Antitumor pharmaceutical compositions based on immune checkpoint blockade and their applications

Abstract

The present invention discloses an antitumor pharmaceutical composition based on immune checkpoint blockade and its application, which comprises paroxetine hydrochloride and a T cell enhancer, which is at least one of vitamin E and thymosin. Through a series of in vitro and in vivo experiments, the present invention has first discovered that cannabidiol and paroxetine hydrochloride can effectively reduce the expression of PD-L1 on the surface of tumor cells, block the PD-1 / PD-L1 signaling pathway, and enhance the tumor cell killing effect of T cells. Based on this, the addition of a T cell enhancer can further increase the number and activity of T cells, significantly enhancing the killing ability of T cells after immunosuppression is released, thereby significantly enhancing the antitumor effect of the drug. The components of the pharmaceutical composition of the present invention, cannabidiol, paroxetine hydrochloride, vitamin E, and thymosin, exert a synergistic effect, improving the antitumor effect.

Description

[Technical Field]

[0001] The present invention relates to the pharmaceutical field, specifically to an antitumor pharmaceutical composition based on immune checkpoint blockade and its application. [Background technology]

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

[0003] Currently, immunotherapy targeting the immune checkpoint PD-1 / PD-L1 has attracted attention. Programmed cell death protein 1 (PD-1) is an important immunosuppressive molecule. It is widely expressed on activated T cells and acts as a surface receptor for activated T cells. Its receptor, programmed death ligand 1 (PD-L1), is barely detectable in normal tissues, but is abundant in various human tumor tissues. When PD-1 binds to PD-L1, it transmits immunosuppressive signals to T cells, suppressing T cell activation and preventing tumor cell killing, leading to tumor immune escape. Blocking the PD-1 / PD-L1 interaction can restore the functional effectiveness of tumor-specific T cells. PD-1 / PD-L1-related immune checkpoint inhibition therapy is an ideal means of tumor immunotherapy. Currently, various monoclonal antibodies against PD-1 / PD-L1 are on the market. Research has found that for solid tumors that respond relatively well to anti-PD-1 / PD-L1 antibodies, such as melanoma, renal cell carcinoma, and non-small cell lung cancer, the therapeutic efficacy rate is positively correlated with the positive expression of PD-L1. However, antibody therapy has many side effects, and currently only a small number of patients benefit from it. Summary of the Invention [Problem to be solved by the invention]

[0004] In response to the problems of significant side effects and limited patient benefits associated with existing anti-PD-1 / PD-L1 antibody immunotherapy, the present invention provides a novel antitumor pharmaceutical composition based on immune checkpoint blockade, which can suppress the expression of PD-L1 protein on the surface of tumor cells, block the PD-1 / PD-L1 signaling pathway, and enhance the tumor cell killing effect of T cells, as well as uses thereof. [Means for solving the problem]

[0005] In order to solve the above technical problems, a first object of the present invention is to provide an antitumor pharmaceutical composition based on immune checkpoint blockade, comprising paroxetine hydrochloride and a T cell enhancer, which is at least one of vitamin E and thymosin.

[0006] Preferably, the antitumor pharmaceutical composition further comprises cannabidiol.

[0007] Preferably, the antitumor pharmaceutical composition consists of cannabidiol, paroxetine hydrochloride, vitamin E and thymosin.

[0008] Preferably, in the pharmaceutical composition, the mass ratio of the amounts of cannabidiol, paroxetine hydrochloride, vitamin E and thymosin used is (20-500):(1-100):(10-400):(1-60).

[0009] Preferably, the pharmaceutical composition further comprises at least one of a pharmaceutically acceptable carrier, excipient, wetting agent, emulsifying agent, pH buffering agent.

[0010] Preferably, the dosage form of the pharmaceutical composition includes at least one of oil, granule, tablet, powder, capsule, microcapsule, pill, powder, oral liquid, sol, spray, and atomized form.

[0011] A second object of the present invention is to provide an antitumor pharmaceutical composition based on immune checkpoint blockade, comprising paroxetine hydrochloride and cannabidiol.

[0012] Another object of the present invention is to provide an application of the above-described anti-tumor pharmaceutical composition based on immune checkpoint blockade in the manufacture of anti-tumor drugs.

[0013] Preferably, the tumor comprises any one of rectal cancer, lung cancer, liver cancer, gastric cancer, bladder cancer, esophageal cancer, breast cancer, and melanoma.

[0014] 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 tumor cell killing effect of T cells. [Effects of the Invention]

[0015] The beneficial effects of the present invention over the prior art include at least the following: (1) Through a series of in vitro cell experiments, the present invention has for the first time discovered and demonstrated that cannabidiol and paroxetine hydrochloride can effectively reduce the expression of PD-L1 on the surface of tumor cells, further block the PD-1 / PD-L1 signaling pathway, and enhance the tumor cell killing effect of T cells.

[0016] (2) The antitumor drug of the present invention is a combination drug based on immune checkpoint inhibition and T cell enhancement, in which cannabidiol and paroxetine hydrochloride effectively suppress the expression of PD-L1 on the surface of tumor cells, significantly reducing the inhibitory effect of T cell activation. Furthermore, the T cell enhancer added based on this can further increase the number and activity of T cells and significantly enhance the killing ability of T cells after immunosuppression is released. The two aspects work together to significantly enhance the antitumor effect of the drug. More importantly, compared to 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 damage to normal tissues by T cells, thereby avoiding the occurrence of side effects.

[0017] (3) Experiments have demonstrated that the components of the pharmaceutical composition of the present invention, cannabidiol, paroxetine hydrochloride, and vitamin E plus thymosin, exert a synergistic effect to improve the antitumor effect. [Brief explanation of the drawings]

[0018] [Figure 1] Figure 1 shows a comparison of cell proliferation in different types of tumor cells after treatment with different concentrations of CBD. [Figure 2] Figure 2 shows the comparison of PD-L1 expression in cells after treatment with different concentrations of CBD in different types of tumor cells, where A is the PD-L1 expression result in RKO cells, B is the PD-L1 expression result in H1975 cells, and C is the PD-L1 expression result in A549 cells. [Figure 3] 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, where A is the PD-L1 expression result on the HCT116 cell membrane, B is the PD-L1 expression result on the RKO cell membrane, C is the PD-L1 expression result on the H460 cell membrane, D is the PD-L1 expression result on the A549 cell membrane, E is the PD-L1 expression result on the H1975 cell membrane, and F is the PD-L1 expression result on the HT29 cell membrane. [Figure 4] Figure 4 shows the comparison of PD-L1 expression on the cell membrane after treating RKO cells with the same concentration of CBD for different periods of time. [Figure 5] FIG. 5 is a comparison of cell viability after co-treatment of RKO cells with CBD and NK92 cells. [Figure 6] Figure 6 shows a comparison of the antitumor effects of rectal cancer mice after treatment with different pharmaceutical compositions, where A is a photograph of the tumor tissue of each group of mice, B is the growth curve of the tumor volume of each group of mice, C is the growth curve of the weight of each group of mice, and D is the growth curve of the tumor weight of each group of mice. [Figure 7] Figure 7 shows a comparison of the antitumor effects of immunodeficient mice with rectal cancer after treatment with different pharmaceutical compositions, where A is a photograph of the tumor tissue of each group of mice, B is the growth curve of the tumor volume of each group of mice, C is the growth curve of the tumor weight of each group of mice, and D is the growth curve of the body weight of each group of mice. [Figure 8] Figure 8 shows a comparison of cell viability after co-treatment of different types of tumor cells with three pharmaceutical compositions and NK92 cells, where A is a comparison of RKO cell viability, B is a comparison of H1975 cell viability, C is a comparison of HepG2 cell viability, and D is a comparison of MGC803 cell viability. [Figure 9] Figure 9 shows a comparison of cell viability after co-treatment of different types of tumor cells with three pharmaceutical compositions and NK92 cells, where E is a comparison of T24 cell viability, F is a comparison of TE-5 cell viability, G is a comparison of MCF-7 cell viability, and H is a comparison of B16-F10 cell viability, and the control group indicates that no drug was administered. [Figure 10] FIG. 10 compares the antitumor effects of CBD + vitamin E + thymosin or CBD + artemisinin + vitamin E + thymosin treatment in mice with rectal cancer. [Figure 11]Figure 11 shows the comparison of antitumor effects after treatment of mice with rectal cancer with CBD, curcumin + piperine, CBD + curcumin + piperine, CBD + curcumin + piperine + vitamin E, or CBD + curcumin + piperine + vitamin E + thymosin. [Figure 12] Figure 12 shows a comparison of the antitumor effects of rectal cancer mice after treatment with different pharmaceutical compositions, where A is a photograph of the tumor tissue of each group of mice, B is the weight growth curve of each group of mice, C is the tumor volume growth curve of each group of mice, and D is the tumor weight of each group of mice. [Figure 13] Figure 13 shows a comparison of cell viability after co-treatment of different types of tumor cells with two pharmaceutical compositions and PD-1-overexpressing JurkaT cells, where A is a comparison of the viability of CBD and PAR in RKO cells, B is a comparison of the viability of CBD and PAR in H1975 cells, C is a comparison of the viability of CBD and PAR in HepG2 cells, and D is a comparison of the viability of CBD and PAR in MGC803 cells. [Figure 14] Figure 14 shows a comparison of cell viability of different types of tumor cells after co-treatment with two pharmaceutical compositions and PD-1-overexpressing JurkaT cells, where A is a comparison of the viability of CBD and PAR in T24 cells, B is a comparison of the viability of CBD and PAR in TE-5 cells, C is a comparison of the viability of CBD and PAR in MCF-7 cells, and D is a comparison of the viability of CBD and PAR in B16-F10 cells. DETAILED DESCRIPTION OF THE INVENTION

[0019] As explained in the background section, conventional anti-PD-1 / PD-L1 antibody immunotherapy has problems with significant side effects and limited patient benefit. PD-L1 expression in tumor cells may affect the clinical efficacy of PD-1 / PD-L1-related immune checkpoint therapy. Small molecules that target and regulate PD-L1 expression are expected to become a new therapeutic approach, and the discovery of small molecules that negatively regulate PD-L1 expression may provide a new approach in immunotherapy.

[0020] Therefore, the present invention aims to explore 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 extensive experimental screening and verification, the present invention finally proposes a novel antitumor pharmaceutical composition based on immune checkpoint blockade, comprising cannabidiol and / or paroxetine hydrochloride and a T cell enhancer. At the same time, another novel antitumor pharmaceutical composition based on immune checkpoint blockade, comprising paroxetine hydrochloride and cannabidiol, is proposed.

[0021] Cannabidiol (CBD), a cannabis extract, is currently being widely developed in areas such as medical skin care products and mood-improving beverages and foods. In recent years, an increasing number of studies have demonstrated that CBD has antitumor activity in various types of tumors. However, the antitumor mechanisms have not yet been fully elucidated, and significant differences exist between different tumor types. However, this study has for the first time discovered that CBD effectively reduces PD-L1 expression in tumors, potentially leading to its application in tumor immunotherapy.

[0022] Paroxetine hydrochloride (PAR) is an antidepressant. Experiments conducted in the present invention have shown that paroxetine hydrochloride also reduces PD-L1 expression in tumor cells. Therefore, paroxetine hydrochloride is also useful as a candidate drug for negatively regulating PD-L1 expression in tumor cells.

[0023] Based on the above research results, the present invention further designs a combination of CBD or PAR with a T cell enhancer. The T cell enhancer has one or more of the following effects: (1) promotes T cell proliferation and differentiation, increasing the proportion or absolute number of T cells, and (2) enhances T cell activity and improves T cell immune function. The concept behind this design is as follows: CBD and PAR can reduce PD-L1 expression in tumor cells, significantly reducing the degree of suppression of T cell activation. Then, by adding a T cell enhancer, the number and activity of T cells can be increased, significantly enhancing the tumor cell killing effect of T cells after immunosuppression is released. It is clear that if a T cell enhancer is added without suppressing PD-L1 expression, even if the T cell enhancer can promote T cell proliferation and differentiation, T cell activation will be suppressed by the PD-1 / PD-L1 pathway, significantly reducing the effect of the T cell enhancer. Therefore, the present invention aims to use CBD or PAR in combination with a T cell enhancer to produce a synergistic effect in the pharmaceutical composition and improve the antitumor activity of the drug.

[0024] In some embodiments, the T cell enhancer comprises 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 differentiation of immune cells, and enhancing the functions of cellular and humoral immunity. Thymosin is a thymus tissue extract derived from calves, pigs, or sheep, and is a soluble polypeptide that enhances T cell immune function and is used to treat congenital or acquired T cell immunodeficiency diseases, autoimmune diseases, and tumors.

[0025] In some embodiments, the antitumor pharmaceutical composition comprises cannabidiol, paroxetine hydrochloride, and vitamin E, wherein the ratio of the amounts of cannabidiol, paroxetine hydrochloride, and vitamin E used is (20-500):(1-100):(10-400).

[0026] In some embodiments, the antitumor pharmaceutical composition comprises cannabidiol, paroxetine hydrochloride, vitamin E, and thymosin, wherein the ratio of the amounts of cannabidiol, paroxetine hydrochloride, vitamin E, and thymosin used is (20-500):(1-100):(10-400):(1-60).

[0027] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, wetting agent, emulsifying agent, or pH buffering agent.

[0028] Suitable pharmaceutically acceptable carriers are well known to those skilled in the art. A thorough description of pharmaceutically acceptable vectors can be found in Remington's Pharmaceutical Sciences. Pharmaceutically acceptable carriers in the composition may include liquids such as water, phosphate buffer, Ringer's solution, physiological saline, balanced salt solutions, glycerin, sorbitol, and the like. These carriers may also contain auxiliary substances, such as lubricants, flow agents, wetting or emulsifying agents, pH buffers, and stabilizers such as albumin. In use, a safe and effective amount of the antitumor drug described in this 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 particular subject will depend on the subject's physical type and health, the nature and severity of the condition, and the choice of therapeutic agent and / or combination of therapeutic agents to be administered. For a given situation, the effective amount can be determined by routine experimentation and is within the discretion of the clinician.

[0029] In some embodiments, the dosage form of the pharmaceutical composition includes oil, granules, tablets, powders, capsules, pills, powders, oral liquids, sols, sprays, atomized formulations, and the like.

[0030] Another aspect of the present invention further provides the application of the above-described anti-tumor pharmaceutical composition based on immune checkpoint blockade in the manufacture of anti-tumor drugs.

[0031] In some embodiments, the tumor comprises any one of rectal cancer, lung cancer, liver cancer, gastric cancer, bladder cancer, esophageal cancer, breast cancer, and melanoma.

[0032] The experimental process and results of the present invention will be described in detail below with reference to the accompanying drawings and examples, to demonstrate in detail that the antitumor 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 tumor cell killing effect of T cells.

[0033] The experimental methods used in the following examples were carried out according to conventional or manufacturer suggested conditions unless otherwise specified.

[0034] Unless otherwise specified, all materials, reagents, etc. used in the following examples are commercially available. In Examples 1 to 4, cannabidiol was purchased from Yuxi Hongbao Biotechnology Co., Ltd. Paroxetine hydrochloride was purchased from MCE Co., Ltd., product number BRL29060A Thymosin enteric-coated tablets were purchased from Heilongjiang Dilong Pharmaceutical Co., Ltd. (National Pharmaceutical Association H20058365) Vitamin E was purchased from Dingrui Chemical (Shanghai) Co., Ltd. (product number A04GS156945) Anti-PD-1 was purchased from Bioxcell Co., Ltd. (Ultra-LEAF TM Purified anti-mouse CD279 (PD-1, BE0146) was purchased from Jin Sun Grain and Oil Company. Corn oil was purchased from Jin Sun Grain and Oil Company. Curcumin was purchased from MCE Company, product number HY-N0005. Capsaicin was purchased from MCE Company, product number HY-10448. Artemisinin was purchased from MCE Company, product number HY-B0094. In Examples 5 and 6, cannabidiol was purchased from Yuxi Hongbao Biotechnology Co., Ltd. Paroxetine hydrochloride drug substance was purchased from Zhejiang Huahai Pharmaceutical Co., Ltd. Vitamin E drug substance was purchased from Zhejiang Xinhe Cheng Co., Ltd. Anti-PD-1 was purchased from Bioxcell Co., Ltd. (Ultra-LEAF TMPurified anti-mouse CD279 (PD-1, BE0146) was purchased from Kintyo Grain Oil Company.

[0035] All experimental cells used in the examples of the present invention were purchased from ATCC (American Type Culture Collection).

[0036] Example 1: Effect of CBD on tumor cells cultured in vitro (1) Experimental process 1. The proliferative effects of CBD on tumor cells cultured in vitro Different types of tumor cells (rectal cancer cells RKO, lung cancer cells H1975, and lung cancer cells A549) in the logarithmic growth phase were seeded into 96-well plates at a seeding density of 5 × 10 cells per well. 3 The cells were then incubated for 24 hours in a 37°C, 5% CO2 incubator. CBD stock solution (10 mM) was diluted with culture medium to 5, 10, and 20 μM, respectively. The old medium in a 96-well plate was aspirated, and 100 μL of medium containing different concentrations of CBD was added per well to set up control wells (0 μM CBD). The incubation continued for 24 hours. After incubation, tumor cell proliferation was detected using an EDU kit (Biyuntian, C0085S). Finally, photographs were taken using a high-content imaging analysis system, and the data were analyzed.

[0037] 2. The effect of CBD on PD-L1 expression in tumor cells Different types of tumor cells (rectal cancer cells RKO, lung cancer cells H1975, and lung cancer cells A549) with good growth conditions and a density of 80% or more were plated on a 6-well plate at a density of 5 × 10 4 The cells were cultured in a 37°C incubator at 2 mL per well. After 12 hours of culture, when the cells had completely adhered to the wall, different concentrations of CBD (0, 10, 20, 30, and 40 μM) were administered. After 24 hours of culture, the culture was terminated and subsequent testing was performed. After the incubation, the cells were harvested and washed twice with cold PBS. After removing the residual PBS, an appropriate amount of cell lysis solution was added to lyse the cells. The cells were then incubated on ice for 15 minutes and centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was carefully aspirated and the protein concentration was measured using the BCA method. An appropriate amount of loading buffer was then added, the cells were shaken evenly, and the cells were incubated in a water bath at 100°C for 10 minutes. Finally, the samples were subjected to Western blot analysis.

[0038] 3. The effect of CBD on PD-L1 expression on tumor cell membranes (1) Different types of tumor cells (colon cancer cells RKO, HCT116, HT29 and lung cancer cells A549, H1975, H460) in the logarithmic growth phase were inoculated into 6-well plates at an inoculation density of 2.5 × 10 per well. 5 The cells were cultured at 2 mL / mL cells per well and then incubated overnight in a cell incubator. The culture medium was aspirated, and CBD at different concentrations (0, 5, 10, and 20 μM) was added for 24 hours. The supernatant was removed, and the cells were washed twice with PBS. After digestion with pancreatin, the cells were centrifuged at 300 g for 5 minutes at 4°C and harvested. The cells were washed twice with pre-chilled PBS and centrifuged at 300 g for 5 minutes at 4°C. The cells were then washed twice with pre-chilled PBS, resuspended in 100 μL of PBS, incubated with anti-PD-L1-Alexa Fluor 647 antibody at room temperature for 30 minutes, centrifuged at 300 g for 5 minutes at 4°C, resuspended in 300 μL of PBS, and analyzed by flow cytometry. The Alexa Fluor 647 signal (excitation / emission wavelengths: 647 nm / 666 nm) of the samples was detected by flow cytometry.

[0039] (2) Rectal cancer cells (RKO) in logarithmic growth phase were seeded into 6-well plates at a density of 2.5 × 10 5The cells were cultured in a cell culture vessel with a volume of 2 mL of suspension. The culture medium was then aspirated, and 20 μM CBD was added for 12 hours. The supernatant was removed, and the cells were washed twice with PBS. After fixation with paraformaldehyde for 15 minutes, 5% BSA was added for 1 hour. After washing twice with PBST, PD-L1 antibody was added and incubated overnight. Red fluorescent antibody was added and incubated at room temperature for 1 hour. DAPI was added for 10 minutes. Finally, images were analyzed using a laser confocal microscope.

[0040] 4. CBD enhanced T cell killing of tumor cells in vitro RKO cells in logarithmic growth phase were seeded into 12-well plates at a density of 5 × 10 4 The cells were grown overnight in a 5% CO2 incubator at 1000 cells / mL. The supernatant was removed, and CBD was added at different concentrations (0, 5, 10, and 20 μM) and incubated together for 12 hours. NK-92 cells (T cells) were added at a 1:500 ratio and cultured for 24 hours. A control group was also set up without NK-92 cells. The supernatant was removed, and the cells were washed twice with PBS. After two more washes with PBS, 4% paraformaldehyde-fixed cells were added. Crystal violet was added and stained for 30 minutes. Finally, excess crystal violet was washed off with PBS, and photographs were taken to collect data.

[0041] (2) Experimental Results 1. CBD had no apparent toxicity to tumor cells in vitro. As shown in Figure 1, there was no obvious difference in the fluorescence intensity of the same type of tumor cells after treatment with different concentrations of CBD. The results indicated that when CBD alone was added to tumor cells cultured in vitro, CBD had no obvious toxicity to the tumor cells. Further research into the antitumor mechanism of CBD is needed.

[0042] 2. CBD reduced the expression of PD-L1 in tumor cells As shown in Figure 2A-C, Western blot experiments showed that CBD significantly suppressed PD-L1 expression in rectal cancer cells (RKO) and lung cancer cells (H1975 and A549), and showed concentration-dependent suppression at 5 μM, 10 μM, and 20 μM.

[0043] 3. CBD reduced PD-L1 expression on cancer cell membranes in a concentration- and time-dependent manner. As shown in Figures 3 and 4, CBD concentration-dependently (5 μM, 10 μM, and 20 μM) reduced PD-L1 expression on the cell membrane of colon cancer cells (RKO, HCT116, and HT29) and lung cancer cells (A549, H1975, and H460). Immunofluorescence results showed that CBD at 20 μM reduced PD-L1 expression on the cell membrane of colon cancer cells (RKO) in a time-dependent manner.

[0044] 4. CBD enhanced T cell killing of cancer cells in vitro Based on the results of experiments 2 and 3, we added CBD and T cells (NK-92 cells) to tumor cells and performed in vitro co-culture to study the effect of CBD on tumor cell killing by T cells.

[0045] As shown in Figure 5, CBD + T cell coculture treatment significantly reduced RKO cell viability and induced apoptosis compared to the control group. The results showed that CBD concentration-dependently enhanced T cell cytotoxicity against RKO cells at 5 μM, 10 μM, and 20 μM by downregulating PD-L1 expression in RKO cells.

[0046] Example 2: In vivo antitumor effects of different pharmaceutical compositions of CBD and paroxetine hydrochloride (1) Experimental process 1. MC38 rectal cancer cells were inoculated into 6-week-old female C57BL / 6 mice (1 × 10 6 A subcutaneous tumor model was constructed by inoculating 100 cells / animal. 3After reaching the target value, the mice were grouped into the following drug combinations and administered: (1) control group (no administration), (2) anti-PD-1 (100 μg / dose) group, (3) anti-CTLA4 (100 μg / dose) group, (4) CBD (100 mg / kg) group, (5) CBD (100 mg / kg) + anti-CTLA4 (100 μg / dose) group, (6) PAR (10 mg / kg) group, (7) PAR (10 mg / kg) + anti-CTLA4 (100 μg / dose) 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) (10) PAR (10 mg / kg) + vitamin E (50 mg / kg) + thymosin (2 mg / kg) group, (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 μg / dose) group, (14) PAR (10 mg / kg) + vitamin E (50 mg / kg) + thymosin (2 mg / kg) + anti-CTLA4 (100 μg / dose) group, (15) CBD (100 mg / kg) + PAR (10 mg / kg) + vitamin E (50 mg / kg) + thymosin (2 mg / kg) + anti-CTLA4 (100 μg / dose) group. Mouse tumor weight, volume, and mouse body weight were monitored every 2 days. The formula for calculating tumor volume was: tumor volume (mm 3 )=0.5×(length)×(width) 2 Fourteen days after administration, the mice were sacrificed and the tumors were dissected.

[0047] The pharmaceutical composition was dissolved by intragastric injection of 100 μL of 10% DMSO plus corn oil per mouse, and the control group was injected with the same volume of solvent.

[0048] 1. Colon cancer cells MC38 were inoculated into 6-week-old female nude mice (1 × 10 6 A subcutaneous tumor model was constructed by inoculating 100 cells / animal. 3After reaching 100 mg / kg, the mice were grouped and administered 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. Mouse tumor weight, volume, and mouse body weight were monitored every two days. The formula for calculating tumor volume was: tumor volume (mm 3 )=0.5×(length)×(width) 2 Fourteen days after administration, the mice were sacrificed and the tumors were dissected.

[0049] (2) Experimental Results 1. Different pharmaceutical compositions of CBD and paroxetine hydrochloride exerted antitumor effects in normal mice. As shown in the AD diagram in Figure 6 and Table 1, the experimental results for comparison groups (1), (2), (4), and (6) showed that the therapeutic effects of CBD and PAR were comparable, and both were comparable to the therapeutic effects of existing common anti-PD-1 antibodies. Furthermore, the experimental results for comparison groups (1), (4), (6), and (8) showed that the combination of CBD and PAR had a stronger tumor-inhibiting effect and a synergistic effect than when CBD or PAR was used alone.

[0050] When treated with a T cell enhancer (vitamin E + thymosin), the experimental results for the comparison groups (1)(4)(9)(10) showed that when vitamin E + thymosin was used alone, there was no clear therapeutic effect, but when CBD and vitamin E + thymosin were used in combination, the therapeutic effect was higher than when CBD or vitamin E + thymosin was used alone, and a synergistic effect was observed.

[0051] Similarly, the experimental results of the comparison groups (1), (6), (9), and (11) showed that the combined use of PAR and vitamin E + thymosin had a stronger therapeutic effect and a synergistic effect than the use of PAR or vitamin E + thymosin alone.

[0052] The experimental results for the comparison groups (1)(8)(9)(12) showed that the combination of CBD+PAR+vitamin E+thymosin had a stronger tumor growth inhibitory effect and a synergistic effect than the use of CBD+PAR or vitamin E+thymosin alone.

[0053] The above results indicate that when either or both of CBD and PAR are used in combination with a T cell enhancer (vitamin E + thymosin) in the pharmaceutical composition of the present invention, a synergistic effect is exerted, resulting in a stronger antitumor effect.

[0054] Table 1: Comparison of tumor volume growth in mice of each group in Example 2

[0055] [Table 1]

[0056] 2. Three compositions of CBD and paroxetine hydrochloride had no antitumor effect in T cell-deficient mice. Based on the above research results, three compositions with excellent antitumor activity were injected into nude mice to explore their effects on tumors in the body of immunodeficient mice.

[0057] The results, as shown in the AD diagrams in Figure 7, showed that there was no significant difference in tumor volume size among the four groups of mice, meaning that none of the three combination drugs (CBD + vitamin E + thymosin, PAR + vitamin E + thymosin, and PAR + CBD + vitamin E + thymosin) had any therapeutic effect on tumors in nude mice.

[0058] The above results indicated that the three compositions were unable to exert a clear antitumor effect when T cells were not present in the body.

[0059] 3. Three compositions of CBD and paroxetine hydrochloride showed superior antitumor effects when combined with other immune checkpoint inhibitors. The present inventors also attempted to obtain pharmaceutical compositions with even more potent antitumor effects by adding other immune checkpoint inhibitors to the three compositions with excellent antitumor effects. In this example, an anti-CTLA4 antibody was used.

[0060] As shown in AD in Figure 6, the experimental results of the comparison groups (10) to (15) showed that when anti-CTLA4 antibody was added to the three types of compositions, the pharmaceutical compositions exerted a synergistic effect and had a more pronounced anti-tumor effect, and among them, the CBD + PAR + vitamin E + thymosin + anti-CTLA4 composition showed the best anti-tumor effect compared to the other compositions.

[0061] Example 3 Three Compositions Enhanced T Cell Killing of Different Tumor Cell Types In Vitro (1) Experimental process Based on the results of Example 2, three compositions with excellent anti-tumor activity were added to different types of tumor cells together with T cells for further study. The specific experimental procedure was as follows: 5 × 10 cells of logarithmic growth phase rectal 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) were cultured. 4 Cells were seeded into 12-well plates at a density of 100 cells / mL and grown overnight in a 37°C, 5% CO2 incubator with the wells attached. The supernatant was removed, and PAR + vitamin E + thymosin, CBD + vitamin E + thymosin, or PAR + CBD + vitamin E + thymosin medium was added. Blank medium was used as a control and incubated for 12 hours. NK-92 cells were added at a 1:500 ratio and cultured for 24 hours. The supernatant was removed, and the cells were washed twice with PBS. Then, 4% paraformaldehyde-fixed cells were added. After two further washes with PBS, crystal violet was added and stained for 30 minutes. Finally, excess crystal violet was washed off with PBS, and photographs were taken to collect data.

[0062] (2) Experimental Results As shown in Figure 8 (A-D) and Figure 9 (E-H), compared with the control group, co-culture of T cells with the three pharmaceutical compositions significantly reduced the viability of rectal 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 (MCF-7), and melanoma (B16-F10) and induced apoptosis in tumor cells. These results indicate that all three pharmaceutical compositions of the present invention can enhance the cytotoxicity of T cells against tumor cells by downregulating PD-L1 expression in tumor cells.

[0063] Example 4 In vivo antitumor effects of CBD and artemisinin compositions and CBD and curcumin compositions (1) Experimental process In the present invention, we also attempted to obtain a different pharmaceutical composition that exerts a synergistic effect by adding other substances that may have the effect of regulating the number or activity of T cells or substances with antitumor effects. In this example, artemisinin, curcumin, and piperine were used. Here, artemisinin has been reported to have antitumor effects, and curcumin and piperine have also been reported to have immunomodulatory effects.

[0064] The specific experimental process was as follows: Colon cancer cells MC38 were inoculated into 6-week-old female C57BL / 6 mice (1 × 10 6 A subcutaneous tumor model was constructed by inoculating 100 cells / animal. 3 After reaching the target value, the mice in the artemisinin group were grouped and administered the following drug combinations: (1) control group, (2) CBD (100 mg / kg) + vitamin E (50 mg / kg) + thymosin (2 mg / kg) group, (3) CBD (100 mg / kg) + artemisinin (20 mg / kg) + vitamin E (50 mg / kg) + thymosin (2 mg / kg) group. The mice in the curcumin group were grouped and administered the following drug combinations. The groups were: (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, (5) CBD (100 mg / kg) + curcumin (50 mg / kg) + piperine (1 mg / kg) + vitamin E (50 mg / kg) + thymosin (2 mg / kg) group.

[0065] The tumor growth volume of mice was monitored every 2 days. The formula for calculating tumor volume was: tumor volume (mm 3 )=0.5×(length)×(width) 2 The mice were sacrificed 14 days after administration and the tumors were dissected. The pharmaceutical composition was dissolved in 10% DMSO + corn oil, which was injected intragastrically at 100 μL per mouse, and the control group was injected with the same volume of solvent.

[0066] (2) Experimental Results As shown in Figure 10, compared with the control group, the tumor growth in 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 antitumor effects of the two groups, indicating that the addition of artemisinin could not further enhance the antitumor effect of the CBD + vitamin E + thymosin composition.

[0067] As shown in Figure 11, compared with the control group, tumor growth in the mice in the CBD group was inhibited to some extent, but tumor growth in the mice in the curcumin + piperine group was not significantly inhibited. In addition, the anti-tumor effects of several pharmaceutical compositions, namely, CBD + curcumin + piperine, CBD + curcumin + piperine + vitamin E, and CBD + curcumin + piperine + vitamin E + thymosin, were comparable, and these pharmaceutical compositions did not show any obvious advantages compared with the effect of CBD alone, indicating that not all combinations synergistically enhance the anti-tumor effect.

[0068] In the present invention, the following experiment was carried out to further verify the therapeutic effect of the pharmaceutical composition when vitamin E alone was used as a T cell enhancer.

[0069] Example 5. In vivo antitumor effects of different pharmaceutical compositions of CBD and paroxetine hydrochloride (1) Experimental process 1. MC38 rectal cancer cells were inoculated into 6-week-old female C57BL / 6 mice (1 × 10 6 A subcutaneous tumor model was constructed by inoculating 100 cells / animal. 3 After reaching 100 μg / dose, mice were grouped and administered the following drug combinations: (1) control group (no administration), (2) anti-PD-1 (100 μg / dose), (3) vitamin E (50 mg / kg), (4) CBD (100 mg / kg), (5) CBD (100 mg / kg) + vitamin E (50 mg / kg), (6) PAR (5 mg / kg), (7) PAR (5 mg / kg) + vitamin E (50 mg / kg), (8) CBD (100 mg / kg) + PAR (5 mg / kg), and (9) PAR (5 mg / kg) + CBD (100 mg / kg) + vitamin E (50 mg / kg). Mouse tumor weight, volume, and mouse body weight were monitored every 2 days. Tumor volume was calculated as follows: tumor volume (mm 3 )=0.5×(length)×(width) 2 Fourteen days after administration, the mice were sacrificed and the tumors were dissected.

[0070] The pharmaceutical composition was dissolved by intragastric injection of 100 μL of 10% DMSO plus corn oil per mouse, and the control group was injected with the same volume of solvent.

[0071] (2) Experimental Results 1. Different pharmaceutical compositions of CBD and paroxetine hydrochloride exerted antitumor effects in normal mice. As shown in the AD diagram in Figure 12 and Table 2, the experimental results for comparison groups (1), (2), (4), and (6) showed that the therapeutic effects of CBD and PAR were comparable, and both were comparable to the therapeutic effects of existing common anti-PD-1 antibodies. Furthermore, the experimental results for comparison groups (4), (8), and (9) showed that the combination of CBD and PAR had a stronger tumor-inhibiting effect and a synergistic effect than when CBD or PAR was used alone.

[0072] When vitamin E was added, the experimental results for comparison groups (1), (3), (4), and (5) showed that there was no clear therapeutic effect when vitamin E was used alone, but when CBD and vitamin E were used together, the therapeutic effect was stronger than when CBD or vitamin E was used alone, and a synergistic effect was observed.

[0073] Similarly, the experimental results of the comparison groups (1), (3), (6), and (7) showed that the combined use of PAR and vitamin E had a stronger therapeutic effect and a synergistic effect than the use of PAR or vitamin E alone.

[0074] The experimental results for comparison groups (1)(3)(8)(9) showed that the combined use of CBD+PAR+vitamin E had a stronger tumor growth inhibitory effect and a synergistic effect than the use of vitamin E and CBD+PAR alone.

[0075] The above results indicate that when either or both of CBD and PAR are used in combination with vitamin E in the pharmaceutical composition of the present invention, a synergistic effect is exerted, resulting in a stronger antitumor effect.

[0076] Table 2: Comparison of tumor volume growth in mice of each group in Example 5

[0077] [Table 2]

[0078] Example 6: PAR + CBD + Vitamin E Composition Enhanced T Cell Killing of Different Tumor Cell Types In Vitro

[0079] (1) Experimental process The study involved adding PAR, CBD, and vitamin E, which have potent anti-tumor effects, to different types of tumor cells along with T cells. The specific experimental process was as follows:

[0080] 5 × 10 cells of logarithmic growth phase rectal 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) were cultured. 4 Cells were seeded into 12-well plates at a density of 1 / mL and grown overnight in a 37°C, 5% CO2 incubator with the walls attached. The supernatant was removed, and CBD + vitamin E, PAR + vitamin E, CBD + PAR, or PAR + CBD + vitamin E were added. These were then incubated for 12 hours with blank medium as a control. PD-1-overexpressing Jurkat T cells were added at a 1:500 ratio and cultured for 24 hours. The supernatant was removed, and the cells were washed twice with PBS. Then, 4% paraformaldehyde-fixed cells were added. After two further washes with PBS, crystal violet was added and stained for 30 minutes. Finally, excess crystal violet was washed off with PBS, and photographs were taken to collect data.

[0081] (2) Experimental Results As shown in the AD diagrams in Figure 13 and 14, compared with the control group, CBD + Vitamin E, PAR + Vitamin E, CBD + PAR, and PAR + CBD + Vitamin E all enhanced the killing ability of T cells and induced apoptosis in rectal 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 (MCF-7), and melanoma (B16-F10). The PAR + CBD + Vitamin E composition showed stronger tumor-killing ability than CBD + Vitamin E, PAR + Vitamin E, or CBD + PAR alone. These results indicate that the CBD + Vitamin E, PAR + Vitamin E, CBD + PAR, and PAR + CBD + Vitamin E compositions of the present invention can enhance the cytotoxicity of T cells 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). The PAR + CBD + Vitamin E composition enhanced the cytotoxic effect of T cells against tumor cells and had a stronger synergistic effect than the other compositions.

[0082] In summary, the present invention provides an antitumor pharmaceutical composition based on immune checkpoint blockade, comprising cannabidiol and / or paroxetine hydrochloride and a T cell enhancer. A series of in vitro and in vivo experiments were conducted to investigate the effects of the pharmaceutical composition on tumor cell membrane PD-L1 expression and tumor cell killing. The results showed that CBD and paroxetine hydrochloride significantly reduced PD-L1 expression on the surface of cancer cells, thereby blocking the PD-1 / PD-L1 signaling pathway and enhancing T cell-mediated tumor cell killing. Experiments also demonstrated that the pharmaceutical ingredients of the present invention, cannabidiol, paroxetine hydrochloride, and vitamin E plus thymosin, exhibit synergistic effects when used in different combinations, improving antitumor efficacy.

[0083] Although the present invention has been described in detail with reference to the preferred embodiments above, it should be recognized that the above description should not be construed as limiting the present invention. After reviewing the above description, those skilled in the art will recognize that multiple modifications and alternatives to the present invention are obvious. Therefore, the scope of protection of the present invention should be limited by the appended claims.

Claims

1. paroxetine hydrochloride and a T cell enhancer which is at least one of vitamin E and thymosin; The present invention relates to an antitumor pharmaceutical composition based on immune checkpoint blockade, characterized in that

2. The antitumor pharmaceutical composition further comprises cannabidiol. The antitumor pharmaceutical composition based on immune checkpoint blockade according to claim 1.

3. The antitumor pharmaceutical composition comprises cannabidiol, paroxetine hydrochloride, vitamin E and thymosin. The antitumor pharmaceutical composition based on immune checkpoint blockade according to claim 2.

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

5. The pharmaceutical composition further comprises at least one of a pharmaceutically acceptable carrier, excipient, wetting agent, emulsifying agent, pH buffering agent, The antitumor pharmaceutical composition based on immune checkpoint blockade according to claim 1.

6. The dosage form of the pharmaceutical composition includes at least one of oil, granule, tablet, powder, capsule, microcapsule, pill, powder, oral liquid, sol, spray, and atomized form; The antitumor pharmaceutical composition based on immune checkpoint blockade according to claim 1.

7. The antitumor pharmaceutical composition comprises paroxetine hydrochloride and cannabidiol. An antitumor pharmaceutical composition based on immune checkpoint blockade, characterized in that

8. The use of the antitumor pharmaceutical composition based on immune checkpoint blockade according to any one of claims 1 to 7 in the manufacture of antitumor drugs.

9. The tumor includes any one of rectal cancer, lung cancer, liver cancer, gastric cancer, bladder cancer, esophageal cancer, breast cancer, and melanoma; 9. The application according to claim 8.

10. The cannabidiol and paroxetine hydrochloride in the drug combination 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 tumor cell killing effect of T cells.

10. The application according to claim 9.

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