No Title

Abstract

An anti-tumor pharmaceutical composition based on immune checkpoint blocking and a use thereof. The anti-tumor pharmaceutical composition comprises: paroxetine hydrochloride and a T cell enhancer, the T cell enhancer being at least one of vitamin E and thymosin. By means of a series of in vivo and in vitro experiments, it was found for the first time that cannabidiol and paroxetine hydrochloride can effectively reduce the expression of PD-L1 on a tumor cell membrane, thereby blocking the PD-1 / PD-L1 signaling pathway and enhancing the effect of T cells in killing tumor cells. T cell enhancer added on this basis can further increase the number and activity of T cells and significantly enhance the killing ability of T cells after relief from immunosuppression, greatly improving the anti-tumor effect of a drug. The components cannabidiol, paroxetine hydrochloride, and vitamin E+thymosin of the pharmaceutical composition can have a synergistic effect to improve the anti-tumor effect.

Description

An anti-tumor drug composition based on immune checkpoint blockade and its application Technical Field

[0001] The present invention relates to the field of medicine, and in particular to an anti-tumor drug composition based on immune checkpoint blockade and its application. Background Art

[0002] With the continuous innovation and development of tumor immunology, cancer immunotherapy (CIT) has achieved remarkable results in the treatment of various types of tumors, bringing hope to cancer patients.

[0003] Currently, therapies targeting the immune checkpoints PD-1 / PD-L1 are attracting significant attention in tumor immunotherapy. Programmed cell death protein 1 (PD-1) is a key immunosuppressive molecule widely expressed in activated T cells and serves as a surface receptor for these cells. The PD-1 receptor, PD-L1 (programmed death ligand 1), is nearly undetectable in normal tissues, but is abundant in various human tumor tissues. Upon binding to PD-L1, PD-1 initiates immunosuppressive signals to T cells, inhibiting their activation and preventing them from killing tumor cells, leading to tumor immune escape. Blocking the PD-1 / PD-L1 interaction can restore the functional effects of tumor-specific T cells. PD-1 / PD-L1-related immune checkpoint inhibition therapy is an ideal means of tumor immunotherapy. Currently, a variety of monoclonal antibodies targeting PD-1 / PD-L1 have entered the market. Studies have found that solid tumors that respond well to anti-PD-1 / PD-L1 antibodies, such as melanoma, renal cancer, and non-small cell lung cancer, have a positive correlation between treatment efficacy and PD-L1 positive expression. However, due to the many side effects of antibody therapy, only a small number of patients currently benefit from it.

[0004] Disclosure of the invention

[0005] In response to the problems of large side effects and small benefits to patients in existing anti-PD-1 / PD-L1 antibody immunotherapy, the present invention provides a new anti-tumor drug composition based on immune checkpoint blockade and its application. The anti-tumor drug composition can inhibit the expression of PD-L1 protein on the surface of tumor cells, thereby blocking the PD-1 / PD-L1 signaling pathway and enhancing the effect of T cells in killing tumor cells.

[0006] To solve the above technical problems, the first object of the present invention is to provide an anti-tumor pharmaceutical composition based on immune checkpoint blockade, wherein the anti-tumor pharmaceutical composition comprises: paroxetine hydrochloride, and a T cell enhancer, wherein the T cell enhancer is at least one of vitamin E and thymosin.

[0007] Preferably, the anti-tumor pharmaceutical composition further comprises cannabidiol.

[0008] Preferably, the anti-tumor pharmaceutical composition consists of cannabidiol, paroxetine hydrochloride, vitamin E and thymosin.

[0009] Preferably, in the pharmaceutical composition, the mass ratio of cannabidiol, paroxetine hydrochloride, vitamin E and thymosin is: (20~500): (1~100): (10~400): (1~60).

[0010] Preferably, the pharmaceutical composition further comprises: at least one of a pharmaceutically acceptable carrier, an excipient, a wetting agent, an emulsifier, and a pH buffer.

[0011] Preferably, the dosage form of the pharmaceutical composition comprises at least one of oil, granules, tablets, powders, capsules, microcapsules, pills, powders, oral liquids, sols, sprays, and atomizers.

[0012] The second object of the present invention is to provide an anti-tumor pharmaceutical composition based on immune checkpoint blockade, wherein the anti-tumor pharmaceutical composition comprises: paroxetine hydrochloride and cannabidiol.

[0013] Another object of the present invention is to provide the use of the aforementioned anti-tumor drug composition based on immune checkpoint blockade in the preparation of anti-tumor drugs.

[0014] Preferably, the tumor includes any one of colorectal cancer, lung cancer, liver cancer, gastric cancer, bladder cancer, esophageal cancer, breast cancer, and melanoma.

[0015] Preferably, cannabidiol and paroxetine hydrochloride in the drug combination can each inhibit the expression of PD-L1 protein on the surface of tumor cells, thereby blocking the PD-1 / PD-L1 signaling pathway and enhancing the effect of T cells in killing tumor cells.

[0016] Compared with the prior art, the beneficial effects of the present invention include at least:

[0017] (1) Through a series of in vitro cell experiments, the present invention discovered and confirmed for the first time that cannabidiol and paroxetine hydrochloride can effectively reduce the expression of PD-L1 on the surface of tumor cells, thereby blocking the PD-1 / PD-L1 signaling pathway and enhancing the effect of T cells in killing tumor cells.

[0018] (2) The anti-tumor drug provided by the present invention is a combination of immune checkpoint inhibition and T cell enhancement. Among them, cannabidiol and paroxetine hydrochloride can effectively inhibit the expression of PD-L1 on the surface of tumor cells, greatly reducing the effect of T cell activation inhibition; in addition, the T cell enhancer added on this basis can further increase the number and activity of T cells and significantly enhance the killing ability of T cells after the release of immunosuppression. The two aspects work together to significantly improve the anti-tumor effect of the drug. More importantly, compared with anti-PD-1 / PD-L1 antibody immunotherapy, CBD and PAR in the drug of the present invention can directly reduce PD-L1 on the surface of tumor cells, which is more targeted and less likely to cause T cells to damage normal tissues, thus avoiding the occurrence of side effects.

[0019] (3) Experiments have shown that the pharmaceutical composition provided by the present invention, comprising cannabidiol, paroxetine hydrochloride, and vitamin E + thymosin, can exert a synergistic effect and enhance the anti-tumor effect.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 shows the comparison of cell proliferation of different types of tumor cells after treatment with different concentrations of CBD.

[0022] Figure 2 shows the comparison of PD-L1 expression in different types of tumor cells after treatment with different concentrations of CBD, where:

[0023] A shows the PD-L1 expression results in RKO cells;

[0024] B shows the PD-L1 expression results in H1975 cells;

[0025] C shows the PD-L1 expression results in A549 cells.

[0026] Figure 3 shows the comparison of PD-L1 expression on the cell membrane of different types of tumor cells after treatment with different concentrations of CBD, among which,

[0027] A shows the expression of PD-L1 on the HCT116 cell membrane;

[0028] B is the expression result of PD-L1 on the RKO cell membrane;

[0029] C is the expression result of PD-L1 on H460 cell membrane;

[0030] D is the expression result of PD-L1 on A549 cell membrane;

[0031] E is the expression result of PD-L1 on H1975 cell membrane;

[0032] F shows the expression results of PD-L1 on the HT29 cell membrane.

[0033] Figure 4 shows the comparison of PD-L1 expression on the cell membrane of RKO cells after being treated with the same concentration of CBD for different time periods.

[0034] Figure 5 is a comparison of cell survival rates of RKO cells after co-treatment with CBD and NK92 cells.

[0035] FIG6 is a comparison of the anti-tumor effects of different pharmaceutical compositions on mice bearing colorectal cancer, wherein:

[0036] A is the photo of tumor tissue of mice in each group;

[0037] B is the growth curve of the tumor volume of mice in each group;

[0038] C is the weight growth curve of mice in each group;

[0039] D is the growth curve of tumor weight of mice in each group;

[0040] FIG7 is a comparison of the anti-tumor effects of different pharmaceutical compositions on colorectal cancer immunodeficient mice, wherein:

[0041] A is the photo of tumor tissue of mice in each group;

[0042] B is the growth curve of the tumor volume of mice in each group;

[0043] C is the growth curve of tumor weight of mice in each group;

[0044] D is the weight growth curve of mice in each group.

[0045] FIG8 is a comparison of cell survival rates of different types of tumor cells after co-treatment with three drug compositions and NK92 cells, wherein:

[0046] A is the comparison of the survival rate of RKO cells;

[0047] B is the comparison of the survival rates of H1975 cells;

[0048] C is the comparison of the survival rate of HepG2 cells;

[0049] D is the comparison of the survival rates of MGC803 cells.

[0050] FIG9 is a comparison of cell survival rates of different types of tumor cells after co-treatment with three drug compositions and NK92 cells, wherein:

[0051] E is the comparison of T24 cell survival rates;

[0052] F is the comparison of the survival rate of TE-5 cells;

[0053] G is the comparison of the survival rate of MCF-7 cells;

[0054] H is the comparison of B16-F10 cell survival rates;

[0055] The Control group did not receive any medication.

[0056] Figure 10 is a comparison of the anti-tumor effects of colorectal cancer mice after treatment with CBD+vitamin E+thymosin or CBD+artemisinin+vitamin E+thymosin.

[0057] Figure 11 shows a comparison of the anti-tumor effects of colorectal cancer mice after treatment with CBD, curcumin + piperine, CBD + curcumin + piperine, CBD + curcumin + piperine + vitamin E, or CBD + curcumin + piperine + vitamin E + thymosin.

[0058] FIG12 is a comparison of the anti-tumor effects of different pharmaceutical compositions on mice bearing colorectal cancer, wherein:

[0059] A is the photo of tumor tissue of mice in each group;

[0060] B is the weight growth curve of mice in each group;

[0061] C is the growth curve of the tumor volume of mice in each group;

[0062] D is the tumor weight of mice in each group.

[0063] FIG13 is a comparison of cell survival rates of different types of tumor cells after co-treatment with two drug compositions and Jurkat cells overexpressing PD-1, wherein:

[0064] A is the comparison of the survival rate of RKO cells by CBD and PAR;

[0065] B is the comparison of the survival rate of H1975 cells by CBD and PAR;

[0066] C is the comparison of the survival rate of HepG2 cells by CBD and PAR;

[0067] D is the comparison of the effects of CBD and PAR on the survival rate of MGC803 cells.

[0068] FIG14 is a comparison of cell survival rates of different types of tumor cells after co-treatment with two drug compositions and Jurkat cells overexpressing PD-1, wherein:

[0069] A is the comparison of the survival rate of T24 cells by CBD and PAR;

[0070] B is the comparison of the survival rate of TE-5 cells by CBD and PAR;

[0071] C is the comparison of the survival rate of MCF-7 cells by CBD and PAR;

[0072] D is the comparison of the effects of CBD and PAR on the survival rate of B16-F10 cells.

[0073] Best Mode for Carrying Out the Invention

[0074] As described in the background, existing anti-PD-1 / PD-L1 antibody immunotherapies suffer from significant side effects and minimal patient benefit. PD-L1 expression in tumor cells may affect the clinical efficacy of PD-1 / PD-L1-related immune checkpoint therapies. Small molecules that target and regulate PD-L1 expression are expected to become a new treatment approach. Finding small molecules that negatively regulate PD-L1 expression may become a new approach in immunotherapy.

[0075] To this end, the present invention attempts to find small molecules that negatively regulate PD-L1 expression in order to block the PD-1 / PD-L1 pathway in tumors, enhance T cell activity, and kill tumor cells. After a large number of experimental screening and verification, the present invention ultimately proposed a new anti-tumor drug composition based on immune checkpoint blockade, including: cannabidiol and / or paroxetine hydrochloride, and a T cell enhancer. At the same time, another new anti-tumor drug composition based on immune checkpoint blockade is also proposed, including: paroxetine hydrochloride and cannabidiol. Among them:

[0076] Cannabidiol (CBD), a cannabis extract, is currently being widely developed for use in medical skincare products, mood-enhancing beverages, and foods. In recent years, while a growing number of studies have demonstrated CBD's anti-tumor activity in various tumor types, its mechanisms remain unclear and vary significantly across tumor types. This study, however, demonstrates for the first time that CBD can effectively reduce PD-L1 expression in tumors, suggesting potential applications in tumor immunotherapy.

[0077] Paroxetine hydrochloride (PAR) is an antidepressant. Experiments conducted by the present invention have shown that paroxetine hydrochloride also reduces PD-L1 expression in tumor cells. Therefore, paroxetine hydrochloride can also be used as a candidate drug for negatively regulating PD-L1 expression in tumor cells.

[0078] Based on the above research findings, the present invention further designs the use of CBD or PAR in combination with a T cell enhancer. The T cell enhancer includes any one or more of the following effects: (1) promoting the proliferation and differentiation of T cells, increasing the percentage or absolute number of T cells; (2) enhancing the activity of T cells and improving the immune function of T cells. The concept of this design is that CBD and PAR can reduce the expression of PD-L1 in tumor cells, greatly reducing the degree of inhibition of T cell activation. On this basis, the addition of a T cell enhancer can increase the number and activity of T cells and significantly enhance the effect of T cells killing tumor cells after the release of immunosuppression. It can be understood that if a T cell enhancer is added when the expression of PD-L1 is not inhibited, even if the T cell enhancer can promote T cell proliferation and differentiation, the activation of T cells will still be inhibited by the PD-1 / PD-L1 pathway, and the effect of the T cell enhancer is thus greatly reduced. Therefore, the present invention combines CBD or PAR with a T cell enhancer in order to achieve a synergistic effect of the pharmaceutical composition and enhance the anti-tumor activity of the drug.

[0079] In some embodiments, the T cell enhancer includes at least one of vitamin E and thymosin. Vitamin E is an important antioxidant and also an effective immunomodulator, promoting the development of immune organs and the differentiation of immune cells, thereby enhancing both cellular and humoral immunity. Thymosin is a soluble polypeptide extracted from thymus tissue of calves, pigs, or sheep that can enhance T cell immune function and is used to treat congenital or acquired T cell immunodeficiency, autoimmune diseases, and tumors.

[0080] In some embodiments, the anti-tumor pharmaceutical composition is composed of cannabidiol, paroxetine hydrochloride and vitamin E. The dosage ratio of cannabidiol, paroxetine hydrochloride and vitamin E is: (20-500): (1-100): (10-400).

[0081] In some embodiments, the anti-tumor pharmaceutical composition comprises cannabidiol, paroxetine hydrochloride, vitamin E, and thymosin, wherein the dosage ratio of cannabidiol, paroxetine hydrochloride, vitamin E, and thymosin is: (20-500): (1-100): (10-400): (1-60).

[0082] In some embodiments, the pharmaceutical composition further comprises: a pharmaceutically acceptable carrier, an excipient, a wetting agent, an emulsifier, and a pH buffer.

[0083] Suitable pharmaceutically acceptable carriers are well known to those skilled in the art. A comprehensive description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences. Pharmaceutically acceptable carriers in compositions may include liquids such as water, phosphate buffered saline, Ringer's solution, physiological saline, balanced salt solution, glycerol, or sorbitol. In addition, these carriers may also contain auxiliary substances such as lubricants, glidants, wetting agents or emulsifiers, pH buffering substances, and stabilizers such as albumin. During use, a safe and effective amount of the anti-tumor drug of the present invention is administered to a mammal (e.g., a human). Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, all of which are within the skill of a skilled physician. The precise effective amount for a given subject depends on the subject's size and health status, the nature and severity of the condition, and the therapeutic agent and / or combination of therapeutic agents selected for administration. For a given condition, the effective amount can be determined through routine experimentation, which is within the judgment of a clinician.

[0084] In some embodiments, the dosage form of the pharmaceutical composition includes oil, granules, tablets, powders, capsules, pills, powders, oral liquids, sols, sprays, atomizers, etc.

[0085] Another aspect of the present invention further provides the use of the aforementioned anti-tumor drug composition based on immune checkpoint blockade in the preparation of anti-tumor drugs.

[0086] In some embodiments, the tumor comprises any one of colorectal cancer, lung cancer, liver cancer, gastric cancer, bladder cancer, esophageal cancer, breast cancer, and melanoma.

[0087] The experimental process and experimental results of the present invention are described in detail below with reference to the accompanying drawings and examples, thereby illustrating in detail that the anti-tumor pharmaceutical composition of the present invention can inhibit the expression of PD-L1 protein on the surface of tumor cells, thereby blocking the PD-1 / PD-L1 signaling pathway and enhancing the effect of T cells in killing tumor cells.

[0088] Unless otherwise specified, the experimental methods used in the following examples were carried out according to conventional conditions or conditions recommended by the manufacturers.

[0089] The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources. Among them, in Examples 1 to 4, cannabidiol was purchased from Yuxi Hongbao Biotechnology Co., Ltd.; paroxetine hydrochloride was purchased from MCE Company, item number BRL29060A; thymosin enteric-coated tablets were purchased from Heilongjiang Dilong Pharmaceutical Co., Ltd. (National Medicine Standard H20058365); vitamin E was purchased from Dingrui Chemical (Shanghai) Co., Ltd. (item number A04GS156945); Anti-PD-1 was purchased from Bioxcell Company (Ultra-LEAF™ Purified anti-mouse CD279 (PD-1, BE0146); corn oil was purchased from Golden Sun Grain and Oil Company; curcumin was purchased from MCE Company, item number HY-N0005; capsaicin was purchased from MCE Company, item number HY-10448; artemisinin was purchased from MCE Company, item number HY-B0094 In Examples 5 and 6, cannabidiol was purchased from Yuxi Hongbao Biotechnology Co., Ltd.; paroxetine hydrochloride API was from Zhejiang Huahai Pharmaceutical Co., Ltd.; vitamin E API was from Zhejiang Xinhecheng Co., Ltd.; anti-PD-1 was purchased from Bioxcell (Ultra-LEAF™ Purified anti-mouse CD279 (PD-1, BE0146); and corn oil was purchased from Golden Sun Grain and Oil Company.

[0090] The experimental cells used in the examples of the present invention were purchased from ATCC (American Type Culture Collection).

[0091] Example 1 Effect of CBD on Tumor Cells Cultured in Vitro

[0092] (I) Experimental process

[0093] 1. Effect of CBD on the proliferation of tumor cells cultured in vitro

[0094] Different types of tumor cells (colorectal cancer cell RKO, lung cancer cell H1975, and lung cancer cell A549) in the logarithmic growth phase were seeded in 96-well plates at a seeding density of 5 × 10 3 The cells were then incubated in a 37°C, 5% CO2 incubator for 24 hours. The CBD stock solution (10 mM) was diluted with culture medium to 5, 10, and 20 μM, respectively. The old culture medium was aspirated from the 96-well plate, and 100 μL of culture medium containing different CBD concentrations was added to each well. A control well (0 μM CBD) was also set up, and the cells were incubated for another 24 hours. Following incubation, the number of tumor cell proliferation was determined using an EDU kit (Beyotime, C0085S). Finally, images were taken and statistically analyzed using a high-content imaging analysis system.

[0095] 2. Effect of CBD on PD-L1 expression in tumor cells

[0096] Different types of tumor cells (colorectal cancer cell RKO, lung cancer cell H1975, lung cancer cell A549) with good growth status and density above 80% were plated into 6-well plates at a density of 5×10 4 Cells were plated in a 2 mL suspension per well and then incubated in a 37°C incubator. After 12 hours of culture and complete cell attachment, CBD was administered at different concentrations (0, 10, 20, 30, and 40 μM). After 24 hours of culture, the cells were terminated and assayed.

[0097] After terminating the culture, the cells were collected and washed twice with cold PBS. After the PBS residue was removed, an appropriate amount of cell lysis buffer was added for lysis and incubated on ice for 15 min. Then, the cells were centrifuged at 12,000 rpm in a 4°C centrifuge for 15 min. The supernatant was carefully aspirated and the protein concentration was determined using the BCA method. Then, the corresponding amount of loading buffer was added, shaken well, and incubated in a 100°C water bath for 10 min. Finally, the samples were subjected to protein immunoblotting experiments.

[0098] 3. Effect of CBD on PD-L1 expression on tumor cell membrane

[0099] (1) Different types of tumor cells (colon cancer cells RKO, HCT116, and HT29, and lung cancer cells A549, H1975, and H460) in the logarithmic growth phase were seeded in 6-well plates at a seeding density of 2.5 × 10 cells per well. 5Cells were plated at 4% PD-L1 / mL, 2 mL per well, and then incubated in a cell culture incubator overnight. The culture medium was aspirated, and CBD at different concentrations (0, 5, 10, and 20 μM) was added for treatment for 24 h. The supernatant was removed, and the cells were washed twice with PBS. After trypsinization, the cells were centrifuged at 300 g and 4°C for 5 min to collect the cells. The cells were washed twice with pre-cooled PBS and centrifuged at 300 g and 4°C for 5 min to collect the cells. The cells were washed twice with pre-cooled PBS and resuspended in 100 μL PBS. Anti-PD-L1-Alexa Fluor 647 antibody was added and incubated at room temperature for 30 min. The cells were centrifuged at 300 g and 4°C for 5 min, and resuspended in 300 μL PBS in a flow cytometer. The Alexa Fluor 647 signal of the samples was detected by flow cytometry (excitation / emission wavelengths: 647 nm / 666 nm).

[0100] (2) Colorectal cancer cells (RKO) in the logarithmic growth phase were seeded in 6-well plates at a density of 2.5×10 5 The cells were plated with 2 mL of suspension per well and incubated in a cell culture incubator overnight. The culture medium was aspirated and 20 μM CBD was added for 12 h. The supernatant was removed and the cells were washed twice with PBS. The cells were fixed with paraformaldehyde for 15 min and then with 5% BSA for 1 h. After washing twice with PBST, PD-L1 antibody was added for overnight incubation. Red fluorescent antibody was added for incubation at room temperature for 1 h and DAPI was added for staining for 10 min. The cells were photographed and analyzed using a laser confocal microscope.

[0101] 4. Study on CBD-enhanced T cell killing of tumor cells in vitro

[0102] RKO cells in the logarithmic growth phase were seeded in 12-well plates at a density of 5 × 10 4 The cells were cultured overnight in a 5% CO2 incubator, and the supernatant was removed. Different concentrations of CBD (0, 5, 10, and 20 μM) were added and incubated for 12 h. NK-92 cells (T cells) were added at a ratio of 1:500 and cultured for 24 h. A control group without NK-92 cells was also set up. The supernatant was removed, the cells were washed twice with PBS, and then fixed with 4% paraformaldehyde. The cells were washed twice with PBS and stained with crystal violet for 30 min. Finally, the excess crystal violet was washed with PBS, and photos were taken to collect data.

[0103] (2) Experimental results

[0104] 1. CBD has no obvious toxicity to tumor cells in vitro

[0105] As shown in Figure 1, there was no significant difference in fluorescence intensity between the same type of tumor cells treated with different concentrations of CBD. This result suggests that when CBD alone is added to tumor cells cultured in vitro, it has no significant toxicity. Further research is needed to understand the anti-tumor mechanism of CBD.

[0106] 2. CBD reduces PD-L1 expression in tumor cells

[0107] As shown in Figure 2 AC, the results of protein immunoblotting experiments showed that CBD could significantly inhibit the expression of PD-L1 in colorectal cancer cells (RKO) and lung cancer cells (H1975 and A549), and was concentration-dependent at 5 μM, 10 μM and 20 μM.

[0108] 3. CBD reduces the expression of PD-L1 on cancer cell membranes in a concentration- and time-dependent manner

[0109] As shown in Figures 3 and 4, flow cytometry results showed that CBD reduced PD-L1 expression on the cell membranes of colon cancer cells (RKO, HCT116, and HT29) and lung cancer cells (A549, H1975, and H460) in a concentration-dependent manner (5 μM, 10 μM, and 20 μM). Immunofluorescence results showed that CBD, at 20 μM, reduced PD-L1 expression on the cell membranes of colon cancer cells (RKO) in a time-dependent manner.

[0110] 4. CBD enhances T cells’ ability to kill cancer cells in vitro

[0111] Based on the above experimental results 2 and 3, CBD and T cells (NK-92 cells) were added to tumor cells for in vitro co-culture to study the effect of CBD on T cells killing tumor cells in vitro.

[0112] The results are shown in Figure 5. Compared with the control group, CBD+T cell co-culture treatment significantly reduced the survival rate of RKO cells and induced apoptosis of RKO cells. This result indicates that CBD enhances T cell cytotoxicity against RKO cells by downregulating PD-L1 expression in RKO cells, and the effect is concentration-dependent at 5 μM, 10 μM, and 20 μM.

[0113] Example 2 In vivo anti-tumor effects of different pharmaceutical combinations of CBD and paroxetine hydrochloride

[0114] (I) Experimental process

[0115] 1. Inoculate colorectal cancer cells MC38 into 6-week-old female C57BL / 6 mice (1×10 6 The subcutaneous tumor model was established in vivo with 10 cells / mouse. The tumor volume reached 50 mm3 Afterwards, the mice were divided into groups and administered with the following drug combinations: (1) control group (no administration), (2) anti-PD-1 (100 μg / each) group, (3) anti-CTLA4 (100 μg / each) group, (4) CBD (100 mg / kg) group, (5) CBD (100 mg / kg) + anti-CTLA4 (100 μg / each) group, (6) PAR (10 mg / kg) group, (7) PAR (10 mg / kg) + anti-CTLA4 (100 μg / each) group, (8) CBD (100 mg / kg) + PAR (10 mg / kg) group, (9) vitamin E (50 mg / kg) + thymosin (2 mg / kg) group, (10) CBD (100 mg / kg) + vitamin E (50 mg / kg) + thymosin (2 mg / kg) group, (11) PAR (10 mg / kg) + vitamin E (50 mg / kg) + thymosin (2 mg / kg), (12) PAR (10 mg / kg) + CBD (100 mg / kg) + vitamin E (50 mg / kg) + thymosin (2 mg / kg), (13) CBD (100 mg / kg) + vitamin E (50 mg / kg) + thymosin (2 mg / kg) + anti-CTLA4 (100 μg / each) group, (14) PAR (10 mg / kg) + vitamin E (50 mg / kg) + thymosin (2 mg / kg) + anti-CTLA4 (100 μg / each) group, (15) CBD (100 mg / kg) + PAR (10 mg / kg) + vitamin E (50 mg / kg) + thymosin (2 mg / kg) + anti-CTLA4 (100 μg / each) group. The tumor weight, volume and body weight of the mice were monitored every 2 days. The formula for calculating tumor volume is: tumor volume (mm 3 ) = 0.5 × (length) × (width) 2 The mice were killed 14 days after administration and the tumors were dissected.

[0116] The drug composition was dissolved in 10% DMSO + corn oil, 100 μL per mouse by gavage, and the control group mice were injected with the same volume of the same solvent.

[0117] 2. Colon cancer cells MC38 were inoculated into 6-week-old female nude mice (1×10 6 The subcutaneous tumor model was established in vivo with 10 cells / mouse. The tumor volume reached 50 mm 3Afterwards, the mice were divided into groups and administered with the following drug combinations: (1) control group, (2) CBD (100 mg / kg) + vitamin E (50 mg / kg) + thymosin (2 mg / kg) group, (3) PAR (10 mg / kg) + vitamin E (50 mg / kg) + thymosin (2 mg / kg) group, and (4) PAR (10 mg / kg) + CBD (100 mg / kg) + vitamin E (50 mg / kg) + thymosin (2 mg / kg) group. The tumor weight, volume, and body weight of the mice were monitored every 2 days. The formula for calculating tumor volume is: tumor volume (mm 3 ) = 0.5 × (length) × (width) 2 The mice were killed 14 days after administration and the tumors were dissected.

[0118] (2) Experimental results

[0119] 1. Different pharmaceutical combinations of CBD and paroxetine hydrochloride exert anti-tumor effects in normal mice

[0120] As shown in Figure 6 AD and Table 1, the experimental results of the comparison groups (1) (2) (4) (6) show that the therapeutic effects of CBD and PAR are comparable, and both are comparable to the therapeutic effects of the commonly used anti-PD-1 antibodies. Further comparison of the experimental results of the comparison groups (1) (4) (6) (8) shows that compared with the use of CBD and PAR alone, the combined use of CBD + PAR has a better tumor inhibition effect and a synergistic effect.

[0121] When T cell enhancer (vitamin E + thymosin) was added, the experimental results of the control group (1) (4) (9) (10) showed that vitamin E + thymosin alone had no obvious therapeutic effect, while when CBD was used in combination with vitamin E + thymosin, the therapeutic effect was better than that when CBD or vitamin E + thymosin was used alone, showing a synergistic effect.

[0122] Similarly, the experimental results of the comparison groups (1)(6)(9)(11) show that when PAR is used in combination with vitamin E + thymosin, the therapeutic effect is better than when PAR or vitamin E + thymosin is used alone, and it also has a synergistic effect.

[0123] The experimental results of the comparison groups (1) (8) (9) (12) show that compared with the use of CBD+PAR or vitamin E+thymosin alone, the combined use of CBD+PAR+vitamin E+thymosin has a stronger inhibitory effect on tumor growth and has a synergistic effect.

[0124] The above results show that in the pharmaceutical composition provided by the present invention, when any one or both of CBD and PAR are used in combination with the T cell enhancer (vitamin E + thymosin), a synergistic effect will be exerted to produce a stronger anti-tumor effect.

[0125] Table 1: Comparison of tumor volume growth in each group of mice in Example 2

[0126]

[0127] 2. The three combinations of CBD and paroxetine hydrochloride had no anti-tumor effect in T cell-deficient mice

[0128] Based on the above research results, three compositions with better anti-tumor effects were injected into nude mice to explore their effects on tumors in immunodeficient mice.

[0129] The results are shown in Figures AD in Figure 7. There was no significant difference in the tumor volume among the four groups of mice. In other words, the three combination drugs (CBD + vitamin E + thymosin; PAR + vitamin E + thymosin; PAR + CBD + vitamin E + thymosin) had no therapeutic effect on tumors in nude mice.

[0130] The above results indicate that when there are no T cells in the body, the three compositions cannot exert significant anti-tumor effects.

[0131] 3. Three combinations of CBD and paroxetine hydrochloride combined with other immune checkpoint inhibitors exert better anti-tumor effects

[0132] The present invention also attempts to add other immune checkpoint inhibitors to the three compositions with better anti-tumor effects in order to obtain a pharmaceutical composition with even better anti-tumor effects. In this example, anti-CTLA4 antibodies are used.

[0133] As shown in Figures AD in Figure 6, the experimental results of the comparison groups (10) to (15) show that after adding anti-CTLA4 antibodies to the three compositions, the drug compositions exert a synergistic effect and have a more significant anti-tumor effect. Among them, the CBD+PAR+vitamin E+thymosin+anti-CTLA4 composition showed the best anti-tumor effect compared with other compositions.

[0134] Example 3 Three compositions enhance T cell killing of different types of tumor cells in vitro

[0135] (1) Experimental process

[0136] Based on the results of Example 2, three compositions with good anti-tumor effects were added together with T cells to different types of tumor cells for further study. The specific experimental process is as follows:

[0137] Colorectal cancer cells (RKO), lung cancer (H1975), liver cancer (HepG2), gastric cancer cells (MGC-803), bladder cancer cells (T24), esophageal cancer cells (TE-5), breast cancer cells (MCF-7), and melanoma cells (B16-F10) in logarithmic growth phase were cultured at a rate of 5×10 4 Cells were seeded at a density of 100 cells / mL in a 12-well plate and allowed to adhere to the plate overnight in a 37°C, 5% CO2 incubator. The supernatant was removed and culture medium containing PAR+vitamin E+thymosin, CBD+vitamin E+thymosin, or PAR+CBD+vitamin E+thymosin was added. A blank culture medium was also set up as a control group and incubated for 12 hours. NK-92 cells were added at a dilution of 1:500 and cultured for 24 hours. The supernatant was removed, the cells were washed twice with PBS, and fixed with 4% paraformaldehyde. After washing twice with PBS, the cells were stained with crystal violet for 30 minutes. The excess crystal violet was washed away with PBS, and the cells were photographed and collected for data.

[0138] (2) Experimental results

[0139] As shown in Figures AD in Figure 8 and EH in Figure 9, compared with the control group, co-culture of the three pharmaceutical compositions with T cells significantly reduced the survival rates of colorectal cancer cells (RKO), lung cancer cells (H1975), liver cancer cells (HepG2), gastric cancer cells (MGC-803), bladder cancer cells (T24), esophageal cancer cells (TE-5), breast cancer cells (MCF-7), and melanoma cells (B16-F10), and induced apoptosis in tumor cells. These results demonstrate that the three pharmaceutical compositions provided by the present invention can enhance T cell cytotoxicity against tumor cells by downregulating PD-L1 expression in tumor cells.

[0140] Example 4 In vivo anti-tumor effects of CBD and artemisinin combinations and CBD and curcumin combinations

[0141] (1) Experimental process

[0142] The present invention also attempts to add other substances that may have a regulatory effect on the number or activity of T cells or substances with anti-tumor effects to obtain different pharmaceutical compositions that exert synergistic effects. This example uses artemisinin and curcumin + piperine. Among them, artemisinin has been reported to have anti-tumor effects, and curcumin and piperine have also been reported to have immunomodulatory effects.

[0143] The specific experimental process is as follows:

[0144] Colon cancer cells MC38 were inoculated into 6-week-old female C57BL / 6 mice (1×10 6 The subcutaneous tumor model was established in vivo with 10 cells / mouse. The tumor volume reached 50 mm3 Afterwards, the mice in the artemisinin-treated group were divided and treated with the following drug combinations: (1) control group, (2) CBD (100 mg / kg) + vitamin E (50 mg / kg) + thymosin (2 mg / kg) group, and (3) CBD (100 mg / kg) + artemisinin (20 mg / kg) + vitamin E (50 mg / kg) + thymosin (2 mg / kg). The mice in the curcumin-treated group were divided and treated with the following drug combinations: (1) control group, (2) CBD (100 mg / kg) group, (3) curcumin (50 mg / kg) + piperine (1 mg / kg) group, (4) CBD (100 mg / kg) + curcumin (50 mg / kg) + piperine (1 mg / kg) group, and (5) CBD (100 mg / kg) + curcumin (50 mg / kg) + piperine (1 mg / kg) + vitamin E (50 mg / kg) + thymosin (2 mg / kg).

[0145] The tumor growth volume of mice was monitored every 2 days. The tumor volume calculation formula is: tumor volume (mm 3 ) = 0.5 × (length) × (width) 2 Fourteen days after administration, the mice were killed and their tumors dissected. The drug composition was dissolved in 10% DMSO + corn oil, with 100 μL administered orally to each mouse. The control group received an equal volume of the same solvent.

[0146] (2) Experimental results

[0147] As shown in Figure 10, compared with the control group, the tumor growth of mice in the CBD + vitamin E + thymosin group and the CBD + vitamin E + thymosin + artemisinin group was inhibited, but there was no significant difference in the anti-tumor effects of the two, indicating that the addition of artemisinin cannot further enhance the anti-tumor effect of the CBD + vitamin E + thymosin combination.

[0148] As shown in Figure 11, compared with the control group, the tumor growth of mice in the CBD group was inhibited to a certain extent, while the tumor growth of mice in the curcumin + piperine group was not significantly inhibited. In addition, the anti-tumor effects of the drug combinations of CBD + curcumin + piperine, CBD + curcumin + piperine + vitamin E, and CBD + curcumin + piperine + vitamin E + thymosin were comparable, and compared with the effect of CBD alone, these drug combinations did not show a significant advantage, indicating that not all combinations can achieve a synergistically enhanced anti-tumor effect.

[0149] In order to further verify the therapeutic effect of the pharmaceutical composition when only vitamin E is used as the T cell enhancer, the present invention conducted the following experiments.

[0150] Example 5 In vivo anti-tumor effects of different pharmaceutical combinations of CBD and paroxetine hydrochloride

[0151] (1) Experimental process

[0152] 1. Inoculate colorectal cancer cells MC38 into 6-week-old female C57BL / 6 mice (1×10 6 The subcutaneous tumor model was established in vivo with 10 cells / mouse. The tumor volume reached 50 mm 3 Afterwards, the mice were divided into groups and administered with the following drug combinations: (1) control group (no drug administration), (2) anti-PD-1 (100 μg / each time) group, (3) vitamin E (50 mg / kg) group, (4) CBD (100 mg / kg) group, (5) CBD (100 mg / kg) + vitamin E (50 mg / kg) group, (6) PAR (5 mg / kg) group, (7) PAR (5 mg / kg) + vitamin E (50 mg / kg) group, (8) CBD (100 mg / kg) + PAR (5 mg / kg) group, (9) PAR (5 mg / kg) + CBD (100 mg / kg) + vitamin E (50 mg / kg) group. The tumor weight, volume and body weight of the mice were monitored every 2 days. The formula for calculating tumor volume is: tumor volume (mm 3 ) = 0.5 × (length) × (width) 2 The mice were killed 14 days after administration and the tumors were dissected.

[0153] The drug composition was dissolved in 10% DMSO + corn oil, 100 μL per mouse by gavage, and the control group mice were injected with the same volume of the same solvent.

[0154] (2) Experimental results

[0155] 1. Different pharmaceutical combinations of CBD and paroxetine hydrochloride exert anti-tumor effects in normal mice

[0156] As shown in Figure 12 AD and Table 2, the experimental results of the comparison groups (1), (2), (4), and (6) show that the therapeutic effects of CBD and PAR are comparable, and both are also comparable to the therapeutic effects of the commonly used anti-PD-1 antibodies. Further comparison of the experimental results of groups (4), (6), and (8) shows that compared with the use of CBD and PAR alone, the combined use of CBD + PAR has a better tumor inhibition effect and a synergistic effect.

[0157] When vitamin E was added, the experimental results of the control groups (1) (3) (4) (5) showed that vitamin E alone had no obvious therapeutic effect, while when CBD and vitamin E were used in combination, the therapeutic effect was better than when CBD or vitamin E was used alone, showing a synergistic effect.

[0158] Similarly, the experimental results of the comparison groups (1)(3)(6)(7) show that when PAR and vitamin E are used in combination, the therapeutic effect is better than when PAR or vitamin E is used alone, and there is also a synergistic effect.

[0159] The experimental results of the comparison groups (1) (3) (8) (9) show that compared with the use of vitamin E and CBD+PAR alone, the combined use of CBD+PAR+vitamin E has a stronger inhibitory effect on tumor growth and has a synergistic effect.

[0160] The above results indicate that, in the pharmaceutical composition provided by the present invention, when either or both of CBD and PAR are used in combination with vitamin E, a synergistic effect is exerted to produce a stronger anti-tumor effect.

[0161] Table 2: Comparison of tumor volume growth in each group of mice in Example 5

[0162]

[0163] Example 6 PAR+CBD+Vitamin E Combination Enhances T Cell Killing of Different Types of Tumor Cells in Vitro

[0164] (1) Experimental process

[0165] PAR+CBD+Vitamin E, which has a good anti-tumor effect, was added to T cells and injected into different types of tumor cells for research. The specific experimental process is as follows:

[0166] Colorectal cancer cells (RKO), lung cancer (H1975), liver cancer (HepG2), gastric cancer cells (MGC-803), bladder cancer cells (T24), esophageal cancer cells (TE-5), breast cancer cells (MCF-7), and melanoma cells (B16-F10) in logarithmic growth phase were cultured at a rate of 5×10 4The cells were seeded at a density of 100 cells / mL in a 12-well plate and allowed to adhere to the plate overnight at 37°C in a 5% CO2 incubator. The supernatant was removed, and CBD + vitamin E, PAR + vitamin E, CBD + PAR, and PAR + CBD + vitamin E were added, respectively. A blank medium control was also set up and incubated for 12 hours. Jurkat cells (T cells) overexpressing PD-1 were added at a dilution of 1:500 and cultured for 24 hours. The supernatant was removed, the cells were washed twice with PBS, and fixed with 4% paraformaldehyde. After washing twice with PBS, the cells were stained with crystal violet for 30 minutes. Finally, the excess crystal violet was washed with PBS, and the data were collected and photographed.

[0167] (2) Experimental results

[0168] As shown in Figures AD in Figures 13 and AD in Figures 14 , compared to the control group, CBD + Vitamin E, PAR + Vitamin E, CBD + PAR, and PAR + CBD + Vitamin E all enhanced T cell cytotoxicity against colorectal cancer cells (RKO), lung cancer cells (H1975), liver cancer cells (HepG2), gastric cancer cells (MGC-803), bladder cancer cells (T24), esophageal cancer cells (TE-5), breast cancer cells (MCF-7), and melanoma cells (B16-F10), and induced tumor cell apoptosis. The PAR + CBD + Vitamin E combination exhibited even greater tumor killing activity than CBD + Vitamin E, PAR + Vitamin E, and CBD + PAR alone. These results demonstrate that the CBD + Vitamin E, PAR + Vitamin E, CBD + PAR, and PAR + CBD + Vitamin E combinations provided by the present invention can enhance T cell cytotoxicity against tumor cells by downregulating PD-L1 expression in tumor cells (rectal cancer cells, lung cancer cells, liver cancer cells, gastric cancer cells, bladder cancer cells, esophageal cancer cells, breast cancer cells, and melanoma cells). Compared with other compositions, the PAR+CBD+vitamin E combination has a stronger synergistic effect in enhancing the cytotoxic effect of T cells on tumor cells.

[0169] In summary, the present invention provides an anti-tumor pharmaceutical composition based on immune checkpoint blockade, which comprises cannabidiol and / or paroxetine hydrochloride, and a T cell enhancer. A series of in vitro and in vivo experiments were conducted to study the effects of the pharmaceutical composition on PD-L1 expression on tumor cell membranes and its tumor cell killing effect. The results showed that CBD and paroxetine hydrochloride can significantly downregulate the expression of PD-L1 on the surface of cancer cells, thereby blocking the PD-1 / PD-L1 signaling pathway and enhancing the effect of T cells on tumor cells. In addition, experiments have shown that the pharmaceutical ingredients cannabidiol, paroxetine hydrochloride, and vitamin E + thymosin provided by the present invention can exert a synergistic effect when combined in different ways, improving the anti-tumor effect.

[0170] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. An anti-tumor drug composition based on immune checkpoint blockade, characterized in that: The anti-tumor drug composition comprises: paroxetine hydrochloride and a T cell enhancer, wherein the T cell enhancer is at least one of vitamin E and thymosin.

2. The anti-tumor drug composition based on immune checkpoint blockade according to claim 1, characterized in that: The anti-tumor pharmaceutical composition also contains cannabidiol.

3. The anti-tumor drug composition based on immune checkpoint blockade according to claim 2, characterized in that: The anti-tumor drug composition consists of cannabidiol, paroxetine hydrochloride, vitamin E and thymosin.

4. The anti-tumor drug composition based on immune checkpoint blockade according to claim 3, characterized in that: In the pharmaceutical composition, the mass dosage ratio of cannabidiol, paroxetine hydrochloride, vitamin E and thymosin is: (20-500): (1-100): (10-400): (1-60).

5. The anti-tumor drug composition based on immune checkpoint blockade according to claim 1, characterized in that: The pharmaceutical composition further comprises: at least one of a pharmaceutically acceptable carrier, an excipient, a wetting agent, an emulsifier, and a pH buffer.

6. The anti-tumor drug composition based on immune checkpoint blockade according to claim 1, characterized in that: The dosage form of the pharmaceutical composition comprises at least one of oil, granules, tablets, powders, capsules, microcapsule preparations, pills, powders, oral liquids, sols, sprays, and atomizers.

7. An anti-tumor drug composition based on immune checkpoint blockade, characterized in that: The anti-tumor drug composition comprises: paroxetine hydrochloride and cannabidiol.

8. Use of the anti-tumor drug composition based on immune checkpoint blockade according to any one of claims 1 to 7 in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that The tumor includes any one of colorectal cancer, lung cancer, liver cancer, gastric cancer, bladder cancer, esophageal cancer, breast cancer, and melanoma.

10. The use according to claim 9, characterized in that Cannabidiol and paroxetine hydrochloride in the drug combination can respectively inhibit the expression of PD-L1 protein on the surface of tumor cells, thereby blocking the PD-1 / PD-L1 signaling pathway and enhancing the effect of T cells in killing tumor cells.