells 143B and U2OS were cultured in complete medium without drugs (0 μM parizurevir); parizurevir treatment group: osteosarcoma cells 143B and U2OS were treated in complete medium containing different concentrations (5, 10, 20 μM) parizurevir.

[0073] To evaluate the effect of parizurevir on the long-term proliferative capacity of osteosarcoma cells, colony formation experiments were conducted on osteosarcoma cells 143B and U2OS from the control group and the parizurevir-treated group.

[0074] Cloning experiment results as follows Figure 1 As shown in D and E, the results indicate that parizurevir treatment significantly reduces the clonogenic ability of osteosarcoma cells.

[0075] Example 2: Control group: Osteosarcoma cells 143B and U2OS were cultured in complete medium without drugs (0 μM parizurevir); Parizurevir treatment group: Osteosarcoma cells 143B and U2OS were treated in complete medium containing different concentrations (5, 10, 20 μM) of parizurevir.

[0076] To evaluate the effect of paliprevir on osteosarcoma cell apoptosis, osteosarcoma cells 143B and U2OS from the control group and the paliprevir-treated group were subjected to immunofluorescence detection of cell apoptosis and Western blotting detection of apoptosis-related protein expression.

[0077] Immunofluorescence assay results of apoptosis are as follows Figure 2As shown in A and B, the results indicate that paliravir treatment can induce significant apoptosis in osteosarcoma cells. The results of Western blotting detection of apoptosis-related proteins are as follows... Figure 2 As shown in C, the results indicate that paliprevir treatment promotes the expression of anti-apoptotic protein (Bcl-XL) while inhibiting the expression of pro-apoptotic protein (BAX), further confirming its apoptosis-inducing effect.

[0078] Example 3: Control group: Osteosarcoma cells 143B and U2OS were cultured in complete medium without drugs (0 μM parizurevir); Parizurevir treatment group: Osteosarcoma cells 143B and U2OS were treated in complete medium containing different concentrations (5, 10, 20 μM) of parizurevir.

[0079] To evaluate the effect of paliprevir on the migration ability of osteosarcoma cells, osteosarcoma cells 143B and U2OS from the control group and the paliprevir-treated group were subjected to Transwell assays.

[0080] The results of the Transwell assay for cell migration ability are as follows: Figure 3 As shown in the figure, the results indicate that parizurevir can significantly inhibit the migration ability of osteosarcoma cells.

[0081] Example 4: The following groups were established: Individual chemotherapy groups were established, in which osteosarcoma cells 143B and U2OS were treated with chemotherapy drugs (cisplatin) alone (0 μM paliprevir); Paliprevir combined with chemotherapy groups were established, in which osteosarcoma cells 143B and U2OS were treated with different concentrations of paliprevir (5, 10, 20 μM) and chemotherapy drugs (cisplatin).

[0082] To assess the effect of pariprevir on the sensitivity of chemotherapy drugs, osteosarcoma cells 143B and U2OS from the chemotherapy alone group and the pariprevir plus chemotherapy group were subjected to cell viability assays using the CCK-8 assay.

[0083] The results of cell viability detection by the CCK-8 assay are as follows: Figure 4 As shown, the results indicate that, compared with cisplatin alone, paliprevir combined with cisplatin significantly reduced osteosarcoma cell activity, suggesting that paliprevir can effectively enhance the sensitivity of osteosarcoma cells to chemotherapeutic drugs.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of paliprevir in the preparation of drugs for treating osteosarcoma.

2. The application as described in claim 1, characterized in that: The drug treats osteosarcoma by inhibiting the proliferation and migration of osteosarcoma cells and promoting their apoptosis.

3. The application as described in claim 2, characterized in that: The drug also includes pharmaceutically acceptable excipients.

4. The application as described in claim 3, characterized in that: The drug is formulated into a clinically acceptable dosage form.

5. The application as described in claim 4, characterized in that: The dosage forms include tablets, pills, capsules, patches, or injections.

6. Application of palirevir in combination with cisplatin in the preparation of drugs for the treatment of osteosarcoma.

7. The application as described in claim 6, characterized in that: The drug also includes pharmaceutically acceptable excipients.

8. The application as described in claim 7, characterized in that: The drug is formulated into a clinically acceptable dosage form.

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