Use of paritaprevir in the preparation of a medicament for treating colorectal cancer, use of paritaprevir in combination with a PD-1 antibody in the preparation of a medicament for treating colorectal cancer

Abstract

The present application relates to the technical field of colorectal cancer treatment, in particular to a kind of paliperidone in preparation colorectal cancer drug, paliperidone and PD-1 antibody combined use in preparation colorectal cancer drug application.The present application is based on virtual screening and molecular docking technology, using FDA approved anticancer drug library identifies the drug Paritaprevir (paliperidone) capable of directly targeting PLS3, and provides Paritaprevir combined with PD-1 targeted antibody treatment regimen, optimizes the dilemma that the effect of PD-1 targeted antibody monotherapy is not ideal, so that more colorectal cancer patients benefit.

Description

Technical Field

[0001] This invention relates to the field of colorectal cancer treatment technology, and particularly to the application of palipvir in the preparation of a drug for colorectal cancer treatment, and the application of palipvir in combination with an antibody in the preparation of a drug for colorectal cancer treatment. Background Technology

[0002] The immunosuppressive microenvironment induced by high expression of the immunosuppressive checkpoints PD-1 / PD-L1 is a key factor in the development and progression of colorectal cancer. However, despite the availability of PD-1 / PD-L1-targeting antibodies in clinical use, their therapeutic efficacy in colorectal cancer patients remains unsatisfactory.

[0003] Immunotherapy combination strategies can better address the challenges of ineffective monotherapy; therefore, focusing on the development of immunotherapy combination strategies and sensitizing existing treatment regimens are important means and approaches for cutting-edge clinical development. Numerous studies have demonstrated that PD-L1 expression levels determine the efficacy of targeted antibody therapy; therefore, elucidating the regulatory mechanisms of PD-L1 expression and developing new combination therapy strategies based on this is crucial for improving immunotherapy. Protein-protein interactions (PPIs) closely regulate specific cellular biological processes by influencing interacting proteins at multiple levels, such as protein stability, post-translational modifications, and cellular sublocalization. Approximately 80% of proteins function by forming complexes through interactions with other proteins. Dysfunction of PPI processes is closely related to cancer development and progression.

[0004] The "drug repurposing" strategy refers to using approved or investigational drugs for new medical indications. This strategy offers several advantages, including shorter development cycles and lower costs. Paritaprevir is a drug used to treat chronic hepatitis C. As a non-structural protein 3 / 4A (NS3 / 4A) protease inhibitor, it blocks viral replication, thereby exerting an antiviral effect. However, its inhibitory efficacy in other diseases has not yet been extensively explored in the field. Summary of the Invention

[0005] Based on virtual screening and molecular docking technology, this invention identifies Paritaprevir, a drug that can directly target PLS3, using an FDA-approved anticancer drug library. It also provides a treatment regimen combining Paritaprevir with a PD-1 targeted antibody, which optimizes the unsatisfactory efficacy of PD-1 targeted antibody monotherapy and benefits more colorectal cancer patients.

[0006] To achieve the above objectives, the present invention provides the use of palipvir in the preparation of a medicament for the treatment of colorectal cancer.

[0007] Preferably, in the preparation of a drug for treating colorectal cancer, the palipvir inhibits the tumor growth of colorectal cancer by targeting and inhibiting the expression of the PLS3 protein.

[0008] Preferably, the concentration of the hepatitis C treatment drug palipvir is 3.8 μg / mL to 22.9 μg / mL.

[0009] Under the same technical concept, the present invention also provides the use of palipvir in combination with PD-1 antibody in the preparation of a medicament for the treatment of colorectal cancer.

[0010] In their research on colorectal cancer, the applicant used proteomics to identify Plastin3 (PLS3), an actin-binding protein that interacts with PD-L1. PLS3 is conserved throughout eukaryotic evolution and is expressed in most tissues of higher eukaryotes. Experiments revealed that PLS3 can promote immune escape from colorectal cancer by interacting with PD-L1 and upregulating PD-L1 expression, thus identifying a novel immunosuppressive factor for colorectal cancer, PLS3, that upregulates PD-L1 expression. Further experiments demonstrated that paritaprevir can directly bind to PLS3 and inhibit the expression of both PLS3 and PD-L1 proteins, producing a synergistic inhibitory effect.

[0011] Preferably, the mass ratio of palipvir to PD-1 antibody is 0.3-2:1.

[0012] Preferably, the PD-1 antibody includes Selleck Biotech's A2122 Anti-mouse PD-1 (CD279)-InVivo.

[0013] Under the same technical concept, the present invention also provides a pharmaceutical composition for treating colorectal cancer, the pharmaceutical composition comprising an effective amount of palipvir and a PD-1 antibody.

[0014] Preferably, the pharmaceutical composition further comprises one or more of a pharmaceutically acceptable excipient, diluent, or carrier.

[0015] The above-described solution of the present invention has the following beneficial effects: (1) This invention provides an application scheme for the hepatitis C treatment drug Paritaprevir in the preparation of a drug for the treatment of colorectal cancer; experiments have shown that Paritaprevir can directly bind to PLS3 protein and inhibit the expression of PLS3 and PD-L1 proteins. Experiments in a C57BL / 6 mouse colorectal cancer subcutaneous tumor model with normal immune function have shown that Paritaprevir alone can inhibit the growth of colorectal cancer tumors, providing a new use for Paritaprevir and a new scheme for the inhibition of colorectal cancer; (2) This invention provides an application scheme for the preparation of a drug for the treatment of colorectal cancer by combining Paritaprevir with a PD-1 targeting antibody, which solves the problem that the existing PD-1 targeting antibody monotherapy for colorectal cancer is not effective; based on the interaction mechanism between PLS3 and PD-L1 proteins, it shows a significant synergistic inhibitory effect on the growth of colorectal cancer. (3) The immunosuppressive microenvironment is considered an important characteristic of colorectal cancer patients. Therefore, screening key immunomodulatory factors can provide a theoretical basis for improving the immunosuppressive microenvironment of colorectal cancer. We identified PLS3 as a key molecule that promotes immune escape of colorectal cancer cells, providing new insights into the formation of the immunosuppressive microenvironment of colorectal cancer. We also screened Paritaprevir, a drug that targets PLS3, which can significantly inhibit PLS3 expression and tumor growth in colorectal cancer. This provides a key basis for future targeted inhibition of the oncogenic effect of PLS3 in other cancer types. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Immunohistochemical staining was performed to detect the expression level of PLS3 in colorectal cancer tissue (left) and adjacent colorectal mucosa tissue (right); Figure 2 Overall survival prognostic analysis of colorectal cancer patients with high and low PLS3 expression groups; Figure 3 Nude mice were used as control and PLS3 overexpression groups to develop subcutaneous tumor models, and tumor volume and weight were measured. Figure 4 To establish C57BL / 6 subcutaneous tumor models in immune normal mice as the control group and the PLS3 overexpression group, tumor volume and weight were measured. Figure 5Western blot was used to detect the expression of PLS3 and PD-L1 proteins in subcutaneous tumor tissues of C57BL / 6 mice; Figure 6 Western blot was used to detect the interaction between exogenous PLS3 and PD-L1 proteins (a) and the interaction between endogenous PLS3 and PD-L1 proteins (b). Figure 7 Western blot was used to detect changes in PD-L1 protein expression after gradient overexpression of PLS3 in SW480 colorectal cancer cells. Figure 8 The survival rate of cancer cells in SW480 cells was detected by crystal violet staining after treatment with activated T cells before and after PLS3 overexpression. Figure 9 For the recovery experiment of C57BL / 6 mouse colorectal cancer subcutaneous tumor model, the control group, PLS3 overexpression group, PD-L1 knockdown group, and PLS3 overexpression + PD-L1 knockdown group were included, and the tumor volume and weight were measured. Figure 10 A schematic diagram of the molecular docking between Paritaprevir and PLS3 protein; Figure 11 Cellular thermal displacement experiments were conducted in HEK-293T cells with a gradually increasing temperature gradient. The expression changes of PLS3 protein were detected with and without the addition of Paritaprevir. Figure 12 RKO cells were treated with different concentrations of Paritaprevir, and the expression of PLS3 and PD-L1 proteins was detected by Western blot. Figure 13 The T-cell killing assay was used to detect the sensitivity of T cells to colorectal cancer cells before and after Paritaprevir treatment, and crystal violet staining was used to detect cancer cell survival rate. Figure 14 A C57BL / 6 mouse subcutaneous tumor model was constructed using MC38 cells. Tumor volume and weight were measured after treatment with PD-1 targeted antibody, Paritaprevir, and a combination of Paritaprevir and PD-1 targeted antibody. Detailed Implementation

[0018] To make the technical problems, solutions, and advantages of this invention clearer, a detailed description will be provided below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0019] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0020] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a locking connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0021] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0022] Example 1: Study on the interaction mechanism between PLS3 and PD-L1 proteins To identify PD-L1 interacting proteins in colorectal cancer, the applicant used proteomic proteometry to identify PLS3, a key PD-L1 interacting protein, in the human colorectal cancer cell line RKO. The interaction between PLS3 and PD-L1 in colorectal cancer is unclear, suggesting that PLS3 may be a key protein crosstalking with PD-L1 in colorectal cancer. Therefore, we focused our research on PLS3. The applicant collected cancer tissue and adjacent colorectal mucosal tissue samples from 25 paired colorectal patients at Xiangya Hospital and performed immunohistochemical (IHC) staining analysis of PLS3. The specific procedures are as follows: 1. Dewaxing of sections: After placing the wax block sections on a glass slide, bake them in an oven at 65℃ for 30 minutes, and then dewax them sequentially with xylene-xylene-100% alcohol-100% alcohol-95% alcohol-90% alcohol-80% alcohol-70% alcohol, leaving each reagent for 5 minutes.

[0023] 2. Antigen retrieval: Prepare 2L of immunohistochemical antigen retrieval buffer (citrate retrieval solution, pH 6.0) (Zhongshan Jinqiao, catalog number: ZLI-9065) and add it to the pressure cooker for antigen retrieval. After the pressure cooker is steamed, cook for 3 minutes. Rinse the pressure cooker with cold water to cool it down and then remove the slide.

[0024] 3. Inactivation: Immediately immerse the slide in 3% hydrogen peroxide solution for 15 minutes, then rinse twice with clean water.

[0025] 4. Oil-based pen stroke organization range (Zhongshan Jinqiao, catalog number: ZLI-9305).

[0026] 5. Wash twice: Place the slide in PBS for 5 minutes, wash twice, and wipe the surrounding tissue dry with PBS solution.

[0027] 6. Serum blocking: Use goat serum (Zhongshan Jinqiao, catalog number: ZLI-9056) for blocking and place in a 37℃ incubator for 30 minutes.

[0028] 7. Add primary antibody: Remove the slide from the incubator, wipe the serum around the tissue on the reverse and front sides of the slide with absorbent paper, dilute the PLS3 primary antibody with antibody dilution buffer (Zhongshan Jinqiao, catalog number: ZLI-9028) (antibody: dilution buffer volume dilution ratio is 1:100, PLS3 antibody purchased from Proteintech Reagent Company, Cat No.12917-1-AP), and incubate overnight at 4°C.

[0029] 8. Incubation with reaction enhancement solution: After removing the slide from the refrigerator, allow it to warm to room temperature for 1 hour. Then wash it three times with PBS for 5 minutes each time. After wiping the PBS around the tissue dry, incubate it in a 37°C incubator for 20 minutes using the reaction enhancement solution from the PV9000 kit from Zhongshan Jinqiao Biotechnology Co., Ltd.

[0030] 9. Add secondary antibody: After removing the slide from the incubator, wash it three times with PBS for 5 minutes each time. After wiping the PBS around the tissue dry, incubate it in a 37°C incubator for 20 minutes using the enhanced enzyme-labeled goat anti-mouse / rabbit IgG polymer (i.e., secondary antibody) from the PV9000 kit of Zhongshan Jinqiao Biotechnology Co., Ltd.

[0031] 10. Add DAB chromogenic solution: After removing the slide from the incubator, wash it three times with PBS for 5 minutes each time. After wiping the PBS around the tissue dry, add DAB chromogenic solution (Zhongshan Jinqiao, catalog number: ZLI-9018) and develop the color for 5-8 minutes. Pay attention to the color development degree; brown indicates a positive result.

[0032] 11. Counterstaining: After rinsing the developed slide with water for a period of time, immerse it in hematoxylin staining solution (Servicebio, product number: G1004) for 3-5 minutes, then rinse with tap water.

[0033] 12. Hematoxylin differentiation: Add the hematoxylin-stained slide to the hematoxylin differentiation solution (Servicebio, product number: G1039) for 3-5 seconds to differentiate, and then rinse with tap water.

[0034] 13. Dehydration: Place the slide in the following order of reagents in 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, 100% ethanol, 100% ethanol, xylene, xylene, and so on for 2 minutes in each reagent.

[0035] 14. Mounting: Apply a drop of neutral resin next to the tissue, cover it with a coverslip, and then place the slide in a fume hood to dry.

[0036] The results showed that PLS3 expression was significantly upregulated in colorectal cancer tissues (see...). Figure 1 Prognostic analysis showed that patients with high PLS3 expression had significantly shorter overall survival than patients with low PLS3 expression (see...). Figure 2 The above results indicate that PLS3 may act as a tumor-promoting factor in colorectal cancer.

[0037] We constructed a stable PLS3 overexpressing cell line and a control cell line using the murine colorectal cancer cell line MC38. Immunodeficient nude mice and immunocompetent C57BL / 6 mice were then subcutaneously injected with the stably overexpressing cell line to establish a subcutaneous colorectal cancer tumor model. Tumor size was monitored 7 days after injection (the monitoring start point was recorded as day 0). The length (mm) and width (mm) of the subcutaneous tumor in mice were measured every 3 days, and the tumor volume was calculated accordingly (calculation formula: volume (mm)). 3 = length x width 2 / 2), after 15 days of monitoring, subcutaneous tumors were collected and weighed in mice. The results showed no significant difference in tumor volume and weight between the PLS3 overexpressing and control groups (see Figure 3 In contrast, in immune-normal C57BL / 6 mice, PLS3 overexpression significantly increased tumor volume and weight compared to the control group (see...). Figure 4 The above results indicate that PLS3 can promote colorectal cancer progression by regulating anti-tumor immunity. Next, we extracted proteins from subcutaneous tumor tissues of C57BL / 6 mice and performed Western blot analysis. The results showed that the PD-L1 expression level in the tumor tissues of the PLS3 stably overexpressing group was also significantly higher than that in the control group (see...). Figure 5 ).

[0038] Next, in vitro cell experiments were performed. After co-transfecting HEK-293T cells with FLAG-PD-L1 and GFP-PLS3 plasmids, we then performed co-immunoprecipitation (Co-IP) experiments, as follows: We co-transfected 1.5 μg of FLAG-PD-L1 overexpression plasmid and 1.5 μg of GFP-PL3 overexpression plasmid into HEK-293T cells in 6 cm cell culture dishes. After 48 h of transfection, cells were collected and lysed for 2 h using Western blot with a mixture of protease inhibitor Cocktail (APExBIO, Cat No: K1007) and 400 μL of IP cell lysis buffer (Beyotime, P0013). The supernatants were collected by centrifugation at 4 °C and used for immunoprecipitation experiments. Cells were divided into three groups: Input (80 μL), IgG (120 μL), and IP (200 μL). The IP group used GFP as bait protein and added GFP antibody (UTIBODY, product number: UM3002), while the IgG group used IgG antibody (HUABIO, product number: HA1027). The antibody-to-lysis buffer volume ratio was 1:100, and the cells were incubated overnight on a rotary shaker at 4 °C. The following day, 30 μL of Protein A / G Plus agarose gel (BIOLINKEDIN, L-2204A) was added and incubated overnight at 4°C on a rotary shaker. Then, 800 μL of Western blot and IP cell lysis buffer were used to wash three times at 4°C for 15 min each time. After centrifugation at 4°C and aspiration of the lysis buffer, 60 μL of 2x protein loading buffer was added to each group. Finally, Western blot experiments were performed to detect the interaction between PLS3 and PD-L1 proteins.

[0039] Experimental results showed an interaction between PLS3 and PD-L1 proteins, which was subsequently confirmed by endogenous Co-IP experiments in RKO cells (see [link to study]). Figure 6 This indicates that PLS3 and PD-L1 proteins can interact.

[0040] Further, PLS3 was overexpressed in the colorectal cell line SW480 cells using a gradient overexpression method, as follows: PLS3 was overexpressed in the SW480 colorectal cancer cell line by transfection with 0 μg, 0.5 μg, 1 μg, and 2 μg of PLS3 overexpression plasmid, respectively. Changes in PD-L1 protein expression were then detected. The results showed that PD-L1 protein expression was upregulated with increasing PLS3 expression levels (see...). Figure 7 This further demonstrates that PLS3 can upregulate the expression of PD-L1 protein in colorectal cancer cells.

[0041] Next, the T-cell killing experiment was conducted as follows: To further clarify the effect of PLS3 overexpression on the sensitivity of T cells to killing colorectal cancer cells, we then activated human peripheral blood mononuclear cells. Activation method: 1. CD3 antibody plating: Prepare PBS containing CD3 antibody (BioLegend, Cat: 317326) for plating 12-well cell culture plates. The concentration of CD3 antibody working solution is 1.25 μg / mL. Add 500 μL of PBS to each well and incubate overnight at 4°C.

[0042] 2. Activation of peripheral blood mononuclear cells: The next day, peripheral blood mononuclear cells were cultured in 12-well plates containing 10% fetal bovine serum in cell culture medium treated with CD28 antibody (BioLegend, Cat: 302934) and interleukin-2 (IL-2, Novoprotein, Cat No: GMP-CD66) for 5-7 days. 1 mL of culture medium was added to each well. The working solution concentration of CD28 antibody was 0.5 μg / mL, and the working solution concentration of IL-2 was 10 ng / mL.

[0043] 3. T cell killing: After PLS3 overexpression and transfection of SW480 cells with control plasmids, tumor cells were seeded into 12-well plates (7×10⁶ cells / wells). 5 (1 cell / well) and cultured for 24 hours. Then, activated peripheral blood mononuclear cells were added at a ratio of 1:5 of tumor cells to peripheral blood mononuclear cells, and cultured for another 24 hours.

[0044] 4. Crystal violet staining: After co-culturing for 24 hours, discard the old culture medium and wash the cells three times with PBS on a shaker for 3 minutes each time. Fix the cells with 4% paraformaldehyde at room temperature for 30 minutes, and then stain with 0.1% crystal violet to assess tumor cell death.

[0045] The results showed that PLS3 overexpression significantly inhibited the killing sensitivity of T cells against colorectal cancer cells (see...). Figure 8 ).

[0046] We continued with in vivo reversion experiments, specifically as follows: We constructed an MC38 cell line stably overexpressing PLS3 combined with PD-L1 knockdown (using shRNA that targets and inhibits murine PD-L1 expression) and a stable MC38 cell line with PD-L1 knockdown alone. The experiment was conducted in four groups: a control group, a PLS3 overexpression group, a PD-L1 knockdown group, and a PLS3 overexpression + PD-L1 knockdown group. Each group consisted of five C57BL / 6 mice, each subcutaneously injected with 1x10... 6 Cells were used for in vivo recovery experiments. Tumor size was monitored 7 days after injection (monitoring start point recorded as day 0). The length (mm) and width (mm) of the tumor were measured every three days, and the tumor volume was calculated using the formula (mm). 3 = length x width 2 / 2, after 15 days of monitoring, the tumor was collected and weighed.

[0047] The results showed that the immune escape effect induced by PLS3 overexpression in colorectal cancer could be reversed by PD-L1 knockdown, indicating that PLS3 promotes immune escape from colorectal cancer cells by upregulating PD-L1 (see...). Figure 9 The above results indicate that PLS3 can induce immune escape in colorectal cancer cells by upregulating PD-L1 expression.

[0048] Example 2: Screening and validation of the PLS3-targeting inhibitor Paritaprevir Based on the molecular regulatory signaling axis identified in Example 1, we further developed interventional measures. Currently, no targeted drugs for PLS3 have been reported. Therefore, we utilized an FDA-approved anticancer drug library, based on virtual screening and molecular docking, to identify targeted drugs for PLS3. The drug library was sourced from Selleck's FDA anticancer drug library (https: / / www.selleck.cn / index.html), catalog number L8000.

[0049] Molecular docking method: The structure of Plastin-3 (PLS3) (ID: P13797) was downloaded from the Uniport website. After hydrogenation, side chain repair, and water molecule addition to the PLS3 protein, energy minimization was performed. Based on the FDA database, PLS3 was selected as the target protein, and the active site center of PLS3 was set to 33.25, 64.25, 40.0 (x, y, z), with box coordinates of 14.5, 20.5, 25.0, and other conditions set to default. Virtual screening was performed using QuickVina2. Docking results are presented as Bindingaffinity, with lower scores indicating higher binding probability. Based on virtual screening and molecular docking technology, the target drug Paritaprevir for PLS3 was identified. Paritaprevir was purchased from Medchemexpress (MCE) reagent company, catalog number: HY-12594. It is a drug used to treat hepatitis C, and its mechanism of action is as a non-structural protein 3 / 4A (NS3 / 4A) protease inhibitor, blocking viral replication and thus exerting an antiviral effect. Molecular docking visualization results suggest that Paritaprevir can bind to the PLS3 protein (see...) Figure 10Further cell thermal shift assay (CETSA) was conducted to verify whether Paritaprevir could bind directly to the PLS3 protein. We first cultured HEK-293T cells in two 10cm cell culture dishes, transfecting each dish with 8μg of PLS3 overexpression plasmid. After 48h of culture, we collected cell clusters and added 1ml of Western blot buffer containing a Cocktail (APExBIO, Cat No: K1007) and IP cell lysis buffer (Beyotime, P0013) to lyse the cells for 2h. After centrifugation at 4℃, we collected the protein supernatant. Then, we added 800μg of paritaprevir and an equal amount of DMSO to two 1ml lysis buffer tubes as controls, and incubated them on a rotating shaker at room temperature for 2h. We then transferred 100μL of supernatant to seven 200μL EP tubes and heated them for 40min using a Bio-RADC1000 Touch PCR instrument at different temperature gradients, including 56℃, 58℃, 61℃, 65℃, 71℃, 76℃, and 79℃. Next, after centrifugation at 4°C, 80 μL of supernatant was transferred to a new EP tube, and protein loading buffer was added. The same volume of protein was added for each temperature and Western blot experiment was performed to detect PLS3 protein expression.

[0050] Compared to the DMSO-treated group, Paritaprevir treatment significantly increased the thermal stability of PLS3 protein (see [link to study]). Figure 11 This indicates that Paritaprevir can bind directly to the PLS3 protein.

[0051] We further validated the regulatory effect of paritaprevir on the expression of PLS3 and PD-L1 proteins. Colorectal cancer cells RKO were treated with different concentrations of paritaprevir (5 μM, 10 μM, 20 μM, and 30 μM). Western blot analysis revealed that paritaprevir inhibited the expression of PLS3 and PD-L1 proteins in colorectal cancer cells in a dose-dependent manner (see...). Figure 12 Subsequently, T-cell killing experiments showed that treatment with Paritaprevir (10 μM) significantly increased the cytotoxic sensitivity of T cells against colorectal cancer cells (see...). Figure 13 ).

[0052] Example 3: Application of Paritaprevir and Paritaprevir + PD-1 targeted antibody in the treatment of colorectal cancer A subcutaneous tumor model was further constructed by subcutaneously injecting MC38 cells into immune-normal C57BL / 6 mice, and the tumor volume was increased to 60-100 mm. 3 Mice were randomly divided into four groups: Group 1: control group; Group 2: PD-1 targeted antibody treatment group (PD-1 antibody was Selleck Biotech's A2122 Anti-mouse PD-1 (CD279)-InVivo); Group 3: Paritaprevir treatment group; and Group 4: Paritaprevir + PD-1 targeted antibody treatment group. Intraperitoneal injections were administered every 3 days for a total of 5 injections. The injection doses were 200 μg of PD-1 targeted antibody and 200 μg of Paritaprevir per mouse. Tumor length (mm) and width (mm) were monitored every 3 days to calculate tumor volume. Day 0 was counted from the injection date, and tumors were collected and weighed on day 15. The results showed that, compared to the control group, both PD-1 targeted antibody and Paritaprevir alone inhibited tumor growth, and the combined use of PD-1 targeted antibody and Paritaprevir showed the strongest anti-tumor effect compared to the groups treated alone (see [link to relevant documentation]). Figure 14 Based on this, we developed a novel immunotherapy strategy in the preparation of drugs for the treatment of colorectal cancer: Paritaprevir + PD-1 targeted antibody.

Claims

1. The use of palipvir in the preparation of drugs for the treatment of colorectal cancer.

2. The application as described in claim 1, characterized in that, In the preparation of a drug for the treatment of colorectal cancer, palipvir inhibits the growth of colorectal cancer tumors by targeting and inhibiting the expression of the PLS3 protein.

3. The application as described in claim 1, characterized in that, The administration concentration of palipvir is 3.8 μg / mL to 22.9 μg / mL.

4. The application of palipvir in combination with PD-1 antibody in the preparation of drugs for the treatment of colorectal cancer.

5. The application as described in claim 4, characterized in that, The mass ratio of palipvir to PD-1 antibody is 0.3-2:

1.

6. The application as described in claim 4, characterized in that, The PD-1 antibody includes Selleck Biotech's A2122 Anti-mouse PD-1 (CD279)-InVivo.

7. A pharmaceutical composition for treating colorectal cancer, characterized in that, The pharmaceutical composition contains an effective amount of palipvir and a PD-1 antibody.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition further comprises one or more of a pharmaceutically acceptable excipient, diluent, or carrier.

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