Use of maprotiline together with CTLA4 antibody in preparation of Anti-tumor drug

Abstract

A use of maprotiline, or a pharmaceutically acceptable salt thereof, together with a CTLA4 antibody in the preparation of an anti-tumor drug, the tumor being selected from colorectal cancers. It has been found that MAP, by means of targeting mTOR, can promote the ubiquitination of PD-L1 in colorectal cancer cells, causing the PD-L1 to degrade, thereby activating the immune system to exert an anti-tumor effect.

Description

Application of maprotiline combined with CTLA4 antibody in the preparation of anti-tumor drugs Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to an application of maprotiline in combination with a CTLA4 antibody in the preparation of an anti-tumor drug. Background Art

[0002] Maprotiline hydrochloride (MAP) is an FDA-approved tetracyclic antidepressant that can alleviate mental retardation and produce antidepressant effects by blocking norepinephrine reuptake at the central presynaptic membrane. It also exhibits anxiolytic, sedative, and visually protective effects. Currently, there are no reports of MAP in cancer immunotherapy.

[0003] Chemical structure of Maprotiline hydrochloride

[0004] Currently, multiple monoclonal antibodies targeting CTLA4, PD-1, or PD-L1 have been approved by the US Food and Drug Administration (FDA) and the China National Medical Products Administration (NMPA) for the clinical treatment of various tumors. These monoclonal antibodies have brought lasting and effective clinical benefits to many patients and transformed the treatment landscape for various tumors. However, the vast majority of cancer patients still do not benefit from these antibodies. Current monoclonal antibody therapies still have many limitations, such as the lack of sensitive predictive biomarkers for clinical application, low overall patient response rates to antibody therapy, the inability of existing efficacy assessment indicators to objectively evaluate tumor immunotherapy, and significant toxic side effects. Small molecule inhibitors are a potential alternative approach because they can not only more easily penetrate cell membranes and directly interfere with the PD-1 / PD-L1 signaling pathway, but also offer numerous advantages such as non-immunogenicity, low cost, oral administration, and easy storage and transportation. These advantages may help overcome some of these therapeutic limitations and provide a wider range of treatment options. Therefore, the screening and development of small molecule inhibitors still holds significant potential. While newly developed small molecule compounds offer greater targeting, their synthesis is complex, and their clinical safety requires further validation. Repurposing approved drugs can reduce costs and significantly shorten research cycles. Since toxicology, pharmacokinetic studies, and clinical trials have already been completed, these proven therapeutic drugs can benefit patients more quickly. Furthermore, to enhance cancer patients' response to PD-L1 / PD-1 inhibitors, some clinical trials are investigating the combination of PD-L1 / PD-1 inhibitors with chemotherapy, targeted therapy, and other immunotherapies to improve therapeutic efficacy. Therefore, identifying drugs with PD-L1 inhibitory activity among already marketed drugs and combining them with CTLA4 antibodies is a novel approach for immunotherapy.

[0005] PD-L1 (programmed death ligand 1), also known as CD274, is a protein that plays a critical role in the immune system. PD-L1 is normally found on the surface of many different cell types, including immune and non-immune cells. Its primary function is to regulate immune system activity by interacting with PD-1 (programmed death receptor), helping to maintain immune balance. In certain cancers, tumor cells overexpress PD-L1. This is a strategy to evade immune attack because when PD-L1 binds to PD-1, the immune system's ability to attack tumor cells is weakened. This phenomenon is known as "immune escape" or "immunosuppression." To overcome the inhibitory effects of PD-L1 in tumor cells, anti-PD-L1 antibodies, such as pembrolizumab and nivolumab, have been developed. These drugs bind to PD-L1, preventing the interaction between PD-L1 and PD-1, thereby releasing T cell suppression and enhancing the immune system's ability to attack cancer cells. This treatment approach is known as immune checkpoint inhibitor therapy. Although PD-L1 antibody therapy can effectively enhance the activity of the immune system, it may also cause some immune-related side effects, such as immune thyroiditis, skin reactions, gastrointestinal problems, etc. Therefore, there are certain risks in receiving PD-L1 antibody treatment.

[0006] In addition, PD-L1 expression levels are regulated by multiple factors, including genetic factors, immune regulation, the microenvironment, drug therapy, protein degradation pathways, and epigenetics. For example, gene mutations, immune system activity and regulatory factors, cells and signaling molecules in the tumor microenvironment, PD-L1 ubiquitination, DNA methylation, and histone modifications all directly affect PD-L1 expression levels. Understanding these factors will help us better understand the role of PD-L1 in immune regulation and tumor immune escape, and is expected to provide useful information for personalized cancer treatment and the development of new therapeutic strategies.

[0007] Clinically, antibody combination immunotherapy has become an important cancer treatment strategy that can enhance the immune system's attack on cancer cells. This combination therapy typically involves the use of antibodies with different mechanisms to enhance the activity of the immune system and improve the therapeutic effect on cancer. The following are some common antibody combination immunotherapy methods and their effects: anti-PD-1 / PD-L1 combined with anti-CTLA4 therapy. Anti-PD-1 / PD-L1 antibodies can remove the PD-1 / PD-L1 inhibitory effect between T cells and cancer cells, while CTLA4 antibodies can enhance the activity of T cells. This combination therapy has achieved significant success in various cancer types, including melanoma; anti-PD-1 / PD-L1 combined with targeted drugs. Sometimes, anti-PD-1 / PD-L1 antibodies can also be used in combination with targeted drugs to enhance the therapeutic effect. For example, for patients with EGFR-mutated non-small cell lung cancer, the combined use of EGFR inhibitors and anti-PD-1 / PD-L1 antibodies may produce better therapeutic effects; anti-PD-1 / PD-L1 combined with anti-tumor antigen (TAA) vaccines, the combination treatment strategy of anti-PD-1 / PD-L1 antibodies and anti-tumor antigen vaccines aims to stimulate the patient's own immune system to produce a specific response to cancer cells. This combination treatment may enhance the immune response induced by the vaccine and improve the immune killing effect on cancer. In addition to the above methods, there are also many other combination strategies of immunotherapy drugs and antibodies, such as anti-LAG-3 antibodies and anti-TIM-3 antibodies, which are also under study. These combination treatments are designed to target different immune regulatory pathways, enhancing the diversity of treatment methods. However, the effect of antibody combination immunotherapy usually varies depending on the cancer type, patient characteristics and treatment history. Therefore, treatment selection usually needs to be formulated based on the patient's specific situation and the latest research results of immunotherapy.

[0008] Patent application publication number CN104546813A discloses the use of maprotiline hydrochloride in the preparation of a drug for inhibiting the metastasis and spread of tumor cells. The drug, prepared using maprotiline hydrochloride, inhibits the metastatic ability of tumor cells. Maprotiline hydrochloride is a low-toxic drug that can significantly block tumor spread. Simultaneously, other related oncology drugs can significantly reduce the death of normal cells. Existing oncology drugs are mostly cytotoxic, killing tumor cells and also a large number of normal cells.

[0009] Summary of the Invention

[0010] The present invention aims to provide an application of maprotiline or a pharmaceutically acceptable salt thereof in combination with a CTLA4 antibody in the preparation of an anti-tumor drug.

[0011] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0012] The first aspect of the present invention provides a use of maprotiline or a pharmaceutically acceptable salt thereof in combination with a CTLA4 antibody in the preparation of an anti-tumor drug.

[0013] The tumor is selected from colorectal cancer.

[0014] The pharmaceutically acceptable salt is an acid addition salt formed by maprotiline with 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.

[0015] The second aspect of the present invention provides a use of maprotiline or a pharmaceutically acceptable salt thereof as a PD-L1 inhibitor in combination with a CTLA4 antibody in the preparation of an anti-tumor drug.

[0016] The tumor is selected from colorectal cancer.

[0017] In the present invention, maprotiline or a pharmaceutically acceptable salt thereof is used as a PD-L1 inhibitor to target mTOR to promote the ubiquitination and degradation of PD-L1 in cancer cells, activate T cell immunity, and exert an anti-cancer effect. There are currently no related reports.

[0018] Under normal circumstances, the main function of the PD-1 / PD-L1 inhibitory co-stimulatory signal is to prevent T cells from being overactivated and attacking normal cells in an uncontrolled manner. However, high levels of PD-L1 expressed on the surface of tumor cells inhibit T cell function and promote T cell exhaustion by binding to PD-1 on the surface of T cells, allowing tumor cells to escape T cell immune attacks. Maprotiline or its pharmaceutically acceptable salt 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 antibodies and PD-L1 antibodies, but CTLA4 terminates the immune response by binding to receptors on the surface of antigen cells. CTLA4 antibodies inhibit CTLA-4 molecules to cause T cells to proliferate in large numbers and attack tumor cells. Therefore, CTLA4 antibodies are selected for combined use with maprotiline or its pharmaceutically acceptable salts.

[0019] The pharmaceutically acceptable salt is an acid addition salt formed by maprotiline with 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.

[0020] The third aspect of the present invention provides a pharmaceutical preparation, which is prepared by combining maprotiline or a pharmaceutically acceptable salt thereof with a CTLA4 antibody.

[0021] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0022] The use of maprotiline or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug, provided by the present invention, has been verified for the first time through in vitro and in vivo experiments to demonstrate its anti-tumor activity through immunomodulatory function. MAP was shown to reduce PD-L1 expression at the cellular level; its T cell killing ability was also verified in vitro at the cellular level; and its immunotherapeutic ability was also verified in animals.

[0023] The use of maprotiline or a pharmaceutically acceptable salt thereof in the preparation of anti-tumor drugs provided by the present invention proves that MAP has immunotherapeutic effects and that the dose producing the anti-tumor effect is safe; it proves that MAP promotes T cell killing of cancer cells by downregulating PD-L1 in vivo and in vitro; the combination of MAP and CTLA4 antibodies can produce a synergistic anti-cancer effect, and the anti-tumor effect is significantly enhanced. This "old drug new use" model provides a new alternative for the clinical treatment of colorectal cancer.

[0024] The present invention provides a use of maprotiline or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug. The breakthrough of the present invention lies in the first discovery that MAP can promote the ubiquitination and degradation of PD-L1 in colorectal cancer cells by targeting mTOR, thereby activating the immune system to exert an anti-tumor effect.

[0025] In order to study whether the combination of MAP and CTLA4 antibodies has a synergistic anti-tumor effect. The present invention subcutaneously inoculated MC38 into C57BL / 6J (female) mice, and treated the mice with corn oil, MAP (40 mg / kg), anti-PD-1 (100 ug / mouse), anti-CTLA4 (100 ug / mouse) and MAP combined with anti-CTLA4. The results showed that compared with the use of MAP alone or anti-CTLA4 alone, the growth rate and volume of the tumor were further improved after the combined treatment of MAP and anti-CTLA4. In addition, the flow cytometry analysis results of tumors in different groups of mice showed that MDSCs (CD11b + Gr-1 + ) and Tregs (CD4 + CD25 + Foxp3 + ) cell population was the smallest and granzyme B levels were the highest. Therefore, the results of this study indicate that MAP shifts the immune microenvironment from immunosuppression to immune activation, and that MAP synergizes with anti-CTLA4 to inhibit the proliferation of colorectal cancer cells and promote anti-tumor effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG1 is a diagram showing the effect of detecting cytotoxicity of RKO and MC38 cells using a CCK-8 kit.

[0027] FIG2 is a schematic diagram of the BSA protein standard curve.

[0028] FIG3 is a schematic diagram showing that MAP can downregulate PD-L1 levels in RKO and MC38 cells.

[0029] FIG4 is a schematic diagram of detecting the expression of PD-L1 on the membrane of colon cancer cells by flow cytometry.

[0030] FIG5 is a schematic diagram showing the results of immunofluorescence detection of the down-regulation effect of MAP on PD-L1 on cancer cell membranes.

[0031] FIG6 is a schematic diagram showing the results of measuring the in vitro killing effect of MAP on T cells by crystal violet staining.

[0032] FIG7 is a schematic diagram showing the anti-tumor effects of different doses of MAP on C57BL / 6J mice.

[0033] FIG8 is a schematic diagram showing the anti-tumor effects of the control group and MAP (40 mg / kg) on ​​nude mice.

[0034] FIG9 is a schematic diagram showing the anti-tumor effect of the combined use of MAP and CTLA4 antibodies.

[0035] FIG10 is a schematic diagram of the immunohistochemical analysis results showing that MAP can be combined with CTLA4 antibodies to produce a synergistic anti-tumor effect. DETAILED DESCRIPTION

[0036] 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.

[0037] Example 1

[0038] Study on the effect of MAP on the vitality of colorectal cancer cells in vitro

[0039] 1.1 Experimental Materials:

[0040] Colorectal cell lines and culture: Human colorectal cancer cells RKO and mouse colorectal cancer cells MC38 were purchased from the Shanghai 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. Maprotiline hydrochloride (Cat. No. HY-B0444) was purchased from MedChemExpress (MCE). Other reagents, including the CCK-8 kit (C0048L) and DMSO (ST1276), were products of Beyotime.

[0041] 1.2 Experimental methods

[0042] The cytotoxicity of MAP to the above-mentioned tumor cell lines was detected by CCK-8. The specific steps are as follows:

[0043] 1. Sample preparation: Dissolve MAP in dimethyl sulfoxide to obtain a 10 μM solution;

[0044] 2. After trypsinization and washing, the cells were suspended in MEM or DMEM 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 / mL;

[0045] 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 8 × 10 3 Culture in a 37°C, 5% CO2 cell culture incubator until the cells are fully adherent;

[0046] 4. Change the medium and add the drug. Add 100 μL of sample diluted in cell growth medium to each well, so that the final reaction concentrations are 1, 5, 10, and 20 μM, respectively. Add the corresponding amount of DMSO to the untreated cells as a control. The wells containing cells without drug and DMSO serve as blanks. Each point is tested in triplicate.

[0047] 5. Incubate the treated cells in a 37°C, 5% CO2 cell culture incubator for 24 hours;

[0048] 6. Replace the culture medium in each well with serum-free medium and add 10 μL of CCK-8 reagent. Continue incubating in the incubator for 2-5 hours. Measure the absorbance (OD value) at a wavelength of 450 nm using a microplate reader.

[0049] 7. Viability calculation: Cell viability (%) = [A1-A3] / [A2-A3] × 100, where A1: OD value of the wells containing cells, CCK-8 solution, and drug solution; A2: OD value of the wells containing cells, CCK-8 solution, and no drug solution; A3: OD value of the wells without cells;

[0050] 8. Use GraphPad Prism 9.0.1 software to perform statistical analysis on the data and draw graphs to obtain the IC 50 .

[0051] 1.3 Experimental Results

[0052] Since the CCK-8 reagent contains WST-8, it is reduced to a highly water-soluble yellow formazan dye by dehydrogenases in the cell mitochondria under the action of an electron carrier (1-Methoxy PMS). The amount of formazan generated is proportional to the number of living cells, so this property can be used to directly analyze cell proliferation and toxicity. If the drug has an inhibitory effect on the cells, the color will be lighter. The results are shown in Figure 1, which is a diagram of the effect of detecting cytotoxicity of RKO and MC38 cells using the CCK-8 kit. The cell survival rate of the control group incubated with CCK-8 reagent (10μL) for 4 hours was the highest, and MAP at 1μM, 5μM, and 10μM had little effect on the cell proliferation of colorectal cancer cells after 24 hours of treatment, indicating that 10μM is a safe concentration of MAP. As can be seen from the figure, the IC of MAP on colorectal cancer cells RKO and MC38 50 The concentrations of MAP were 17.71μM and 22.94μM, respectively. This indicated that MAP had no toxic effect on RKO and MC38 cells at 10μM.

[0053] This paper takes colon cancer cells (RKO and MC38) as an example to further illustrate that MAP exerts anti-tumor effects by reducing the expression of PD-L1 in tumor cells.

[0054] Example 2

[0055] Study on downregulation of PD-L1 expression in colon cancer cells by MAP

[0056] 2.1 Experimental Materials

[0057] Colorectal cell lines and culture: Human colorectal cancer cells RKO and mouse colorectal cancer cells MC38 were purchased from the Shanghai 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 kit was from Beyotime (P0009).

[0058] 2.2 Experimental methods

[0059] 2.2.1 Protein immunoblotting

[0060] Cells in good growth condition were cultured at a rate of 4 × 10 5 Cells were seeded at a density of 100 μg / well in a 6-well plate, with a suspension volume of 2 mL per well. The plate was placed in a 37°C incubator for incubation. After cells were fully adhered, MAP was administered at 5, 10, or 20 μM. After 24 hours of incubation, cells were harvested for use in experiments.

[0061] The collected cells were rinsed twice with cold PBS, the PBS was aspirated, and the appropriate amount of lysis buffer containing PMSF was added based on the different culture plates and the actual cell number level. After incubation on ice for 15 minutes, the cells were centrifuged at 12,000 rpm in a 4°C centrifuge for 15 minutes. The supernatant was gently aspirated and transferred to a fresh pre-cooled centrifuge tube and placed on ice. This is the protein sample, and the precipitate was discarded. Protein quantification was performed using the BCA method. Protein denaturation is generally performed using 5× SDS gel reduction loading buffer (5× Loading Buffer), which is mixed with the sample at a ratio of 1:4 and incubated in a metal bath at 95°C for 10 minutes. After cooling, the sample was stored at -20°C.

[0062] The BCA assay method is as follows (Biyuntian BCA kit):

[0063] Based on the number of samples, prepare a working solution of Reagent A and Reagent B in the kit at a ratio of 50:1. Mix thoroughly and add 200 μL of BCA working solution to each well of a flat-bottom 96-well plate, protecting from light. Prepare a 25 mg / mL protein standard solution with the standard sample in the kit. Plot a standard curve using the BSA concentration (mg / mL) on the horizontal axis and the absorbance on the vertical axis, according to the concentrations shown in the table below.

[0064] Table 1 Standard preparation of koji protein

[0065] The standard curve is shown in FIG2 , which is a schematic diagram of the BSA protein standard curve.

[0066] When quantifying protein in samples, take 2 μL of each sample and add it to 198 μL of BCA working solution. Then shake it in a 37°C incubator for 30 minutes. Finally, measure the absorbance of the sample on a microplate reader and substitute the value into the protein standard curve to calculate the protein concentration of the sample.

[0067] Prepare the protein sample and perform electrophoresis on a protein gel corresponding to the molecular weight of the target protein. Initially, run the gel at 80V for approximately 30 minutes. Once the indicator bromophenol blue enters the separating gel, switch to 120V. Turn off the power when the indicator reaches approximately 0.5 cm from the bottom of the gel. Remove the gel and transfer it to a methanol-activated PVDF membrane. Incubate the membrane at 250 mA for 1.5 hours in an ice bath. After transfer, quickly remove the PVDF membrane and block it in 5% skim milk for 1 hour. Remove the membrane and wash it three times with TBST (5 minutes each) on a shaker. Incubate with the corresponding primary antibody (PD-L1) overnight at 4°C. After the primary antibody incubation, wash the membrane three times with TBST (5 minutes each). Then, incubate with the secondary antibody for 2 hours at 4°C or 1 hour at room temperature (22-25°C) with shaking. Finally, wash the membrane three times with TBST before developing it in a developer.

[0068] 2.3 Experimental Results

[0069] RKO and MC38 cells were treated with 5μM, 10μM, and 20μM MAP for 24 hours. The results are shown in Figure 3, which illustrates the downregulation of PD-L1 levels in RKO and MC38 cells by MAP. Figure A shows that after 24-hour treatment of RKO cells with 5μM, 10μM, and 20μM MAP, PD-L1 protein levels in RKO cells decreased with increasing MAP concentrations. Figure B shows that after 24-hour treatment of MC38 cells with 5μM, 10μM, and 20μM MAP, PD-L1 protein levels in MC38 cells decreased with increasing MAP concentrations. Figure C shows that RKO cells were treated with 10μM MAP for 3, 6, 9, 12, and 24 hours, respectively. The results demonstrate that MAP can time-dependently reduce PD-L1 expression in RKO cells. Figure D shows that after 3, 6, 9, 12, and 24-hour treatment of MC38 cells with 10μM MAP, respectively. The results showed that MAP could time-dependently reduce the expression of PD-L1 in MC38 cells.

[0070] As can be seen from A and B, the effects of 5μM, 10μM and 20μM MAP on reducing PD-L1 in colorectal cancer cells (RKO and MC38) increased successively, indicating that it was dose-dependent; as can be seen from C and D, the downregulation of PD-L1 in colorectal cancer cells (RKO and MC38) by 10μM MAP was time-dependent. The results showed that MAP significantly reduced the expression of PD-L1, and this effect was related to the concentration of MAP. 10μM MAP was used to treat RKO cells and treated for 3 hours, 6 hours, 9 hours, 12 hours and 24 hours respectively. The results showed that MAP was able to time-dependently reduce the expression of PD-L1 in cells.

[0071] Example 3

[0072] Flow cytometry detection of the downregulation effect of MAP on PD-L1 on cancer cell membrane

[0073] 3.1 Experimental Materials

[0074] Colorectal cell lines and culture: Human colorectal cancer cells RKO, DLD1, and HT29, and mouse colorectal cancer cells MC38 were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Cells were cultured in MEM, RPMI1640, McCoy's 5A, and DMEM (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.

[0075] 3.2 Experimental methods

[0076] 3.2.1 Flow cytometry analysis of tumor cell membranes revealed PD-L1 abundance

[0077] Colorectal cancer cells in good condition were seeded at a rate of 2.5×10 5Cells were seeded at a density of 100 μL per well in a 12-well plate, with 1 mL of cell suspension per well. After cells were fully adhered, MAP was added for 24 hours. The medium was then removed, and the cells were trypsinized and harvested using an EP tube. 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.

[0078] 3.3 Experimental Results

[0079] PD-L1 expression on the membrane plays a crucial role in immune regulation, particularly in regulating T cell activity and immune system balance. Cancer cells primarily escape the immune system by binding PD-L1 on their membranes to PD-1 on the surface of T cells. Therefore, only by reducing PD-L1 expression on cancer cell membranes can anti-cancer effects be achieved through immune responses.

[0080] The results are shown in FIG4 , which is a schematic diagram of flow cytometry detection of PD-L1 expression on the membrane of colon cancer cells. In the figure, A indicates that after RKO cells were treated with 5μM, 10μM, 15μM and 20μM MAP for 24 hours, the abundance of PD-L1 on the membrane surface of RKO cells decreased with the increase of MAP administration concentration; B indicates that after DLD1 cells were treated with 5μM, 10μM, 15μM and 20μM MAP for 24 hours, the abundance of PD-L1 on the membrane surface of DLD1 cells decreased with the increase of MAP administration concentration; C indicates that after HT29 cells were first treated with 50ng / mL IFN-γ for 2 hours and then treated with 5μM, 10μM, 15μM and 20μM MAP for 24 hours, the abundance of PD-L1 on the membrane surface of HT29 cells decreased with the increase of MAP administration concentration; D indicates that after MC38 cells were treated with 5μM, 10μM, 15μM and 20μM MAP for 24 hours, the abundance of PD-L1 on the membrane surface of MC38 cells decreased with the increase of MAP administration concentration.

[0081] As shown in Figures AD, MAP reduces PD-L1 expression on the cell membranes of colon cancer cells (RKO, DLD1, HT29, and MC38) in a concentration-dependent manner (5μM, 10μM, 15μM, and 20μM). In this experiment, flow cytometry demonstrated that MAP can reduce PD-L1 abundance on the cell membranes of colorectal cancer cells RKO, DLD1, HT29, and MC38 in a concentration-dependent manner, blocking the binding of PD-L1 to PD-1 and achieving an immunotherapy effect.

[0082] Example 4

[0083] Immunofluorescence detection of MAP downregulation of PD-L1 on cancer cell membranes

[0084] 4.1 Experimental Materials

[0085] Colorectal cell lines and culture: Human colorectal cancer cells RKO and DLD1, and mouse colorectal cancer cells MC38 were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Cells were cultured in MEM, RPMI1640, and DMEM (MeilunBio) supplemented with 10% fetal bovine serum (Biological Industries) at 37°C and 5% CO2. Anti-PD-L1 antibody for immunofluorescence was purchased from Proteintech (Cat. No. 66248-1-Ig).

[0086] 4.2 Experimental methods

[0087] 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. MAP was added for 24 h, the supernatant was removed, and the cells were washed three times with PBS.

[0088] 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 for 5 minutes each, add 5% BSA to the sample wells of the 12-well plate for 1 hour, and wash twice with PBS;

[0089] 3. Primary antibody incubation: Add diluted PD-L1 primary antibody, incubate overnight at 4°C, and wash three times with TBST, 5 minutes each time;

[0090] 4. 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. Wash twice with TBST buffer, 5 minutes each time.

[0091] 5. Analyze the results after laser confocal microscopy.

[0092] 4.3 Experimental Results

[0093] The intensity of the red color in immunofluorescence staining 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 shows a schematic diagram of the immunofluorescence assay results for MAP's downregulation of PD-L1 on the cancer cell membrane. After MAP treatment of colorectal cancer cells, the red color of the immunofluorescence staining can reflect the expression of PD-L1. The higher the concentration of MAP, the less red the immunofluorescence staining, indicating that MAP reduces the abundance of PD-L1 on the cell membrane of colorectal cancer cells (RKO, DLD1, and MC38) in a concentration-dependent manner. Immunofluorescence results showed that after MAP treatment of colorectal cancer cells, the red fluorescence decreased in a concentration-dependent manner (5μM, 10μM, 15μM, and 20μM), further demonstrating that MAP can reduce PD-L1 expression on the cell membrane of colon cancer cells (RKO, DLD1, and MC38) and prevent PD-L1 translocation to the plasma membrane.

[0094] Example 5

[0095] Study on the effect of MAP on enhancing the killing effect of T cells on colon cancer cell line RKO in vitro

[0096] 5.1 Experimental Materials

[0097] Human colorectal cell lines and culture: Colorectal cancer cells RKO were purchased from the Shanghai Institute of Biochemistry and 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 obtained from Biyuntian (Catalog No. C0121).

[0098] 5.2 Experimental methods

[0099] The in vitro killing effect of MAP on RKO cell line was measured by NK-92 method. The specific steps are as follows:

[0100] 1. Cell culture and washing: Cancer cells in good condition were cultured at a rate of 3×10 5 The cells were seeded at a density of 1 mL / mL in a 12-well plate, 1 mL per well, and placed in a cell culture medium until the cells were completely attached, and then treated with MAP for 24 h;

[0101] 2. 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;

[0102] 3. Discard the supernatant, wash three times with PBS, fix the cells with 4% paraformaldehyde, and wash three times with PBS. Stain with crystal violet solution for 25 minutes, then wash away excess crystal violet with PBS. Invert the 12-well plate and allow it to dry naturally.

[0103] 4. Use Cytation5 to acquire images and data, and use ImageJ software to perform statistical analysis on the remaining tumor cells.

[0104] 5.3 Experimental Results

[0105] To further evaluate the in vitro antitumor effect of MAP, RKO cells were co-cultured with NK92 cells, and viable tumor cells were detected by crystal violet staining. The results are shown in Figure 6, which shows a schematic diagram of the crystal violet staining assay for MAP-enhanced T cell cytotoxicity in vitro. Figure A shows that after RKO cells were treated with 5μM and 10μM MAP for 24 hours and then added to NK-92 cells, MAP increased the cytotoxicity of NK-92 cells against tumor cells in a concentration-dependent manner. The results in Figure A show that MAP also enhanced the cytotoxicity of NK92 cells against RKO colorectal cancer cells in a concentration-dependent manner. Figure B shows a statistical diagram of apoptotic cells in Figure A. As can be seen from Figures A and B, MAP increased the cytotoxicity of NK-92 cells against tumor cells in a concentration-dependent manner. The results show that, at safe and effective concentrations, MAP significantly enhanced T cell cytotoxicity against tumor cells and reduced tumor cell survival. Figure 6 demonstrates that MAP enhances T cell cytotoxicity against cancer cells by downregulating PD-L1 expression in colorectal cancer cells, inducing cell apoptosis.

[0106] Example 6

[0107] Anti-tumor effect of MAP in vivo

[0108] 6.1 Experimental Materials

[0109] Tumor line: Mouse colorectal cancer cells (MC38) were purchased from the Shanghai 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.

[0110] Animals: C57BL / 6J mice (female) were purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., with certificate number (20220009006037).

[0111] Other reagents: Maprotiline hydrochloride (product number: HY-B0444) 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.).

[0112] Sample: MAP was dissolved in DMSO and corn oil at a 10% corn oil dose.

[0113] 6.2 Experimental methods

[0114] 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 / mouse. The tumor volume reached 50 mm 3 Afterwards, the mice were divided into 4 groups: (1) control group; (2) low MAP (10 mg / kg) group; (3) medium MAP (20 mg / kg) group; (4) high MAP (40 mg / kg) group. The drug was administered orally every day for 16 days, and 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.

[0115] 6.3 Experimental Results

[0116] MAP treatment showed a significant inhibitory effect on the growth of mice inoculated with MC38 tumors, and was accompanied by concentration dependence. The anti-tumor effect of MAP was the best when the dosage concentration was 40 mg / kg. The results are shown in Figure 7, which is a schematic diagram of the anti-tumor effect of different doses of MAP on C57BL / 6J mice, among which 40 mg / kg had the best anti-tumor effect. Among them, A is a comparison of the actual tumors of mice after treatment in different groups. The results show that the anti-tumor effect of MAP is concentration-dependent; B is a schematic diagram of the tumor volume curves of different groups. The results show that MAP inhibits tumor growth in a concentration-dependent manner; C is a comparison of tumor weights of various groups. The results show that as the dosage concentration of MAP increases, the weight of the tumor decreases; D is a schematic diagram of the weight changes of mice in different groups. The results show that MAP has no significant effect on the weight of mice; E is a diagram of Tregs (CD4 + CD25 + Foxp3 + ) flow cytometry analysis diagram of cell populations; F is a statistical diagram of Foxp3 in different groups, E and F show that with the increase of MAP dosage, Tregs (CD4 + CD25 + Foxp3 +) cell populations decreased in order; G is the activated MDSCs (CD11b + Gr-1 + ) Schematic diagram of flow cytometry analysis of cell populations; H is a statistical diagram of Gr-1 in different groups. G and H show that with the increase of MAP dosage, MDSCs (CD11b + Gr-1 + ) The proportion of the cell population decreases successively; I is a flow cytometry analysis diagram of granzyme B in tumors of different groups; J is a statistical diagram of granzyme B in different groups, I and J show that with the increase of MAP administration dose, the proportion of granzyme B in tumor cells increases successively.

[0117] As shown in Figure 7A, the inhibitory effect of MAP on mouse tumors gradually increased with increasing doses. The inhibition rates at MAP doses of 10, 20, and 40 mg / kg were 22.19%, 53.11%, and 70.16%, respectively (Figure 7B). Comparison of tumor weights between groups (Figure 7C) further validated the anti-tumor efficacy of MAP. MAP administration had little effect on mouse body weight (Figure 7D), demonstrating that MAP has no systemic toxic effects on mice. Furthermore, within the tumor microenvironment, myeloid-derived suppressor cells (MDSCs) and Tregs strongly suppress T lymphocyte immunity, promoting tumor immune escape. Activated MDSCs and Tregs express high levels of PD-L1, which interacts with PD-1 on T cells, ultimately leading to their depletion. Flow cytometric analysis of tumor tissue from different groups of mice revealed that levels of granzyme B, a marker of cytotoxic T cell activation, increased with increasing MAP dose, indicating that MAP stimulates cytotoxic T cell activity (Figure 7I). Furthermore, compared with tumors in the control group, activated MDSCs (CD11b + Gr-1 + ) and Tregs (CD4 + CD25 + Foxp3 + ) cell population decreased significantly with the increase of MAP (Figure 7EH). These results indicate that MAP can effectively activate tumor-infiltrating T cells to exert anti-tumor effects.

[0118] Example 7

[0119] Antitumor effect of MAP in nude mice

[0120] 7.1 Experimental Materials:

[0121] Tumor line: Mouse colorectal cancer cells (MC38) were purchased from the Shanghai 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.

[0122] Animals: Nude mice were purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., with certificate number (20220009007529).

[0123] Other reagents: Maprotiline hydrochloride (product number: HY-B0444) 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.).

[0124] Sample: MAP was dissolved in DMSO and corn oil at a 10% corn oil dose.

[0125] 7.2 Experimental methods

[0126] 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 (MAP 40 mg / kg). The mice were given MAP 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.

[0127] 7.3 Experimental Results

[0128] To further confirm that MAP exerts its anti-tumor effects through immune pathways, this study used immunodeficient mice to verify its anti-tumor effects. The results are shown in Figure 8, which shows the anti-tumor effects of the control group and MAP (40 mg / kg) in nude mice. Figure A is a comparison of the actual tumor sizes in the control and treatment groups, demonstrating that MAP has no anti-tumor effect in immunodeficient nude mice; B is a schematic diagram of the tumor growth curves for the two groups of mice, showing that MAP has no effect on tumor growth in nude mice; C is a statistical diagram of tumor weights in the two groups of mice, demonstrating that MAP has no effect on nude mouse tumor weights; and E is a schematic diagram of H&E staining of organs in the two groups of mice, showing that MAP has no effect on the morphology of the heart, liver, spleen, lung, and kidney.

[0129] As shown in Figure 8, MAP lost its inhibitory effect on MC38 tumors in T cell-deficient nude mice, indicating that the anti-tumor effect of MAP is primarily through activation of the immune system and increased T cell infiltration. Furthermore, comparison of HE staining results of the viscera of mice in different groups confirmed that MAP was non-toxic to mice at this dose.

[0130] Example 8

[0131] MAP can combine with CTLA4 antibodies to produce synergistic anti-tumor effects

[0132] 8.1 Experimental Materials:

[0133] Tumor line: Mouse colorectal cancer cells (MC38) were purchased from the Shanghai 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.

[0134] Animals: C57BL / 6J mice (female) were purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., with certificate number (20220009009804).

[0135] Other reagents: Maprotiline hydrochloride (catalog number: HY-B0444) was purchased from MedChemExpress (MCE); anti-mouse-PD-1 antibody (Invivogen, catalog number: BE0146); anti-mouse-CTLA4 (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.).

[0136] Sample: MAP was dissolved in DMSO and corn oil at a 10% corn oil dose.

[0137] 8.2 Experimental methods

[0138] To investigate whether the combination of MAP and anti-CTLA4 (CTLA4 antibody) has a synergistic anti-tumor effect, MC38 colon cancer cells were inoculated into 6-8 week old female C57BL / 6J mice (8×10 5 cells / mouse) to establish a subcutaneous tumor model. 3Afterwards, the mice were divided into five groups: (1) control group; (2) anti-PD-1 group; (3) anti-CTLA4 group; (4) MAP (40 mg / kg) group; (5) MAP (40 mg / kg) + anti-CTLA4 group (100 μg / mouse). The administration period was 16 days. MAP was administered by gavage, and the antibody was injected intraperitoneally every five days. A total of three injections of the antibody were given, 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.

[0139] 8.3 Experimental Results

[0140] Due to the significant adverse reactions associated with the combined use of PD-1 and CTLA4 antibodies, the combination of small molecule drugs and CTLA4 antibodies has emerged as a cancer immunotherapy strategy aimed at enhancing the immune system's ability to attack tumors. This combination therapy aims to improve therapeutic efficacy by simultaneously intervening in multiple immune regulatory pathways.

[0141] Figure 9 is a schematic diagram of the anti-tumor effect of the combined use of MAP and CTLA4 antibodies. Among them, A is a schematic diagram of the actual tumor size of mice in different groups, and the results show that the combination of MAP and CTLA4 antibodies has the best anti-tumor effect; B is a schematic diagram of the tumor volume curve of mice in different groups, and the results show that the combination of MAP and CTLA4 antibodies has the most obvious inhibitory effect on tumor growth; C is a schematic diagram of the tumor weight comparison of each group, and the results show that the tumor weight of the combination of MAP and CTLA4 antibodies is the lightest; D is a schematic diagram of the weight change of mice in different groups, and the results show that the combination of MAP and CTLA4 antibodies has a certain effect on the weight of mice; E is a schematic diagram of Tregs (CD4 + CD25 + Foxp3 + ) flow cytometry analysis diagram of cell populations; F is a statistical diagram of Foxp3 in different groups; E and F show the expression of Tregs (CD4 + CD25 + Foxp3 + ) cell population accounted for the smallest proportion; G is the activated MDSCs (CD11b + Gr-1 + ) Schematic diagram of flow cytometry analysis of cell populations; H is a statistical diagram of Gr-1 in different groups; G and H show the expression of MDSCs (CD11b + Gr-1 +) cell population accounted for the smallest proportion; I is a flow cytometry analysis diagram of granzyme B in different groups of tumors; J is a statistical diagram of granzyme B in different groups; I and J show that MDSCs (CD11b + Gr-1 + ) cell population has the highest proportion.

[0142] Figure 10 is a schematic diagram of immunohistochemical analysis results demonstrating the synergistic anti-tumor effect of MAP in combination with CTLA4 antibodies. Panel A is a schematic diagram of the immunohistochemical analysis results of tumor tissues from different groups, and Panel B is a schematic diagram of the quantification results of Panel A. The results of Panels A and B show that the levels of CD3 (a T cell marker), CD4 (regulatory helper T cells), CD8 (cytotoxic T cells), and C-caspase-3 (cleaved caspase-3) were highest in the tumors of the MAP and CTLA4 antibody group, while levels of Ki-67 (a proliferation marker), PD-L1, and FOXP3 (an immunosuppressive molecule) were decreased, indicating that MAP induces significant tumor cell apoptosis in mice. This demonstrates that the combination of MAP and CTLA4 antibodies significantly enhances T cell infiltration in tumor tissues, with a greater effect than the PD-1 antibody, MAP, or CTLA4 antibody alone.

[0143] The results showed that compared with the use of MAP alone or CTLA4 antibody alone, the growth rate and volume of tumors were further improved after the combined treatment of MAP and CTLA4 antibody (as shown in Figure 9, AC). However, the combined treatment had a certain effect on the weight of mice (as shown in Figure 9, D). In addition, the flow cytometry analysis of tumors in different groups of mice showed that the MDSCs (CD11b + Gr-1 + ) and Tregs (CD4 + CD25 + Foxp3 + ) cell population was the smallest and granzyme B levels were the highest (Figure 9, EJ). Therefore, the results indicate that MAP shifts the immune microenvironment from immunosuppression to immune activation, and that MAP synergizes with CTLA4 antibodies to inhibit the proliferation of colorectal cancer cells and promote anti-tumor effects.

[0144] Colorectal cancer is a highly prevalent malignant tumor in humans and poses a serious threat to human health. At present, the clinical treatment of colorectal cancer is still mainly based on surgery, chemotherapy, and targeted drugs, but the overall efficacy is limited. Therefore, there is an urgent need to explore safe and effective new treatment strategies. Blocking the combination of PD-1 and PD-L1 is an important tumor immunotherapy method, but the expression of PD-L1 in tumor cells may affect the clinical efficacy of PD-1 / PD-L1 related immune checkpoint therapy. Therefore, finding small molecules that target and regulate PD-L1 expression is expected to become a new treatment method. The present invention found through protein immunoblotting, flow cytometry and immunofluorescence experiments that maprotiline hydrochloride (MAP) can not only reduce the total protein level of PD-L1 in cancer cells, but also reduce the expression of PD-L1 on the cancer cell membrane, thereby activating the immune system to exert an anti-tumor effect. In addition, it was found that the anti-tumor effect of maprotiline hydrochloride combined with CTLA4 antibodies was significantly enhanced, providing a new alternative for the clinical treatment of colorectal cancer.

[0145] Advantages of the present invention: Small molecule drugs have many advantages, such as non-immunogenicity, low price, oral administration, easy storage and transportation, etc. These advantages may help to overcome the challenges faced by immune checkpoint inhibitors in some cases, including immune-related side effects, high treatment costs and the need for intravenous injection. Therefore, the screening and development of small molecule inhibitors still have important potential. In addition, compared with newly developed small molecule compounds whose synthesis process is more complicated and whose clinical safety also needs further verification, the reuse of approved and marketed drugs can reduce costs and significantly shorten the research cycle. Since the drug toxicology, pharmacokinetic studies and clinical trials of marketed drugs have been completed, these drugs with confirmed efficacy can benefit patients more quickly. Therefore, this "new use of old drugs" model has great prospects in future tumor immunotherapy.

[0146] 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 maprotiline or a pharmaceutically acceptable salt thereof in combination with a CTLA4 antibody in the preparation of an anti-tumor drug, characterized in that: The tumor is selected from colorectal cancer.

2. Use of maprotiline 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 maprotiline 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 maprotiline or a pharmaceutically acceptable salt thereof as a PD-L1 inhibitor in combination with a CTLA4 antibody in the preparation of an anti-tumor drug.

4. A pharmaceutical preparation, characterized in that The pharmaceutical preparation is prepared by combining maprotiline or a pharmaceutically acceptable salt thereof with a CTLA4 antibody.

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