Anti-tumor pharmaceutical composition based on immune checkpoint blocking and use thereof

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

[19] (3) An experiment revealed that the components of the pharmaceutical composition provided by the present invention, namely, cannabidiol, paroxetine hydrochloride, vitamin E, and thymosin, can exert synergistic effects and improve the anti-tumor effects. BRIEF DESCRIPTION OF THE DRAWINGS

[20] Fig. 1 is a comparison of the proliferation of different types of tumor cells treated with different concentrations of CBD.

[21] Fig. 2 is a comparison of the expression of PD-L1 in different types of tumor cells treated with different concentrations of CBD.

[22] A is a result of the expression of PD-L1 in RKO cells;

[23] B is a result of PD-L1 expression in H1975 cells; and

[24] C is a result of the expression of PD-L1 in A549 cells.

[25] Fig. 3 is a comparison of the expression of PD-L1 on the cell membrane in different types of tumor cells treated with different concentrations of CBD.

[26] A is a result of the expression of PD-L1 on the HCT116 cell membrane;

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

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

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

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

[31] F is a result of the expression of PD-L1 on the HT29 cell membrane.

[32] Fig. 4 is a comparison of the expression of PD-L1 on the cell membrane in RKO cells treated with the same concentration of CBD for different time. 2024314973   08 Jun 2026

[33] Fig. 5 is a comparison of the viability of RKO cells co-treated with CBD and NK92 cells.

[34] Fig. 6 is a comparison of anti-tumor effects of mice with colorectal cancer treated with different pharmaceutical compositions, wherein,

[35] A shows tumor tissue photographs of each group of mice;

[36] B shows growth curves of tumor volumes of each group of mice;

[37] C shows growth curves of body weight of each group of mice; and

[38] D shows growth curves of tumor weight of each group of mice.

[39] Fig. 7 is a comparison of anti-tumor effects of colorectal cancer-immunodeficient mice treated with different pharmaceutical compositions, wherein,

[40] A shows tumor tissue photographs of each group of mice;

[41] B shows growth curves of tumor volumes of each group of mice;

[42] C shows growth curves of tumor weight of each group of mice; and

[43] D shows growth curves of body weight of each group of mice.

[44] Fig. 8 is a comparison of the viability of different types of tumor cells co-treated with 3 pharmaceutical compositions and NK92 cells, wherein,

[45] A is a comparison of the viability of RKO cells;

[46] B is a comparison of the viability of H1975 cells;

[47] C is a comparison of the viability of HepG2 cells; and

[48] D is a comparison of the viability of MGC803 cells.

[49] Fig. 9 is a comparison of the viability of different types of tumor cells co-treated with 3 pharmaceutical compositions and NK92 cells, wherein,

[50] E is a comparison of the viability of T24 cells;

[51] F is a comparison of the viability of TE-5 cells;

[52] G is a comparison of the viability of MCF-7 cells;

[53] H is a comparison of the viability of B16-F10 cells; and

[54] wherein a control group refers to the group in which no drug is administrated.

[55] Fig. 10 is a comparison of the anti-tumor effects on mice with colorectal cancer treated with CBD+vitamin E+thymosin or CBD+artemisinin+vitamin E+thymosin.

[56] Fig. 11 is a comparison of anti-tumor effects on mice with colorectal cancer treated with CBD, curcumin+piperine, CBD+curcumin+piperine, CBD+curcumin+piperine+vitamin E, or CBD+curcumin+piperine+vitamin E+thymosin.

[57] Figure 12 is a comparison of anti-tumor effects of mice with colorectal cancer treated with different pharmaceutical compositions, wherein,

[58] A shows tumor tissue photographs of each group of mice;

[59] B shows growth curves of body weight of each group of mice;

[60] C shows growth curves of tumor volumes of each group of mice;

[61] D shows the tumor weights of each group of mice.

[62] Figure 13 is a comparison of the viability of different types of tumor cells co-treated 2024314973   08 Jun 2026 with 2 pharmaceutical compositions and Jurkat cells overexpressing PD-1, wherein,

[63] A shows a comparison of viability of RKO cells co-treated with CBD and PAR;

[64] B shows a comparison of viability of H1975 cells co-treated with CBD and PAR;

[65] C shows a comparison of viability of HepG2 cells co-treated with CBD and PAR;

[66] D shows a comparison of viability of MGC803 cells co-treated with CBD and PAR.

[67] Figure 14 is a comparison of the viability of different types of tumor cells co-treated with 2 pharmaceutical compositions and Jurkat cells overexpressing PD-1, wherein,

[68] A shows a comparison of viability of T24 cells co-treated with CBD and PAR;

[69] B shows a comparison of viability of TE-5 cells co-treated with CBD and PAR;

[70] C shows a comparison of viability of MCF-7 cells co-treated with CBD and PAR;

[71] D shows comparison of viability of B16-F10 cells co-treated with CBD and PAR. DETAILED DESCRIPTION

[72] As described in the background, existing anti-PD-1 / PD-L1 antibody immunotherapy has several problems, such as severe side effects and limited benefits for patients. The expression of PD-L1 in tumor cells may affect the clinical efficacy of PD-1 / PD-L1 related immune checkpoint therapy. A micromolecule that regulates the expression of PD-L1 in a targeted manner is expected to become a new therapeutic mode, and identifying a micromolecule that negatively regulates the expression of PD-L1 could become a new solution in the process of immunotherapy.

[73] Therefore, the present invention aimed to identify a micromolecule that negatively regulates the expression of PD-L1 to block the PD-1 / PD-L1 pathway in tumors, enhance the activity of T cells, and kill tumor cells. Through a large number of experimental screens and verifications, the present invention finally provides a novel anti-tumor pharmaceutical composition based on immune checkpoint blockade, comprising cannabidiol and / or paroxetine hydrochloride, and a T-cell enhancer. At the same time, the present invention also provides another novel anti-tumor pharmaceutical composition based on immune checkpoint blockade, comprising paroxetine hydrochloride and cannabidiol. Among them,

[74] Cannabidiol (CBD) is a Cannabis sativa extract that has been widely used in the fields of medical skin-care products, beverages and foods capable of improving mood. In recent years, although an increasing number of studies have shown that CBD has an anti-tumor activity in different types of tumors, the anti-tumor mechanism is not completely understood and greatly differs among different types of tumors. In addition, the present invention is the first to report that CBD can effectively reduce the expression of PD-L1 in tumors and is expected to be used in cancer immunotherapy.

[75] Paroxetine hydrochloride (PAR) is an antidepressant. The present invention revealed 2024314973   08 Jun 2026 that paroxetine hydrochloride also reduces the expression of PD-L1 in tumor cells, indicating that paroxetine hydrochloride can also be used as a candidate drug for negatively regulating the expression of PD-L1 in tumor cells.

[76] On the basis of the above studies, the present invention further designs the combined use of CBD or PAR with a T-cell enhancer. The T-cell enhancers include any one or more of the following effects: (1) promoting the proliferation and differentiation of T cells and increasing the percentage or absolute quantity of T cells and (2) enhancing the activity of T cells and improving the immune functions of T cells. The design concept is as follows: CBD and PAR can reduce the expression of PD-L1 in tumor cells and greatly reduce the degree of inhibition of T cells activation. On this basis, the T-cell enhancer is added, the quantity and activity of T cells can be improved, and the ability of T cells to kill tumor cells is markedly increased after immunosuppression is relieved. If the T-cell enhancer is added when the expression of PD-L1 is not inhibited, even if the T-cell enhancer can promote the proliferation and differentiation of T cells, the activation of T cells can still be inhibited by the PD-1 / PD-L1 pathway, and the effect of the T-cell enhancer is greatly reduced. Therefore, CBD or PAR can be combined with the T-cell enhancer for use because the pharmaceutical composition can exert a synergistic effect and improve the anti-tumor activity of the drug.

[77] 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 that can promote the development of immune organs in organisms and the differentiation of immune cells and improve the functions of cellular immunity and humoral immunity in these organisms. Thymosin is derived from a thymus tissue extract of a calf, a pig or a sheep and is a soluble polypeptide that can enhance the immune functions of T cells and can be used for treating congenital or acquired T cells immunodeficiency diseases, autoimmune diseases, and tumors.

[78] In some embodiments, the anti-tumor pharmaceutical composition comprises cannabidiol, paroxetine hydrochloride, and vitamin E. Cannabidiol, paroxetine hydrochloride, and vitamin E have a dose ratio of (20-500):(1-100):(10-400).

[79] In some embodiments, the anti-tumor pharmaceutical composition comprises cannabidiol, paroxetine hydrochloride, vitamin E, and thymosin. Cannabidiol, paroxetine hydrochloride, vitamin E, and thymosin have a dose ratio of (20-500):(1-100):(10-400):(1-60).

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

[81] Suitable pharmaceutically acceptable carriers are well known to be skilled in the art. A 2024314973   08 Jun 2026 sufficient description of pharmaceutically acceptable carriers can be found in Remington’s Pharmaceutical Sciences. The pharmaceutically acceptable carriers in the compositions may comprise liquids such as water, phosphate buffered saline, ringer’s solution, physiological saline, a balanced salt solution, glycerol or sorbitol, and the like. In addition, auxiliary substances such as a lubricant, a glidant, a wetting agent or an emulsifier, a pH buffering substance and a stabilizer, such as albumin and the like, may also be present in these carriers. In use, a safe and effective amount of the anti-tumor drug of the present invention is administered to a mammal (e.g., a human). The specific dose also takes into account factors such as the route of administration and the health of a patient, and the like, which are within the skill range of a skilled physician. The precise effective amount for a subject will depend upon the size and health of the subject, the nature and extent of the disorder, and the combination of a therapeutic agent and / or a therapeutic agent selected for administration. For a given condition, the effective amount can be determined via a routine experiment and can be determined by a clinician.

[82] In some embodiments, the pharmaceutical composition has a dosage form of an oil, a granule, a tablet, a pulvis, a capsule, a pill, a powder, an oral liquid, a sol, a spray and an atomizing agent.

[83] Another aspect of the present invention is the use of anti-tumor pharmaceutical composition based on immune checkpoint blockade in the preparation of an anti-tumor drug.

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

[85] The experimental process and experimental results of the present invention are described in detail below with reference to the accompanying drawings and embodiments so that the anti-tumor pharmaceutical composition of the present invention can inhibit the expression of PD-L1 proteins on the surface of tumor cells, thereby blocking the PD-1 / PD-L1 signaling pathway and enhancing the ability of T cells to kill tumor cells.

[86] Unless otherwise specified, the experimental methods used in the following embodiments are conducted conventionally or according to the conditions recommended by the manufacturers.

[87] The materials, reagents, etc., used in the following embodiments are all commercially available unless otherwise specified. Among them, in Embodiments 1 to 4, cannabidiol was purchased from Yuxi Hongbao Biotechnology Co., Ltd.; paroxetine hydrochloride was purchased from MCE company with the article number BRL29060A; thymosin enteric-coated tablets were purchased from Heilongjiang Dilong Pharmaceutical Co., Ltd. (NMPA approval number H20058365); vitamin E was purchased from Shanghai Sunny Biotech Co., Ltd. 2024314973   08 Jun 2026 (article number A04GS156945); anti-PD-1 was purchased from Bioxcell company (Ultra-LEAF™ purified anti-mouse CD279 (PD-1, BE0146)); corn oil was purchased from GOLD SUN Grain and Oil Co., Ltd.; curcumin was purchased from the MCE company with the article number HY-N0005; capsaicin was purchased from the MCE company with the article number HY-10448; and artemisinin was purchased from the MCE company with the article number HY-B0094. In Embodiments 5 and 6, cannabidiol is purchased from Yuxi Hongbao Biotechnology Co., Ltd.; the active pharmaceutical ingredient of paroxetine hydrochloride is from Zhejiang Huahai Pharmaceutical Co., Ltd.; the active pharmaceutical ingredient of vitamin E is from Zhejiang NHU Co., Ltd.; anti-PD-1 is purchased from Bioxcell company (Ultra-LEAF™ Purified anti-mouse CD279 (PD-1, BE0146)); and corn oil is purchased from GOLD SUN Grain and Oil Co., Ltd.

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

[89] Embodiment 1 Effects of CBD on in vitro cultured tumor cells

[90] (I) Experimental process

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

[92] Different types of tumor cells (RKO colorectal cancer cells, H1975 lung cancer cells, and A549 lung cancer cells) in the logarithmic growth phase were respectively inoculated into a 96-well plate at an inoculum density of 5x103 per well, and then, the tumor cells were placed into an incubator at 37OC and 5% CO2 for incubation for 24 h; a CBD mother solution (10 mM) was respectively diluted with culture medium to 5 uM, 10 uM, or 20 pM, and the culture medium in the 96-well plate was aspirated off, 100 pL of culture medium containing different concentrations of CBD was added to each well, a control well (0 uM CBD) was set, and the cells were continuously incubated for 24 h. After the culture was completed, the quantity of proliferating tumor cells was detected with an EdU kit (Beyotime, C0085S). Finally, a high-content imaging analysis system was used for photographing and the data were counted.

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

[94] Different types of well-grown tumor cells (RKO colorectal cancer cells, H1975 lung cancer cells, and A549 lung cancer cells) with a density of 80% or more were respectively spread on a 6-well plate at a density of 5x104 cells / well, the volume of the suspension in each well was 2 mL, and the cells were subsequently placed in an incubator at 37OC for culture. After 12 h of culture and when the cells had completely adhered to the wall, different concentrations (0 uM, 10 uM, 20 uM, 30 uM, and 40 uM) of CBD were added. After the cells were continuously cultured for 24 h, the culture was terminated, and subsequent detection was conducted. 2024314973   08 Jun 2026

[95] After the culture was terminated, the cells were collected and washed with cold PBS 2 times. After the PBS residue was removed, a proper amount of cell lysate was added for cracking, and the cells were incubated on ice for 15 min and then centrifuged on a 4OC centrifuge at 12,000 rpm for 15 min. The supernatant was carefully aspirated, the protein concentration was measured via the BCA method, a corresponding amount of loading buffer was added, the cells were evenly shaken and incubated in a I00C water bath for 10 min, and finally, the sample was subjected to a protein immunoblotting experiment.

[96] 3. Effect of CBD on the expression of PD-L1 on the membrane of tumor cells

[97] (1) Different types of tumor cells (RKO, HCT116 and HT29 colon cancer cells, and A549, H1975 and H460 lung cancer cells) in the logarithmic growth phase were respectively inoculated on a 6-well plate at an inoculum density of 2.5*105 cells / well with 2 mL / well and then placed in a cell incubator overnight incubation. The culture mixture was removed, CBD at different concentrations (0 uM, 5 uM, 10 uM, and 20 uM) was added for treatment for 24 h, the supernatant was removed, and the cells were washed with PBS 2 times, digested with pancreatin, centrifuged at 300 * g and 4C for 5 min, and collected. The cells were washed with precooled PBS 2 times, centrifuged at 300 * g and 4C for 5 min, and collected. The cells were washed 2 times with precooled PBS, 100 uL of PBS was added to resuspend the cells, an anti-PD-L1-Alexa Fluor 647 antibody was added, the cells were incubated at room temperature for 30 min and centrifuged at 300 * g and 4C for 5 min, 300 uL of PBS was added to resuspend the cells in a flow tube, and an Alexa Fluor 647 signal was detected from the sample using a flow cytometer (excitation / emission wavelength of 647 nm / 666 nm).

[98] (2) The colorectal cancer cells (RKO) in the logarithmic growth phase were inoculated into a 6-well plate at a density of 2.5*105 cells / well, with a 2 mL volume of the suspension in each well. The plate was placed into a cell incubator for incubation overnight, the culture mixture was removed, 20 uM CBD was added for 12 h, the supernatant was removed, PBS was used for washing 2 times, paraformaldehyde was added for fixing for 15 min, 5% BSA was added for fixing for 1 h, PBST was used for washing 2 times, a PD-L1 antibody was added overnight incubation, a red fluorescent antibody was added for incubation at room temperature for 1 h, DAPI was added for staining for 10 min, and finally, a laser scanning confocal microscope was used for photographing and analysis.

[99] 4. Research of CBD on enhancing in vitro killing of tumor cells by T cells

[100] RKO cells in the logarithmic growth phase were inoculated into a 12-well plate at a density of 5*104 cells / mL and placed in a 5% CO2 incubator for growth adhering to a wall overnight. The supernatant was removed, CBD at different concentrations (0 uM, 5 uM, 10 uM, and 20 uM) was added, and the cells were coincubated for 12 h. NK-92 cells (T cells) were added at 1:500, the cells were cultured for 24 h, and a control group without the NK-92 cells was set at the same time. The supernatant was removed, PBS was used for washing 2 times, and 4% paraformaldehyde was added to fix the cells. Then, PBS was used for washing 2 times, crystal violet was added for dyeing for 30 min, and finally, redundant crystal violet 2024314973   08 Jun 2026 was washed away with PBS, and photographing was performed to collect the data.

[101] (II) Experimental results

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

[103] As shown in Fig. 1, the fluorescence intensity of the same type of tumor cells treated with different concentrations of CBD was not significantly different. This result indicated that when only CBD was added to tumor cells cultured in vitro, CBD was not significantly toxic to tumor cells. Further research on the anti-tumor mechanism of CBD is needed.

[104] 2. CBD reduces the expression of PD-L1 in tumor cells

[105] As shown in A-C of Fig. 2, the Western blotting results revealed that CBD significantly inhibited the expression of PD-L1 in RKO colorectal cancer cells and H1975 and A549 lung cancer cells in a concentration-dependent manner at concentrations of 5 pM, 10 pM, and 20 pM.

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

[107] As shown in Figs. 3 and 4, the results of the flow assay revealed that CBD decreased the expression of PD-L1 on the cell membranes of RKO, HCT116 and HT29 colon cancer cells and A549, H1975 and H460 lung cancer cells in a concentration-dependent manner (5 pM, 10 pM, and 20 pM). The result of immunofluorescence showed that when at 20 pM, CBD time-dependently decreased the expression of PD-L1 on RKO colon cancer cell membranes.

[108] 4. CBD enhances in vitro killing of cancer cells by T cells

[109] On the basis of the above experimental results 2 and 3, CBD and T cells (NK-92 cells) were added to tumor cells for in vitro coculture. The effect of CBD on the in vitro killing of tumor cells by T cells was studied.

[110] As shown in Fig. 5, compared with the control, CBD+T cells coculture treatment significantly reduced the viability of RKO cells and induced their apoptosis. The results indicated that CBD enhanced the cytotoxicity of T cells to RKO cells by downregulating the expression of PD-L1 in RKO cells in a concentration-dependent manner at concentrations of 5 pM, 10 pM, and 20 pM.

[111] Embodiment 2 In-vivo anti-tumor effects of different pharmaceutical compositions of CBD and paroxetine hydrochloride

[112] (I) Experimental process

[113] 1. MC38 colorectal cancer cells were inoculated into 6-week-old female C57BL / 6 mice (1x106 cells / mouse) to establish a subcutaneous tumor model. After the tumor volume reached 50 mm3, the mice were grouped and administered according to the following 2024314973   08 Jun 2026 pharmaceutical combinations: (1) control group (no administration), (2) anti-PD-1 (100 pg time) group, (3) anti-CTLA4 (100 pg time) group, (4) CBD (100 mg / kg) group, (5) CBD (100 mg / kg)+anti-CTLA4 (100 pg / time) group, (6) PAR (10 mg / kg) group, (7) PAR (10 mg / kg)+anti-CTLA4 (100 pg / kg) 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) group, (13) CBD (100 mg / kg)+vitamin E (50 mg / kg)+thymosin (2 mg / kg)+anti-CTLA4 (100 pg / time) group, (14) PAR (10 mg / kg)+vitamin E (50 mg / kg)+thymosin (2 mg / kg)+anti-CTLA4 (100 pg / time) group, and (15) CBD (100 mg / kg)+PAR (10 mg / kg)+vitamin E (50 mg / kg)+thymosin (2 mg / kg)+anti-CTLA4 (100 pg / time) group. The tumor weight and volume, and body weight of the mice were monitored once every 2 days. The tumor volume was calculated as follows: tumor volume (mm3) = 0.5x(length)x(width)2. The mice were sacrificed 14 days after drug administration, and the tumors were dissected.

[114] The dissolving mode of the pharmaceutical composition was as follows: the pharmaceutical composition was dissolved in 10%DMSO+corn oil, the mice were gavaged with the pharmaceutical composition at 100 pl / mouse, and the mice in the control group were injected with the same volume of the solvent.

[115] 2. MC38 colorectal cancer cells were inoculated into 6-week-old female nude mice (1x106 cells / mouse) to establish a subcutaneous tumor model. After the tumor volume reached 50 mm3, the mice were grouped and administered according to the following pharmaceutical 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 and volume, and the body weight of the mice were monitored once every 2 days. The tumor volume was calculated as follows: tumor volume (mm3) = 0.5x(length)x(width)2. The mice were sacrificed 14 days after drug administration, and the tumors were dissected.

[116] (II) Experimental results

[117] 1. Anti-Tumor Effects of Different Drug Combinations of CBD and Paroxetine Hydrochloride in Normal Mice

[118] As shown in A-D of Figure 6 and Table 1, the results of the experiments in comparative groups (1), (2), (4) and (6) revealed that CBD and PAR had equivalent therapeutic effects and that CBD and PAR had equivalent therapeutic effects to those of an anti-PD-1 antibody commonly used in the prior art. The results of the experiments in comparative groups (1), (4), (6) and (8) revealed that compared with the single use of CBD 2024314973   08 Jun 2026 and PAR, the combined use of CBD+PAR had a better tumor inhibition effect and synergistic effect.

[119] After the T-cell enhancer (vitamin E+thymosin) was added for treatment, the results of the experiments in the comparison groups (1), (4), (9) and (10) showed that the use of vitamin E+thymosin had no obvious treatment effect. However, when CBD was combined with vitamin E+thymosin, the treatment effect was better than that when CBD and vitamin E+thymosin were used alone, and a synergistic effect existed.

[120] Similarly, the results of the experiments in comparative groups (1), (6), (9) and (11) revealed that when PAR was combined with vitamin E+thymosin, the treatment effect was better than that when PAR and vitamin E+thymosin were used alone, and a synergistic effect also existed.

[121] The results of the experiments in comparative groups (1), (8), (9) and (12) showed that, compared with the single use of CBD+PAR, and vitamin E+thymosin, when CBD+PAR+vitamin E+thymosin was combined, the tumor growth inhibition effect was stronger and a synergistic effect was detected.

[122] The results showed that in the pharmaceutical composition provided by the present invention, when any one or two of CBD and PAR were combined with a T-cell enhancer (vitamin E+thymosin), a synergistic effect was achieved, and a stronger anti-tumor effect was achieved.

[123] Table 1: Comparison of Tumor Volume Growth in Each Group of Mice in Embodiment 2 2024314973   08 Jun 2026 Group DaA-nm^ control anti-PD-1 anti-CTLA4 CBD CBD+anti-CTLA4 PAR PAR+anti-CTLA4 CBD+PAR vitamin E + thymosin CBD+vitamin E +thymosin PAR+vitamin E +thymosin CBD+PAR+ vitamin E +thymosin CBD+vitamin E+thymosin + anti-CTLA4 PAR+vitamin E +thymosin* anti-CTLA4 CBD+PAR+vitamin E+thymosin+anti-CTLA4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 55.98 4 64.65 4 57.73 4 54 99 4 44.534 42.94 4 49.724 47.02 4 52554 46.144 33.924 34.59 4 41324 2 22,43 14.49 1608 12.84 14.07 43.63 ±7.93 1338 1394 10.49 20.78 t9.8S 886 922 2036 2935 £ 11.76 103.33 4 92.70 4 81.72 4 70.96 4 68.09 4 56.86 4 59.944 118 464 72394 69.944 4724 + 65.93 4 59.814 4 5594 45.54 23.73 22.44 57.45 £ 7 94 3726 1333 21.26 4521 36.22 3280 16.57 29.11 27.42 38.83 £ 13.02 17155 i 106 33 4 96.63 4 87 63 4 89204 8354 4 86.99 4 68 644 133534 84294 79.074 55 48 + 7450 4 64314 6 84 69 55.83 48 65 33.43 39.07 43.99 3437 24.32 48.94 34.21 3519 2393 51.10 2875 42.67 £19.94 361.19 £ 185 21 £ 20780 £ 178.79 £ 145.23£ 17763± 135 18± 91.37£ 336,44 £ 142 92 £ 124,00 £ 9060* 104 02± 9094 £ 8 240.97 9253 144.33 175.83 77.16 133 19 7723 2311 192.71 63.44 56.04 46 10 59.03 42.46 55.41 £3224 501.79 4 338 24 4 326.57 4 279.84 4 182.774 263.45 4 18321 4 132.23 4 501.184 197.75 4 182.78 4 132 98* 165 63 4 13191 4 10 254.17 212.09 245.38 225.91 106 13 204.90 112.75 87.77 248.85 121.38 10214 6588 113.07 86.70 69.75 4 54.87 652.97 £ 388 92 £ 42207 ± 399.70 £ 245.72£ 393.48 £ 228 57 £ 192 45 £ 648.74± 271.35 £ 241.07 £ 175.95* 174 65 £ 138.O5£ 12 382.81 24251 299.81 297.10 18345 342 99 147.22 114.78 358.80 20835 213.23 9263 120.74 8528 74.43 4 61.86 77552 4 493.44 ±3 53133 ± 493.09 4 267.53 4 510.894 238.68 4 239.76± 75528 4 321.91 4 272.21 £ 258.00* 189 56 4 169 83 4 14 441.20 57.79 271.20 337.47 220.33 445.89 181.66 127.33 328.56 305 13 285.09 141 74 147.37 158.35 11531459 13 925264 570 04 4 577.00 4 600.09 4 326 42 4 603 614 291.74 4 333.174 885244 376.62 4 338 31 4 301 46* 236 0 4 4 248 16 4 16 454.61 51234 311 69 465 197.05 526 33 180.14 154 66 3208 23381 23280 167.60 142.57 142.17 14239 ±67.54 2024314973   08 Jun 2026

[124] 2. 3 compositions of CBD and paroxetine hydrochloride have no anti-tumor effect in T cells-deficient mice

[125] Based on the research results, 3 compositions with a better anti-tumor effect were injected into nude mice to explore the effect of the compositions on tumors in the immunodeficient mice.

[126] The result was shown in A-D of Fig. 7, there was no significant difference in the size of the tumor volume among the 4 groups of mice; that is, 3 combined drugs (CBD+vitamin E+thymosin; PAR+vitamin E+thymosin; and PAR+CBD+vitamin E+thymosin) had no therapeutic effect on the tumors of the nude mice.

[127] The results indicated that the 3 compositions failed to exert a significant anti-tumor effect when no T cells were present in vivo.

[128] 3. 3 compositions of CBD and paroxetine hydrochloride combined with other immune checkpoint inhibitors have better antitumor effects

[129] The present invention also attempted to add other immune checkpoint inhibitors to the 3 compositions with a better anti-tumor effect so as to obtain a pharmaceutical composition with a more excellent anti-tumor effect. An anti-CTLA4 antibody was used in the present embodiment.

[130] As shown in A-D of Fig. 6, the results of the experiments in comparative groups (10) to (15) showed that the pharmaceutical composition exerted a synergistic effect and had a more significant anti-tumor effect after the anti-CTLA4 antibody was added to the 3 compositions, wherein the CBD+PAR+vitamin E+thymosin+anti-CTLA4 composition exhibited the optimal anti-tumor effect compared with the other compositions.

[131] Embodiment 3: the 3 compositions enhance the in vitro killing of different types of tumor cells by T cells

[132] (I) Experimental process

[133] Based on the research results of Embodiment 2, 3 compositions with a better anti-tumor effect were added to different types of tumor cells together with T cells. The specific experimental process was as follows:

[134] RKO colorectal cancer cells, H1975 lung cancer cells, HepG2 liver cancer cells, MGC-803 gastric cancer cells, T24 bladder cancer cells, TE-5 esophageal cancer cells, MCF-7 breast cancer cells, and B16-F10 melanoma cells in the logarithmic growth phase were inoculated into a 12-well plate at a density of 5*104 / mL, the cells were placed in an incubator at 37°C and 5% CO2 for growth adhering to a wall overnight, the supernatant was removed, the PAR+vitamin E+thymosin composition, a CBD+vitamin E+thymosin composition or a PAR+CBD+vitamin E+thymosin composition culture medium were respectively added, a blank culture medium was used as a control group, the cells were 2024314973   08 Jun 2026 coincubated for 12 h, the NK-92 cells were added at a ratio of 1:500, the cells were cultured for 24 h, the supernatant was removed, the PBS was used for washing 2 times, the 4% paraformaldehyde mixture was added to fix the cells, the PBS was used for washing 2 times, the crystal violet mixture was added for dyeing for 30 min, the excess crystal violet was washed away with PBS, and the photographing was performed to collect data.

[135] (II) Experimental results

[136] As shown in A-D of Fig. 8 and E-H of Fig. 9, compared with the control group, the co-culture treatment of the 3 pharmaceutical compositions and the T cells significantly reduced the viability of RKO colorectal cancer cells, H1975 lung cancer cells, HepG2 liver cancer cells, MGC-803 gastric cancer cells, T24 bladder cancer cells, TE-5 esophageal cancer cells, MCF-7 breast cancer cells, and B16-F10 melanoma cells and induced the apoptosis of tumor cells. These results indicated that all 3 pharmaceutical compositions provided by the present invention can increase the cytotoxicity of T cells to tumor cells by down-regulating the expression of PD-L1 in tumor cells.

[137] Embodiment 4 In-vivo anti-tumor effects of CBD and artemisinin composition, and CBD and curcumin composition

[138] (I) Experimental process

[139] The present invention also involves the addition of other substances that may have a regulatory effect on the quantity or activity of T cells or have an anti-tumor effect to obtain different pharmaceutical compositions that exert synergistic effects. Artemisinin and curcumin+piperine were used in the present example. Among them, artemisinin has been reported to have anti-tumor effects, and curcumin and piperine have also been reported to have immunomodulatory effects.

[140] The specific experimental process was as follows:

[141] MC38 colon cancer cells were inoculated into 6-week-old female C57BL / 6 mice (1X106 cells / mouse) to establish a subcutaneous tumor model. After the tumor volume reached 50 mm3, the mice in the artemisinin-administered groups were grouped and administered with the following pharmaceutical 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 curcumin-administrated group were grouped and administered with the following pharmaceutical 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 group (1 mg / kg), and (5) CBD (100 mg / kg)+curcumin (50 mg / kg)+piperine (1 mg / kg)+vitamin E (50 mg / kg)+thymosin (2 mg / kg).

[142] The tumor growth volume of the mice was monitored once every 2 days. The tumor volume was calculated as follows: tumor volume (mm3) = 0.5X(length)X(width)2. The mice 2024314973   08 Jun 2026 were sacrificed 14 days after drug administration, and the tumors were dissected. The dissolving mode of the pharmaceutical composition was as follows: the pharmaceutical composition was dissolved in 10%DMSO+corn oil, the mice were gavaged with the pharmaceutical composition at 100 ul mouse, and the mice in the control group were injected with the same volume of the solvent.

[143] (II) Experimental results

[144] As shown in Fig. 10, compared with the control group, the tumor growth of the 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, indicating that the addition of the artemisinin did not further improve the anti-tumor effect of the CBD + vitamin E + thymosin composition.

[145] As shown in Fig. 11, compared with the control group, the tumor growth of the mice in the CBD group was inhibited to a certain degree, whereas the tumor growth of the mice in the curcumin + piperine group was not significantly inhibited. In addition, the pharmaceutical compositions of CBD + curcumin + piperine, CBD+curcumin+piperine+vitamin E, and CBD+curcumin+piperine+vitamin E+thymosin had equivalent anti-tumor effects. Compared with CBD alone, the pharmaceutical compositions did not have obvious advantages, indicating that not all combinations can achieve synergistic anti-tumor effects.

[146] To further verify the treatment effect of the pharmaceutical composition, when the T-cell enhancer was only vitamin E, the following experiment was conducted.

[147] Embodiment 5 In-vivo anti-tumor effects of different pharmaceutical compositions combining CBD and paroxetine hydrochloride

[148] (I) Experimental process

[149] MC38 colon cancer cells were inoculated into 6-week-old female C57BL / 6 mice (1x106 cells / mouse) to establish a subcutaneous tumor model. After the tumor volume reached 50 mm3, the mice were grouped and administered with the following pharmaceutical combinations: (1) control group (no drug administration), (2) anti-PD-1 (100 ug per 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, and (9) PAR (5 mg / kg) + CBD (100 mg / kg) + vitamin E (50 mg / kg) group.

[150] The tumor weight, tumor volume, and mouse body weight were monitored once every 2 days. The tumor volume was calculated as follows: tumor volume (mm3) = 0.5x(length)x(width)2. The mice were sacrificed 14 days after drug administration, and the tumors were dissected. 2024314973   08 Jun 2026

[151] The dissolving mode of the pharmaceutical composition was as follows: the pharmaceutical composition was dissolved in 10%DMSO+corn oil, the mice were gavaged with the pharmaceutical composition at 100 pl / mice, and the mice in the control group were injected with the same volume of the solvent.

[152] (II) Experimental results

[153] 1. Anti-Tumor Effects of Different Drug Combinations of CBD and Paroxetine Hydrochloride in Normal Mice

[154] As shown in A-D of Figure 12 and Table 2, the results of the experiments in comparative groups (1), (2), (4), and (6) revealed that CBD and PAR had equivalent therapeutic effects and that CBD and PAR had equivalent therapeutic effects to those of an anti-PD-1 antibody commonly used in the prior art. The results of the experiments in comparative groups (4), (6), and (8) showed that compared with the single use of CBD and PAR, the combined use of CBD+PAR had a better tumor inhibition effect and synergistic effect.

[155] When vitamin E was added, a comparison of the experimental results of groups (1), (3), (4), and (5) revealed that vitamin E alone had no significant therapeutic effect. However, when CBD was combined with vitamin E, the therapeutic effect was better than CBD or vitamin E alone, showing a synergistic effect.

[156] Similarly, comparing the experimental results of groups (1), (3), (6), and (7), when PAR was combined with vitamin E, the therapeutic effect was also better than PAR or vitamin E alone, again demonstrating a synergistic effect.

[157] Comparing the experimental results of groups (1), (3), (8), and (9), it was observed that the combination of CBD+PAR+vitamin E had a stronger inhibitory effect on tumor growth compared to vitamin E alone or CBD + PAR, indicating a synergistic effect.

[158] These results indicate that in the pharmaceutical compositions provided by this invention, the combination of CBD and / or PAR with vitamin E exerts a synergistic effect, producing a stronger anti-tumor effect.

[159] Table 2: Comparison of Tumor Volume Growth in Mice from Each Group in Embodiment 5 2024314973   08 Jun 2026 \\ Group DaV\ X Control PD-1 VE CBD CBD+VE PAR PAR+VE CBD+PAR CBD+PAR+VE 0 0 0 0 0 0 0 0 0 0 4 61.06 + 24,82 54.69 ±13.35 58.77 ±13.71 63.99 ±18.84 42.65 ±8.348 44.74 ± 18.47 40.34 ±17.99 50.10 ± 15.02 52.10 ±12.95 6 166.54 ±55.49 91.09 ±21.88 124.89 ±22.51 113.24±27.18 90.20 ±17.20 61.50 ±24.22 65.91 ±19.43 54.79 ± 20.25 54.20 ±17.48 8 226.51 ±55.18 104.39 ±42.39 191.75+59.69 120.48± 19.56 108.84 ± 39.41 85.83 ±32.97 91.64 ±17.96 68.63 ± 32.77 55.44 ±23.03 10 420.06±110.72 118.77 ±21.50 284.59 ± 64.79 176.51 ±42.50 163.69± 65.98 136.25 ±49.31 120.16± 49.37 115.55± 53.97 78.20 ±17.74 12 513.38 ±161.01 163.17 ± 68.73 459.67± 130.48 241.75± 97.54 204.74 ± 92.50 176.62 ±57.57 147.65± 15.84 135.08±71.17 99.84 ±22.86 14 638.49 ±206.40 188.79 ±75.52 532.81 ±179.11 314.83 ± 103.70 264.94 ±140.64 262.06± 41.51 217.65 ± 67.02 189.38± 79.76 131.40± 50.64 16 687.21 + 177.98 211.82 ±71.23 624.47 ±203.01 359.72± 95.70 336.945+ 136.10 302.90 + 117.83 274.61 + 69.01 225.75 ±82.25 171.56± 53.86

[160] Embodiment 6: PAR+CBD+vitamin E combination enhances in-vitro killing of different types of tumor cells by T cells

[161] (I) Experimental process

[162] The combination of PAR+CBD+vitamin E, which exhibits good anti-tumor effects, was co-incubated with T-cells and different types of tumor cells for investigation. The specific experimental process was as follows:

[163] RKO colorectal cancer cells, H1975 lung cancer cells, HepG2 liver cancer cells, MGC-803 gastric cancer cells, T24 bladder cancer cells, TE-5 esophageal cancer cells, MCF-7 breast cancer cells, and B16-F10 melanoma cells in the logarithmic growth phase were inoculated into a 12-well plate at a density of 5^104 cells / mL. The cells were then placed in an incubator at 37°C and 5% CO2 for growth adhering to a wall overnight, and the supernatant was removed. CBD+vitamin E, PAR+vitamin E, CBD+PAR, and PAR+CBD+vitamin E were added to each well, a blank culture medium was set as a control group, and the cells were co-incubated for 12 h. Jurkat cells overexpressing PD-1 (T-cells) were added at 1:500, and the cells were cultured for 24 h. The supernatant was removed, PBS was used for washing 2 times, and 4% paraformaldehyde was added to fix the cells. PBS was then used for washing 2 times, crystal violet was added for 30 min, and finally, redundant crystal violet was washed away by PBS, and the photographing was performed to collect data.

[164] (II) Experimental results

[165] As shown in A-D of Fig. 13 and A-D of Fig. 14, compared with the control group, CBD + vitamin E, PAR + vitamin E, CBD+PAR, and PAR+CBD+vitamin E all enhanced the ability of T cells to kill RKO colorectal cancer cells, H1975 lung cancer cells, HepG2 liver cancer cells, MGC-803 gastric cancer cells, T24 bladder cancer cells, TE-5 esophageal cancer cells, MCF-7 breast cancer cells, and B16-F10 melanoma cells and induced the apoptosis of tumor cells. Furthermore, the PAR + CBD + vitamin E combination exhibited stronger tumor-killing ability compared to CBD+vitamin E, PAR+vitamin E, and CBD+PAR alone. These results indicate that the CBD+vitamin E, PAR+vitamin E, CBD+PAR, and PAR+CBD+vitamin E combinations provided by this invention can increase the cytotoxicity of T cells against tumor cells by downregulating the expression of PD-L1 in tumor cells 2024314973   08 Jun 2026 (including colorectal cancer, lung cancer, liver cancer, gastric cancer, bladder cancer, esophageal cancer, breast cancer, and melanoma). Compared to other combinations, PAR+CBD+vitamin E combination enhances the cytotoxicity of T cells against tumor cells and has a stronger synergistic effect.

[166] In summary, the present invention provides an anti-tumor pharmaceutical composition based on immune checkpoint blockade, which comprises cannabidiol and / or paroxetine hydrochloride, as well as a T-cell enhancer. A series of in vitro and in vivo experiments have investigated the effects of pharmaceutical compositions on the expression of PD-L1 on the tumor cell membrane and its tumor-killing activity. The results showed that CBD and paroxetine hydrochloride can significantly down-regulate the expression of PD-L1 on the surface of cancer cells, thereby blocking the PD-1 / PD-L1 signaling pathway and enhancing the ability of T cells to kill tumor cells. In addition, the present study demonstrated that the pharmaceutical ingredients provided by the present invention, namely, cannabidiol, paroxetine hydrochloride, and vitamin E+thymosin, can exert a synergistic effect and improve the anti-tumor effect.

[167] Although the content of the present invention has been described in detail through the aforementioned preferred embodiments, it should be recognized that the above description should not be considered as limiting the present invention. Upon reading the aforementioned content, it will be apparent to those skilled in the art that various modifications and alternations to the present invention can be made. Therefore, the claimed scope of the present invention should be defined by the appended claims.

Claims

1. An anti-tumor pharmaceutical composition based on immune checkpoint blockade, comprising paroxetine hydrochloride and a T-cell enhancer, wherein the T-cell enhancers are vitamin E and thymosin.

2. The anti-tumor pharmaceutical composition based on immune checkpoint blockade according to claim 1, wherein the anti-tumor pharmaceutical composition further comprises cannabidiol.

3. The anti-tumor pharmaceutical composition based on immune checkpoint blockade according to claim 2, wherein the anti-tumor pharmaceutical composition consists of cannabidiol, paroxetine hydrochloride, vitamin E, and thymosin.

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

5. The anti-tumor pharmaceutical composition based on immune checkpoint blockade according to claim 1, wherein the pharmaceutical composition further comprises at least one of a pharmaceutically acceptable carrier, an excipient, a wetting agent, an emulsifier, and a pH buffering agent.

6. The anti-tumor pharmaceutical composition based on immune checkpoint blockade according to claim 1, wherein the dosage form of the pharmaceutical composition comprises at least one of an oil, a granule, a tablet, a pulvis, a capsule, a microcapsule preparation, a pill, a powder, an oral liquid, a sol, a spray, and an atomizing agent.

7. A use of the anti-tumor pharmaceutical composition based on immune checkpoint blockade according to any one of claims 1-6 in the preparation of an anti-tumor drug.

8. The use according to claim 7, wherein the tumor comprises any one of the following: colorectal cancer, lung cancer, liver cancer, gastric cancer, bladder cancer, esophageal cancer, breast cancer, or melanoma.

9. The use according to claim 8, wherein in the pharmaceutical composition, cannabidiol and paroxetine hydrochloride can respectively inhibit the expression of PD-L1 proteins on the surface of tumor cells, thereby blocking the PD-1 / PD-L1 signaling pathway and enhancing the effect of T cells on killing tumor cells.

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