Use of paroxetine in preparation of antitumor drug

Abstract

A use of paroxetine (PAR) or a pharmaceutically acceptable salt thereof in the preparation of an anti-colorectal cancer drug, and a use of paroxetine or a pharmaceutically acceptable salt thereof in combination with a CTLA4 antibody in the preparation of an anti-colorectal cancer drug. It is found for the first time that paroxetine hydrochloride can yield an immune anti-tumor effect by reducing the expression of PD-L1 on colonic cells. Reduction of the pression level of PD-L1 by PAR is verified at the cellular level, enhancement of the in vitro killing ability of T cells by PAR is verified at the cellular level, and the immune killing ability of PAR and the combination of PAR and the CTLA4 antibody is verified at the animal level.

Description

Application of paroxetine in the preparation of anti-tumor drugs Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to an application of paroxetine in the preparation of anti-tumor drugs. Background Art

[0002] Paroxetine hydrochloride (PAR) is an antidepressant that selectively inhibits 5-HT reuptake, increasing 5-HT concentration in the synaptic cleft, thereby enhancing central 5-HTergic nerve function and exerting its antidepressant effect. Currently, there are few reports on the anti-tumor effects of paroxetine hydrochloride.

[0003] PD-1 (programmed cell death protein 1) is an important immunosuppressive molecule belonging to the immunoglobulin superfamily. It was originally cloned from the apoptotic mouse T-cell hybridoma 2B4.11. Immune regulation targeting PD-1 is crucial for combating tumors, infections, autoimmune diseases, and organ transplant survival. Its ligand, PD-L1, can also be targeted, and corresponding antibodies can achieve the same effect. Binding of PD-1 and its ligand, PD-L1, inhibits T cell activation, leading to downregulation of autoimmune function. Blocking PD-1 / PD-L1 can reactivate the anti-tumor activity of cytotoxic T cells and is an important approach for tumor immunotherapy. PD-1 / PD-L1 immunotherapy can treat a wide range of tumor types, effectively improving overall survival and revolutionizing cancer treatment. However, clinical practice has shown that while PD-1 / PD-L1 therapy is highly effective, it can vary widely from patient to patient, with some patients experiencing significant responses while others fail to respond. Furthermore, PD-L1 expression in tumor cells may affect the clinical efficacy of PD-1 / PD-L1-related immune checkpoint therapy. Reducing PD-L1 expression on tumor cell membranes through transcriptional regulation, fundamentally blocking the binding of PD-L1 to PD-1, can reduce side effects for patients. In other words, small molecules that target and regulate PD-L1 expression in tumor cells may become a new treatment approach. Therefore, identifying small molecules that can reduce PD-L1 expression will become a new approach in immunotherapy.

[0004] In recent years, immunotherapy has rapidly developed and attracted increasing attention in clinical practice due to its promising anti-tumor effects, further fueling efforts in CRC. Compared to traditional therapies, immunotherapy can kill cancer cells by activating anti-tumor immunity and specifically targeting cancer cell antigens to protect normal cells from attack. Programmed cell death protein 1 (PD-1) is the most important receptor for activating T cells and mediating immunosuppression. Programmed cell death ligand 1 (PD-L1) participates in programmed cell death 1 (PD-1), leading to T cell apoptosis or dysfunction. Therefore, PD-1 / PD-L1 inhibitors can prevent T cell apoptosis and dysfunction, further enhancing T cell activation. The anti-tumor immune process primarily involves immune elimination, immune balance, and immune escape. PD-1 and PD-L1 are a pair of important immune checkpoints (ICs) that act as brakes in the immune system and play a key role in tumor immune escape. Upon binding of PD-1 and PD-L1, tumor cells exploit T cell receptor recognition to further suppress immunity and evade immune surveillance. In 2002, evidence first emerged that the PD-1 pathway mediates tumor immunity, demonstrating that overexpression of PD-L1 impairs the lytic activity of T cells, significantly promoting tumor development and invasion. Interestingly, this effect can be reversed by the use of monoclonal antibodies targeting PD-L1. PD-L1 is highly expressed on the surface of many tumor cells. It also induces immune cells (particularly T helper lymphocytes, or Th cells) to secrete immunosuppressive factors, further inhibiting the killing effect of anti-tumor immunity. Anti-PD-1 / PD-L1 therapy can bind to PD-1 and PD-L1 accordingly, further preventing PD-1 on the surface of T cells from binding to PD-L1 on the surface of tumor cells. This function can reverse the suppressive effect of tumor cells on the immune system and restore anti-tumor immunity. High PD-L1 expression is associated with an enhanced response to PD-1 / PD-L1-targeted therapies, but currently only a small number of patients have benefited from this approach. PD-1 / PD-L1 immunotherapies also have various side effects and adverse reactions. As PD-1 / PD-L1 immunotherapy drugs have been used clinically for longer periods of time, the side effects and adverse reactions of this type of anticancer drug have become increasingly clear. The immune side effects of PD-1 anticancer drugs can be divided into two categories: common side effects and rare side effects. There are four common side effects: 1. Skin toxicity is the most common immune-related side effect, experienced by one-third of patients, but the vast majority are relatively mild and easily managed. Maculopapular rash and pruritus are more common, while lichenification, eczema, and psoriasis may also occur. 2. Gastrointestinal toxicity is also a very common side effect of immunotherapy, experienced by approximately 20% of patients, with diarrhea being the most common. 3. Hepatotoxicity is also a relatively classic immune side effect, generally appearing 6-14 weeks after the start of treatment. Clinically, it manifests as a significant increase in transaminases, and most patients also experience a fever, with an incidence of approximately 5%.4. The most common pulmonary toxicity is pneumonitis. When PD-1 drugs are used alone, the incidence is low, with the proportion of serious side effects being less than 1%. However, with more complex treatments, especially with various combination therapies, the incidence of pneumonitis is gradually increasing. Rare side effects of PD-1 anticancer drugs include cardiovascular toxicity, hematologic toxicity, nephrotoxicity, neurotoxicity, ocular toxicity, and skeletal muscle toxicity.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide an application of paroxetine or a pharmaceutically acceptable salt thereof in the preparation of anti-tumor drugs.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides a use of paroxetine or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug.

[0009] The tumor is selected from colorectal cancer.

[0010] The pharmaceutically acceptable salt is an acid addition salt formed by paroxetine and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

[0011] The second aspect of the present invention provides a use of paroxetine or a pharmaceutically acceptable salt thereof as a PD-L1 inhibitor in the preparation of an anti-tumor drug.

[0012] The tumor is selected from colorectal cancer.

[0013] In the present invention, paroxetine or a pharmaceutically acceptable salt thereof acts as a PD-L1 inhibitor to promote the lysosomal degradation of PD-L1 in cancer cells, activate T cell immunity, and exert anti-cancer effects. Currently, no relevant reports exist. Under normal conditions, the primary function of the PD-1 / PD-L1 inhibitory co-stimulatory signal is to prevent uncontrolled overactivation of T cells and attack of normal cells. However, high levels of PD-L1 expressed on the surface of tumor cells bind to PD-1 on the surface of T cells, inhibiting T cell function and promoting T cell exhaustion, allowing tumor cells to evade T cell immune attack. Paroxetine or a pharmaceutically acceptable salt thereof in the present invention acts as a PD-L1 inhibitor to prevent tumor cell immune escape. Its mechanism of action is consistent with that of PD-1 and PD-L1 antibodies. However, CTLA4 terminates the immune response by binding to receptors on the surface of antigenic cells. CTLA4 antibodies, by inhibiting the CTLA-4 molecule, promote massive T cell proliferation and attack on tumor cells. Therefore, CTLA4 antibodies are selected for combined use with paroxetine or a pharmaceutically acceptable salt thereof.

[0014] The third aspect of the present invention provides a use of paroxetine or a pharmaceutically acceptable salt thereof in combination with a CTLA4 antibody in the preparation of an anti-tumor drug.

[0015] The tumor is selected from colorectal cancer.

[0016] The fourth aspect of the present invention provides a pharmaceutical preparation, which is prepared by combining paroxetine or a pharmaceutically acceptable salt thereof with a CTLA4 antibody.

[0017] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0018] The present invention is the first to discover that paroxetine hydrochloride can exert an immune anti-tumor effect by reducing the expression of PD-L1 on colon cells. The present invention verifies at the cellular level that PAR reduces PD-L1 expression, that PAR can enhance the in vitro killing ability of T cells, and that the immune killing ability of PAR and the combination of PAR and CTLA4 antibodies is effective in animals.

[0019] The present invention discovered for the first time that PAR has immune and anti-tumor effects in addition to its antidepressant effect; PAR is not toxic to normal cells; PAR can reduce the expression of PD-L1 in tumor cells; PAR can enhance the ability of T cells to kill tumor cells; and PAR combined with CTLA4 antibodies can exert a better anti-tumor effect.

[0020] The present invention discovered for the first time that PAR can exert anti-tumor activity by reducing PD-L1 in tumor cells, and that the combination of PAR and CTLA4 antibodies exerts a better anti-tumor effect and exhibits minimal toxicity to normal epithelial cells and animals. It can be used clinically as a combination drug for anti-tumor purposes. It was also found that after treatment of colorectal cancer cells, T cells can have a significant killing effect on tumor cells. Therefore, the combination of PAR and antibodies is expected to become a new and effective means of tumor treatment.

[0021] To investigate whether the combination of PAR and CTLA4 antibodies has a synergistic anti-tumor effect, the present invention subcutaneously inoculated MC38 into C57BL / 6J (female) mice and treated them with corn oil, PAR (20 mg / kg), anti-PD-1 (100 μg / mouse), anti-CTLA4 (100 μg / mouse), and PAR combined with anti-CTLA4. The results showed that compared with PAR or anti-CTLA4 alone, the combined treatment of PAR and anti-CTLA4 further improved tumor growth rate and volume. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG1 is a schematic diagram of the results of detecting cell viability of RKO and MC38 cells using a CCK8 kit.

[0023] FIG2 is a schematic diagram showing the effect of detecting cell proliferation of RKO and MC38 cells using an EDU kit.

[0024] FIG3 is a schematic diagram showing that PAR reduces PD-L1 expression in RKO and MC38 cells.

[0025] FIG4 is a schematic diagram of detecting the expression of PD-L1 on the membrane of colon cancer cells by flow cytometry.

[0026] FIG5 is a schematic diagram of the results of immunofluorescence detection of the down-regulation effect of PAR on PD-L1 on the cancer cell membrane.

[0027] FIG6 is a schematic diagram showing the results of the crystal violet staining method for determining the in vitro killing effect of PAR on T cells.

[0028] FIG7 is a schematic diagram showing the anti-tumor effects of the control group and PAR (10 and 20 mg / kg) on ​​C57 tumor-bearing mice.

[0029] FIG8 is a schematic diagram showing the anti-tumor effects of the control group and PAR (20 mg / kg) on ​​tumor-bearing nude mice.

[0030] FIG9 is a schematic diagram showing the anti-tumor effect of the combined use of PAR and CTLA4 antibodies on C57 mice. DETAILED DESCRIPTION

[0031] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0032] Example 1

[0033] Study on the effect of PAR on the proliferation of colorectal cancer cells in vitro

[0034] 1.1 Experimental Materials:

[0035] Colorectal cell lines and culture: Human colorectal cancer cells RKO and mouse colorectal cancer cells MC38 were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Cells were cultured in MEM and DMEM (MeilunBio), respectively, supplemented with 10% fetal bovine serum (Biological Industries) at 37°C and 5% CO2. PAR was purchased from MCE (Cat. No. HY-B0492); CCK8 was purchased from Tongren (Cat. No. CK04); and EdU (C0071S) kit and DMSO (ST1276) were all products of Beyotime.

[0036] 1.2 Experimental methods

[0037] The cytotoxicity of compound PAR against the above-mentioned tumor cell lines was measured using CCK8 and EdU assays. The specific steps of the CCK8 and EdU assays are as follows:

[0038] 1. Sample preparation: Dissolve the compound in dimethyl sulfoxide to obtain a 10 μM solution;

[0039] 2. After trypsinization and washing, the cells were suspended in MEM medium containing 10% fetal bovine serum. The number of viable cells was counted by trypan blue exclusion method, and the cell suspension density was adjusted to 1×10 5 pieces / mL;.

[0040] 3. Add 100 μl of cells to each well of a flat-bottom 96-well plate. The total number of cells in each well is 5 × 10 3 In a flat-bottom 12-well plate, 1 ml of cells per well is used, and the total number of cells per well is 2.5×10 5 , cultured overnight in a 37°C, 5% CO2 cell culture incubator;

[0041] 4. Add 5 μl of sample diluted in cell growth medium to each well, resulting in final concentrations of 1, 5, and 10 μM. Add the corresponding amount of DMSO to untreated cells as a control, and wells containing cells without drug or DMSO as background. Perform three replicates for each point.

[0042] 5. Incubate the drug-treated cells in a 37°C, 5% CO2 cell culture incubator for 24 hours, followed by CCK8 and EdU detection.

[0043] 6. CCK8 assay: Add 10 μl of CCK8 solution to each well and continue to incubate in the incubator for 2 hours. Then, detect with a microplate reader at a wavelength of 450 nm. Calculate cell viability using the following formula: Cell viability (%) = (measured value - blank value) / (control value - blank value) × 100%.

[0044] 7. EdU detection: Add 10 μl of EdU solution to each well and continue to incubate in the incubator for 2 hours. The generated green fluorescence is photographed at a wavelength of 488 nm using high-content imaging. The intensity of the green fluorescence is counted using ImageJ software and the effect on the proliferation of each tumor cell is calculated.

[0045] 1.3 Experimental Results

[0046] 1.3.1 CCK8 detection of colorectal cancer cell viability

[0047] After RKO and MC38 cells were treated with PAR for 24 hours, CCK8 was added to detect the viability of colon cancer cells. The results are shown in Figure 1. Figure 1 is a schematic diagram of the results of RKO and MC38 cell viability detection using a CCK8 kit. After 24 hours of treatment of colorectal cancer cells with PAR at 1μM, 5μM, and 10μM, the CCK8 results showed that PAR had almost no effect on the viability of colorectal cancer cells (RKO and MC38). PAR had almost no toxicity to RKO cells and was slightly toxic to MC38 cells, but it did not affect the subsequent immune experiments. This suggests that PAR's anti-colorectal cancer effect is not achieved directly through a killing effect.

[0048] 1.3.2 EDU toxicity assay for colorectal cancer cells

[0049] After treatment with the EdU kit, the proliferating cells exhibit very bright green fluorescence under a fluorescence microscope. If the drug has an inhibitory effect on the cells, the fluorescence will weaken. The results are shown in Figure 2, which is a schematic diagram of the effect of detecting cell proliferation of RKO and MC38 cells using the EDU kit. The control group incubated with EdU (10 μM) for 2 hours had bright green fluorescence, and after PAR was treated with colorectal cancer cells at 5 μM, 10 μM and 20 μM for 24 hours, there was no significant change in green fluorescence, indicating that PAR had almost no toxicity to colorectal cancer cells (RKO and MC38). There was no obvious difference in the fluorescence displayed by PAR on RKO and MC38 at various concentrations, suggesting that they had no obvious toxicity to colorectal cancer cells (RKO and MC38) respectively. The present invention takes colon cancer cells (RKO and MC38) as an example to further illustrate that PAR achieves anti-tumor activity by reducing the expression of PD-L1 in tumor cells.

[0050] Example 2

[0051] Study on the effect of PAR on reducing PD-L1 in colon cancer cells

[0052] 2.1 Experimental Materials

[0053] Colorectal cell lines and culture: Human colorectal cancer cells RKO and mouse colorectal cancer cells MC38 were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Cells were cultured in RPMI-MEM (MeilunBio) and DMEM (MeilunBio), respectively, supplemented with 10% fetal bovine serum (Biological Industries) at 37°C and 5% CO2. PD-L1 antibody was purchased from Abcam, catalog number ab203103; the BCA assay kit was purchased from Beyotime (P0009).

[0054] 2.2 Experimental methods

[0055] 2.2.1 Protein immunoblotting

[0056] RKO and MC38 cells with good growth status and density above 80% were used for plating. 5×10 5 The cell suspension was plated into a six-well plate with a volume of 2 mL per well. The plate was placed in a 37°C incubator for incubation. After cells were fully adhered, PAR was administered at concentrations of 1, 5, or 10 μM. The culture was continued for 24 hours before termination and use in experiments.

[0057] The collected cells were washed twice with cold PBS. After the PBS residue was removed, the corresponding amount of lysis buffer containing PMSF was added according to the different culture plates and the actual cell number. The cells were incubated on ice for 15 minutes. The cells were centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was gently aspirated and transferred to a freshly pre-cooled centrifuge tube and placed on ice. This was the protein sample, and the precipitate was discarded. Protein quantification was performed using the BCA method. Protein denaturation was generally performed using 5× SDS gel reduction loading buffer (5× Loading Buffer). After mixing with the sample at a ratio of 1:4, the cells were incubated in a metal bath at 95°C for 10 minutes. After cooling, the samples were stored at -20°C.

[0058] Prepared protein samples were electrophoresed on the corresponding protein gel according to the molecular weight of the protein. At the beginning, the sample was concentrated with 80V voltage. After the sample completely ran out of the concentrated gel and the molecular weight on the marker was completely separated, the voltage was adjusted to 120V. When the required target protein was separated, the electrophoresis was stopped and the membrane was transferred to a PVDF membrane pretreated with methanol in an ice bath at a constant current of 300mA for 2h. After the transfer was completed, the membrane was blocked with 5% skim milk solution at room temperature for 1h, and then washed three times with TBST solution for 5min each time. Incubate with the corresponding primary antibody (PD-L1) in a 4°C refrigerator for 18h. After the primary antibody incubation was completed, the membrane was washed three times with TBST, and then incubated with the corresponding secondary antibody at room temperature for 1h. Finally, the membrane was washed three times with TBST and the corresponding protein bands were detected using an instrument.

[0059] 2.3 Experimental Results

[0060] PAR was treated with 1μM, 2μM, 5μM, and 10μM of RKO and MC38 cells for 24 hours. The results showed that PAR could reduce PD-L1 expression in a concentration-dependent manner. The results are shown in Figure 3, which is a schematic diagram of PAR reducing PD-L1 expression on RKO and MC38 cells. PAR at 1μM, 2μM, 5μM, and 10μM can dose-dependently reduce PD-L1 expression in colorectal cancer cells (RKO and MC38).

[0061] Example 3

[0062] Flow cytometry was used to detect the downregulation effect of PAR on PD-L1 on cancer cell membranes

[0063] 3.1 Experimental Materials

[0064] Human colorectal cell lines and their culture: Colorectal cancer cells (HCT116, MC38, and RKO) were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Cells were cultured in MEM (MeilunBio) supplemented with 10% fetal bovine serum (Biological Industries) at 37°C and 5% CO2. Flow cytometry antibodies were purchased from Biolegend, including mouse and human PD-L1 antibodies, catalog numbers 124307 and 329706, respectively.

[0065] 3.2 Experimental methods

[0066] 3.2.1 Flow cytometry analysis of tumor cell membranes revealed PD-L1 abundance

[0067] Cancer cells in the logarithmic growth phase were cultured at a rate of 2.5×10 5 Cells were seeded at a density of 100 μL / mL in a 6-well plate, with 2 mL per well. The cells were incubated overnight in a cell culture incubator. The culture medium was aspirated and treated with PAR for 24 hours. The culture medium was then removed and the cells were harvested using an EP tube after trypsinization. The cells were centrifuged at 1200 rpm at 4°C for 3 minutes. The supernatant was removed and the cells were washed twice with pre-chilled PBS and centrifuged again at 1200 rpm at 4°C for 3 minutes. The supernatant was removed and 100 μL of PBS-diluted PE anti-human CD274 antibody or PE anti-mouse CD274 antibody was added to each tube and incubated at 4°C for 30 minutes. After incubation, the cells were washed with 400 μL of PBS and centrifuged at 1200 rpm at 4°C for 3 minutes. The supernatant was removed and the cells were resuspended in 500 μL of PBS and transferred to a flow cytometer for analysis.

[0068] 3.3 Experimental Results

[0069] Since Western Blot results show the effect of the compound on the total PD-L1 protein in the cytoplasm and membrane, and immune checkpoint inhibitors work by blocking the binding of PD-1 and PD-L1, mainly through membrane receptors, only PAR can reduce membrane proteins to be used for immunotherapy. The present invention used flow cytometry to detect the expression of PD-L1 on the cell membrane after PAR acted on RKO, MC38 and HCT116 cells. The results are shown in Figure 4, which is a schematic diagram of flow cytometry detection of PD-L1 expression on the membrane of colon cancer cells. PAR reduced the expression of PD-L1 on the cell membrane of colon cancer cells in a concentration-dependent manner (1μM, 5μM, 15μM and 20μM).

[0070] Example 4

[0071] Immunofluorescence detection of PAR downregulation of PD-L1 on cancer cell membranes

[0072] 4.1 Experimental Materials

[0073] Colorectal cell lines and culture: Human colorectal cancer cell lines DLD1 and mouse colorectal cancer cell lines MC38 were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Cells were cultured in RPMI1640 and DMEM (MeilunBio) medium supplemented with 10% fetal bovine serum (Biological Industries), respectively, at 37°C and 5% CO2. Anti-PD-L1 antibody for immunofluorescence was purchased from Proteintech (Cat. No. 66248-1-Ig).

[0074] 4.2 Experimental methods

[0075] 4.2.1 Immunofluorescence detection of PD-L1 expression on cancer cell membranes

[0076] 1. Cell culture and washing: Cancer cells in good condition were cultured at a rate of 2.5×10 5 Cells were seeded into 12-well plates at a density of 1 mL per well, and cell culture medium was added until the cells were completely attached. PAR was added for 24 h, the supernatant was removed, and the cells were washed three times with PBS.

[0077] 2. Fixation and blocking: Fix the cells in the culture plate with 4% paraformaldehyde solution for 15 minutes, remove the fixative, wash with PBS three times, 5 minutes each time, add 5% BSA to the sample wells of the 12-well plate for 1 hour, and wash twice with PBS;

[0078] 3. Primary antibody incubation: Add diluted PD-L1 primary antibody and incubate overnight at 4°C;

[0079] 4. Washing: Wash 3 times with TBST, 5 minutes each time;

[0080] 5. Secondary antibody incubation: Finally, add red fluorescent antibody and incubate at room temperature in the dark for 1 hour. Add DAPI staining for 10 minutes before taking pictures.

[0081] 6. Washing: Remove the secondary antibody working solution and wash with TBST buffer 3 times, 5 minutes each time;

[0082] 7. Analyze the results after taking laser confocal microscopy photos.

[0083] 4.3 Experimental Results

[0084] The intensity of the red color in immunofluorescence represents the expression of PD-L1 on the cell membrane. Stronger fluorescence indicates higher PD-L1 expression, while weaker fluorescence indicates weaker PD-L1 expression. The results are shown in Figure 5, which is a schematic diagram of the immunofluorescence detection of the downregulation of PD-L1 on the cancer cell membrane by PAR. The immunofluorescence results showed that after PAR treatment of colorectal cancer cells, the red color of the immunofluorescence staining can reflect the expression of PD-L1. After PAR treatment, the abundance of red fluorescence decreased in a dose-dependent manner (5μM, 10μM, 15μM, and 20μM), indicating that PAR reduces the expression of PD-L1 on the cell membrane of colon cancer cells in a concentration-dependent manner.

[0085] Example 5

[0086] Study on the effect of PAR on T cells' killing of colon cancer cells RKO in vitro

[0087] 5.1 Experimental Materials

[0088] Human colorectal cell lines and culture: Colorectal cancer cells RKO were purchased from the Institute of Biochemical Cell Biology, Chinese Academy of Sciences. Cells were cultured in MEM medium (purchased from MeilunBio) supplemented with 10% fetal bovine serum (Biological Industries) at 37°C and 5% CO2. NK-92 cells were purchased from ATCC and cultured in a specialized NK cell culture medium (Shanghai Xuhe Biological, Catalog No. S-22). Crystal violet solution was purchased from Biyuntian (Catalog No. C0121).

[0089] 5.2 Experimental methods

[0090] The in vitro killing effect of compound PAR on the above tumor cell lines was measured by NK-92 assay, and the specific steps are as follows:

[0091] 1. Sample preparation: Dissolve compound PAR in dimethyl sulfoxide to obtain a 10 μM solution;

[0092] 2. The cells were trypsinized and washed, then suspended in MEM medium containing 10% fetal bovine serum. The number of viable cells was counted by placental blue exclusion method, and the cell suspension density was adjusted to 1×10 5 cells / mL;

[0093] 3. Add 100 μl of cells to each well of a flat-bottom 12-well plate. The total number of cells in each well is 3 × 10 4 Culture overnight in a 37°C, 5% CO2 cell culture incubator;

[0094] 4. Add 5 μl of sample diluted in cell growth medium to each well, resulting in final reaction concentrations of 1, 5, and 10 μM. Add the corresponding amount of DMSO to untreated cells as a control, and wells containing cells without drug or DMSO as background. Perform three replicates for each point.

[0095] 5. Incubate the drug-treated cells in a 37°C, 5% CO2 cell culture incubator for 24 hours;

[0096] 6. Add NK-92 cells at a ratio of 1:0.5 and culture for an appropriate amount of time. During this period, pay attention to the killing situation to avoid excessive killing;

[0097] 7. Remove the supernatant, wash three times with PBS, and fix the cells with 4% paraformaldehyde. Then, wash three more times with PBS, stain with crystal violet for 30 minutes, and finally wash away the excess crystal violet with PBS.

[0098] 8. Take photos to collect data, and use ImageJ software to calculate the intensity of crystal violet for statistical analysis.

[0099] 5.3 Experimental Results

[0100] To investigate the role of PAR in T cell-mediated tumor cell killing, RKO cells were co-cultured with activated NK-92 cells, and the anti-tumor effect of PAR was evaluated by measuring viable tumor cells using crystal violet staining. The results are shown in Figure 6, which is a schematic diagram of the results of crystal violet staining to determine the in vitro PAR-enhanced T cell killing effect. After 24 hours of PAR treatment of colorectal cancer cells, PD-L1 expression in colorectal cancer cells was first downregulated, and then the T cell killing ability was tested. The results showed that PAR treatment of colorectal cancer cells enhanced the killing effect of T (NK92) cells against RKO colorectal cancer cells. Compared with the control group, PAR treatment significantly reduced the survival rate of RKO cells and induced cell apoptosis. These results indicate that PAR enhances T cell cytotoxicity against cancer cells by downregulating PD-L1 expression in colorectal cancer cells, and this effect is concentration-dependent at 5 μM, 10 μM, and 20 μM.

[0101] Example 6

[0102] Anti-tumor effects of PAR in vivo

[0103] 6.1 Experimental Materials

[0104] Tumor line: Mouse colorectal cancer cells (MC38) were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Cells were cultured and passaged in DMEM (MeilunBio) supplemented with 10% fetal bovine serum (Biological Industries) at 37°C and 5% CO2.

[0105] Animals: C57BL / 6J mice (female) were purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., certificate number: (20220009004717).

[0106] Other reagents: Paroxetine hydrochloride (product number HY-B0492) was purchased from MedChemExpress (MCE); DMSO (batch number: 20030415) was a product of Shanghai Chemical Reagent Company; corn oil (Golden Sun Grain and Oil Co., Ltd.).

[0107] Sample: PAR was dissolved in DMSO and corn oil at a 10% corn oil dose.

[0108] 6.2 Experimental methods

[0109] Colon cancer cells MC38 were inoculated into 6-8 week old female C57BL / 6J mice (1×10 6 cell / ) to establish a subcutaneous tumor model. The tumor volume reached 50mm 3 Afterwards, the mice were divided into three groups: (1) control group; (2) low PAR (10 mg / kg) group; (3) high PAR (20 mg / kg) group. The volume of the mouse tumor was measured once a day. The tumor volume calculation formula is: tumor volume (mm 3 ) = (length) × (width) 2 ×1 / 2. Mice were sacrificed 18 days after administration and tumors were dissected.

[0110] 6.3 Experimental Results

[0111] The results are shown in Figure 7, which graphically illustrates the antitumor effects of the control group and PAR (10 and 20 mg / kg) in C57 tumor-bearing mice. The 20 mg / kg dose exhibited the greatest antitumor effect. Figure A shows photographs of actual tumors in mice after treatment in each of the three groups. Figure B shows a graphical representation of tumor volume curves for the control group and each treatment group. Figure C shows a graphical representation of tumor weights for the control group and each treatment group. Figure D shows a graphical representation of weight changes for the mice in the control group and each treatment group. As shown in Figure A, PAR (20 mg / kg) exhibited the greatest antitumor effect in C57 tumor-bearing mice. As shown in Figure 7B, PAR treatment significantly inhibited the growth of mice inoculated with MC38 tumors in a concentration-dependent manner, with the 20 mg / kg PAR dose exhibiting the greatest antitumor effect. PAR was administered once daily for 18 days via oral gavage. Comparison of in vitro tumor observations (Figure 7A) and tumor weights (Figure 7C) further confirmed that the 20 mg / kg PAR dose exhibited the greatest antitumor effect. The results shown in FIG7D indicate that the PAR compound has almost no effect on the body weight of mice, suggesting that PAR has no obvious toxic effect on mice.

[0112] Example 7

[0113] Anti-tumor effect of PAR in nude mice

[0114] 7.1 Experimental Materials:

[0115] Tumor line: Mouse colorectal cancer cells (MC38) were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Cells were cultured and passaged in DMEM (MeilunBio) supplemented with 10% fetal bovine serum (Biological Industrie) at 37°C and 5% CO2.

[0116] Animals: Female nude mice were purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., with the qualification certificate number: (20220009007529).

[0117] Other reagents: Paroxetine hydrochloride (product number HY-B0492) was purchased from MedChemExpress (MCE); DMSO (batch number: 20030415) was a product of Shanghai Chemical Reagent Company; corn oil (Golden Sun Grain and Oil Co., Ltd.).

[0118] Sample: PAR was dissolved in DMSO and corn oil at a 10% corn oil dose.

[0119] 7.2 Experimental methods

[0120] Colon cancer cells MC38 were inoculated into 6-8 week old female nude mice (8×10 5 cells / mouse) in the armpit to establish a subcutaneous tumor model. When the tumor volume is about 50 mm 3 The mice were randomly divided into a control group and a treatment group (20 mg / kg) of PAR. The mice were given PAR by gavage every day for 12 days, and the tumor volume and body weight of the mice were measured every other day. The tumor volume calculation formula is: tumor volume (mm 3 ) = (length) × (width) 2 ×1 / 2.

[0121] 7.3 Experimental Results

[0122] To further confirm that PAR's anti-tumor effects are mediated through immune pathways, this study used immunodeficient mice to verify PAR's anti-tumor effects. Interestingly, in nude mice lacking T cells, as shown in Figure 8, there was no significant difference in tumor size between the two groups, indicating that the tumor-suppressing effect of PAR on MC38 tumor-bearing mice was abolished. This suggests that PAR's anti-tumor effects can be attributed to its activation of T cell immunity in vivo. Therefore, it can be concluded that PAR has no anti-tumor effect in nude mice lacking T cells.

[0123] The results are shown in Figure 8, which illustrates the antitumor effects of the control group and PAR (20 mg / kg) in tumor-bearing nude mice. Figure A is a diagram showing the tumor size in the control group and each treatment group, demonstrating that the antitumor effect of PAR is concentration-dependent. Figure B is a diagram showing the tumor growth curves for each group, demonstrating that PAR inhibits tumor growth in a concentration-dependent manner. Figure C is a diagram showing the statistical weight of each group, demonstrating that tumor weight decreases with increasing PAR concentration. Figure D is a diagram showing the changes in mouse body weight in different groups, demonstrating that PAR has no significant effect on mouse body weight.

[0124] Example 8

[0125] Combination of PAR and CTLA4 antibodies produces synergistic anti-tumor effects

[0126] 8.1 Experimental Materials:

[0127] Tumor line: Mouse colorectal cancer cells (MC38) were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Cells were cultured and passaged in DMEM (MeilunBio) supplemented with 10% fetal bovine serum (Biological Industries) at 37°C and 5% CO2.

[0128] Animals: C57BL / 6J mice (female) were purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., certificate number: (20220009008655).

[0129] Other reagents: Paroxetine hydrochloride (catalog number HY-B0492) was purchased from MedChemExpress (MCE); anti-mouse-PD-1 antibody (Invivogen, catalog number BE0146); anti-mouse-CTLA4 antibody (Invivogen, catalog number BP0032); DMSO (batch number 20030415) was a product of Shanghai Chemical Reagent Company; corn oil (Golden Sun Grain and Oil Co., Ltd.).

[0130] Sample: PAR was dissolved in DMSO and corn oil at a 10% corn oil dose.

[0131] 8.2 Experimental methods

[0132] Colon cancer cells MC38 were inoculated into 6-8 week old female C57BL / 6J mice (8×10 5 The subcutaneous tumor model was established with 10 cells. The tumor volume reached 50 mm 3Afterwards, the mice were divided into 5 groups: (1) control group; (2) anti-PD-1 group (100 μg / mouse); (3) anti-CTLA4 group (100 μg / mouse); (4) PAR group (20 mg / kg); (5) PAR (20 mg / kg) + anti-CTLA4 group (100 μg / mouse). The administration period was 16 days. PAR was administered by gavage, and the antibody was injected intraperitoneally every five days, for a total of three injections of the antibody, each time with 100 μg injected intraperitoneally. The tumor volume and body weight of the mice were measured every other day. The formula for calculating tumor volume is: tumor volume (mm 3 ) = (length) × (width) 2 ×1 / 2. The mice were sacrificed 16 days after administration and the tumors were dissected.

[0133] 8.3 Experimental Results

[0134] Because the combined use of PD-1 and CTLA4 antibodies has significant adverse reactions, the combination of small molecule drugs and CTLA4 antibodies has become a cancer immunotherapy strategy aimed at enhancing the immune system's attack on tumors. The results showed that the therapeutic effects of the anti-PD-1, anti-CTLA4, and PAR groups were comparable, and the anti-CTLA4 + PAR group showed a significant difference from the PAR group alone (Figure 9, AC), indicating that PAR and anti-CTLA4 have a synergistic anti-cancer effect.

[0135] The results are shown in Figure 9, which is a schematic diagram of the anti-tumor effect of PAR combined with CTLA4 antibodies on C57 mice. A is a schematic diagram of the tumor size of the control group and each treatment group, and the results show that the combination of PAR and CTLA4 antibodies has the best anti-tumor effect. B is a schematic diagram of the tumor growth curve of each group, and the results show that the combination of PAR and CTLA4 antibodies has the most significant inhibitory effect on tumor growth. C is a statistical diagram of the tumor weight of each group, and the results show that the tumor weight of the combination of PAR and CTLA4 antibodies is the lightest. D is a schematic diagram of the weight changes of mice in each group, and the results show that the combination of PAR and CTLA4 antibodies has a certain effect on the weight of mice. From the above results, it can be concluded that PAR and CTLA4 antibodies synergistically inhibit the proliferation of colorectal cancer cells and promote anti-tumor effects, providing a new alternative for the clinical treatment of colorectal cancer.

[0136] The present invention, through Western blotting, flow cytometry, and immunofluorescence assays, confirmed that PAR can not only reduce intracellular PD-L1 expression but also membrane expression. These results suggest that PAR may be a promising candidate compound for reducing PD-L1 in immunotherapy. The immunosuppressive effects of PD-1 / PD-L1 depend on cell surface membrane receptors, suggesting that PAR may have a role in relieving immunosuppression. In vivo and in vitro experiments revealed that PAR can both enhance immune killing in vitro and enhance T cell cytotoxicity in tumor-bearing mice. Since PAR only works in immunocompetent mice and has no tumor-suppressing effect in T cell-deficient nude mice, PAR may enhance T cell cytotoxicity by downregulating PD-L1 expression in tumor cells. Furthermore, animal studies showed that the anti-PD-1, anti-CTLA4, and PAR groups had comparable therapeutic effects in mice, while the anti-CTLA4 + PAR group showed significant differences compared to the PAR alone group, suggesting that PAR and anti-CTLA4 have a synergistic effect, and that the combination of PAR and the antibody can achieve a more effective tumor treatment.

[0137] The present invention has the following advantages:

[0138] First, while PD-1 / L1 monoclonal antibodies have made considerable progress in the clinical treatment of various tumors, significantly extending survival for some cancer patients and even achieving complete remission in some, PD-1 / L1 monoclonal antibodies still have limitations in terms of tumor response rates. Most patients have a low response to PD-1 / L1 monoclonal antibodies, and responses vary significantly between different tumor types. This may be due to the complex tumor microenvironment. Studies have shown that in tumor tissue, factors such as leaky blood vessels, collagen clotting, and increased hyaluronic acid can affect the penetration and distribution of large molecule drugs such as PD-1 / L1 monoclonal antibodies. In solid tumors, PD-1 / L1 monoclonal antibodies have poor penetration, and their effectiveness may be limited to cancer cells immediately adjacent to blood vessels, resulting in a low objective response rate (ORR). Compared to PD-1 / L1 antibodies with molecular weights exceeding 140,000 Da, small molecule inhibitors offer inherent advantages in this regard.

[0139] Secondly, the immunogenicity of PD-1 / L1 monoclonal antibodies is also a prominent issue. Once biomacromolecules enter the human body, they can easily trigger a cytokine storm, resulting in a strong immune response and a variety of serious adverse reactions. However, due to their smaller molecular weight, the immunogenicity of small molecule PD-L1 inhibitors is negligible.

[0140] Thirdly, there are the issues of cost, compliance, and accessibility. Monoclonal antibodies are difficult to prepare, and inconvenient to store and transport, which results in high costs. In addition, the current administration method of PD-1 / L1 monoclonal antibodies is still mainly intravenous, once every 2-3 weeks, and patient compliance with treatment is far lower than that of oral small molecule targeted drugs. Oral PD-L1 small molecule inhibitors are easy to transport and store, and have good stability. Currently, research on the combination of small molecule targeted drugs and PD-1 / L1 monoclonal antibodies is in full swing. In the future, if CTLA4 antibodies and PD-L1 small molecule inhibitors are taken orally at the same time, it will greatly improve patient compliance and quality of life.

[0141] In this invention, repurposing approved and marketed drugs (PARs) not only reduces the cost of developing new drugs but also significantly shortens the research cycle, allowing patients to benefit more quickly. Through a series of biological experiments, it was verified in vitro that paroxetine not only effectively promotes the degradation of PD-L1 in cancer cells but also increases the killing effect of T cells on cancer cells. Animal experiments further demonstrated paroxetine's inhibitory effect on tumor growth. Furthermore, paroxetine can be used in combination with CTLA4 antibodies to exert a synergistic anti-cancer effect, providing a new alternative for the clinical treatment of cancer.

[0142] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with this patent can make slight changes or modifications to equivalent embodiments using the above technical content without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the solution of the present invention.

Claims

1. A use of paroxetine or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug, characterized in that: The tumor is selected from colorectal cancer.

2. The use of paroxetine or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug according to claim 1, characterized in that: The pharmaceutically acceptable salt is an acid addition salt formed by paroxetine and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

3. Use of paroxetine or a pharmaceutically acceptable salt thereof as a PD-L1 inhibitor in the preparation of an anti-tumor drug.

4. Use of paroxetine or a pharmaceutically acceptable salt thereof in combination with a CTLA4 antibody in the preparation of an anti-tumor drug.

5. The use of paroxetine or a pharmaceutically acceptable salt thereof in combination with a CTLA4 antibody in the preparation of an anti-tumor drug according to claim 4, characterized in that: The tumor is selected from colorectal cancer.

6. A pharmaceutical preparation, characterized in that The pharmaceutical preparation is prepared by combining paroxetine or its pharmaceutically acceptable salt with CTLA4 antibody.

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