Antitumor drug 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] This invention relates to the pharmaceutical field, and more specifically to an antitumor drug composition based on immune checkpoint blockade and its applications. [Background technology]

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

[0003] Currently, in tumor immunotherapy, related therapies targeting the immune checkpoint PD-1 / PD-L1 are attracting attention. Programmed cell death protein 1 (PD-1) is an important immunosuppressive molecule that is widely expressed on activated T cells and is the surface receptor for activated T cells. The receptor for PD-1, PD-L1 (programmed death ligand 1), is hardly detectable in normal tissues, but is abundant in various human tumor tissues. When PD-1 binds to PD-L1, it sends an immunosuppressive signal to T cells, suppressing T cell activation, preventing the killing of tumor cells, and causing tumor immune evasion. By blocking the interaction between PD-1 and PD-L1, the functional effects of tumor-specific T cells can be restored. PD-1 / PD-L1 related immune checkpoint suppression therapy is an ideal approach to tumor immunotherapy, and various monoclonal antibodies against PD-1 / PD-L1 are currently on the market. Studies have shown that while the efficacy of anti-PD-1 / PD-L1 antibodies is positively correlated with PD-L1 positivity in solid tumors that respond relatively well to anti-PD-1 / PD-L1 antibodies, such as melanoma, renal cancer, and non-small cell lung cancer, antibody therapy has many side effects, and currently only a small number of patients benefit from it. [Overview of the project] [Problems that the invention aims to solve]

[0004] This invention addresses the problems of existing anti-PD-1 / PD-L1 antibody immunotherapy, which suffers from significant side effects and limited patient benefits, by providing a novel antitumor drug composition and its applications based on immune checkpoint blocking, 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 by T cells. [Means for solving the problem]

[0005] To solve the above technical problems, the first object of the present invention is to provide an antitumor drug 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 drug composition further comprises cannabidiol.

[0007] Preferably, the antitumor drug composition comprises 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 pharmaceutically acceptable carriers, excipients, wetting agents, emulsifiers, and pH buffers.

[0010] Preferably, the dosage form of the pharmaceutical composition includes at least one of the following: oil, granules, tablets, powder, capsule, microcapsule, pill, powder, oral solution, sol, spray, and atomizer.

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

[0012] Another object of the present invention is to provide the application of the previously described immune checkpoint blockade-based antitumor drug composition in the manufacture of antitumor drugs.

[0013] Preferably, the tumor includes one of the following: rectal cancer, lung cancer, liver cancer, stomach cancer, bladder cancer, esophageal cancer, breast cancer, or melanoma.

[0014] Preferably, cannabidiol and paroxetine hydrochloride in the drug combination can suppress 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] Compared to conventional technologies, the beneficial effects of the present invention include at least the following: (1) Through a series of in vitro cell experiments, the present invention has discovered and demonstrated for the first time that cannabidiol and paroxetine hydrochloride can effectively reduce PD-L1 expression 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 anti-tumor drug according to the present invention is a combined drug combination based on immune checkpoint inhibition and T cell enhancement. Here, cannabidiol and paroxetine hydrochloride can effectively inhibit the expression of PD-L1 on the surface of tumor cells and can significantly reduce the inhibitory effect on T cell activation. In addition, the T cell enhancer added based on this can further increase the quantity and activity of T cells, and can significantly enhance the killing ability of T cells after immune suppression is lifted. The two aspects act together to significantly enhance the anti-tumor effect of the drug. More importantly, CBD and PAR in the drug of the present invention can directly reduce PD-L1 on the surface of tumor cells compared with anti-PD-1 / PD-L1 antibody immunotherapy, have higher targeting, are less likely to cause damage to normal tissues by T cells, and can avoid the occurrence of side effects.

[0017] (3) Experimentally, it has been proven that cannabidiol, paroxetine hydrochloride and vitamin E + thymosin, which are components of the pharmaceutical composition according to the present invention, exert a synergistic effect and improve the anti-tumor effect.

Brief Description of the Drawings

[0018] [Figure 1] Figure 1 is a comparison of cell proliferation after treatment with different concentrations of CBD on different types of tumor cells. [Figure 2] Figure 2 is a comparison of the expression of PD-L1 in cells after treatment with different concentrations of CBD on different types of tumor cells. Here, 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 is a comparison of the expression of PD-L1 on the cell membrane after treatment with different concentrations of CBD on different types of tumor cells. Here, 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 a comparison of PD-L1 expression on the cell membrane at different time intervals after treating RKO cells with the same concentration of CBD. [Figure 5] Figure 5 shows a comparison of cell viability after co-treatment of RKO cells with CBD and NK92 cells. [Figure 6] Figure 6 compares the antitumor effects of different pharmaceutical compositions on mice with rectal cancer, 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 body 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 compares the antitumor effects of different pharmaceutical compositions on rectal cancer immunodeficiency mice, where A is a tumor histogram of each group of mice, B is the growth curve of tumor volume for each group of mice, C is the growth curve of tumor weight for each group of mice, and D is the body weight growth curve for each group of mice. [Figure 8] Figure 8 compares the cell viability of three different types of tumor cells after co-treatment with NK92 cells using three different pharmaceutical compositions, 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 compares the cell viability of NK92 cells after co-treatment with three different pharmaceutical compositions for different types of tumor cells, where E is the viability comparison for T24 cells, F is the viability comparison for TE-5 cells, G is the viability comparison for MCF-7 cells, and H is the viability comparison for B16-F10 cells. Here, the control group is the group that did not receive any drug. [Figure 10] Figure 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 compares the antitumor effects of CBD, curcumin + piperine, CBD + curcumin + piperine, CBD + curcumin + piperine + vitamin E, or CBD + curcumin + piperine + vitamin E + thymosin in mice with rectal cancer. [Figure 12] Figure 12 compares the antitumor effects of different pharmaceutical compositions on mice with rectal cancer, where A is a photograph of the tumor tissue of each group of mice, B is the body 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 compares the cell viability of different types of tumor cells after co-treatment with two different pharmaceutical compositions and Jurka T cells overexpressing PD-1, where A is a comparison of the viability of CBD and PAR against RKO cells, B is a comparison of the viability of CBD and PAR against H1975 cells, C is a comparison of the viability of CBD and PAR against HepG2 cells, and D is a comparison of the viability of CBD and PAR against MGC803 cells. [Figure 14] Figure 14 compares the cell viability of different types of tumor cells after co-treatment with two different pharmaceutical compositions and Jurka T cells overexpressing PD-1, where A is a comparison of the viability of CBD and PAR against T24 cells, B is a comparison of the viability of CBD and PAR against TE-5 cells, C is a comparison of the viability of CBD and PAR against MCF-7 cells, and D is a comparison of the viability of CBD and PAR against B16-F10 cells. [Modes for carrying out the invention]

[0019] Conventional anti-PD-1 / PD-L1 antibody immunotherapy, as explained in the background technology section, suffers from significant side effects and limited patient benefits. PD-L1 expression in tumor cells may influence the clinical efficacy of PD-1 / PD-L1-related immune checkpoint therapy. Small molecules that target and regulate PD-L1 expression are expected to represent a new therapeutic approach, and the search for small molecules that negatively regulate PD-L1 expression could offer a novel approach in the immunotherapy process.

[0020] Therefore, the present invention seeks to find small molecules that negatively regulate PD-L1 expression in order to block the PD-1 / PD-L1 pathway in tumors, enhance T cell activity, and kill tumor cells. After numerous experimental screenings and validations, the present invention ultimately proposes a novel antitumor drug composition based on immune checkpoint blockade, comprising cannabidiol and / or paroxetine hydrochloride and a T cell enhancer. Simultaneously, the present invention proposes another novel antitumor drug composition based on immune checkpoint blockade, comprising paroxetine hydrochloride and cannabidiol.

[0021] Cannabidiol (CBD) is a cannabis extract that is currently being widely developed in fields such as medical skincare products and mood-enhancing beverages and foods. In recent years, an increasing number of studies have shown that CBD has antitumor activity in different types of tumors, but the antitumor mechanism is not yet fully understood, and there are significant differences in activity across different tumor types. However, research in this invention has for the first time discovered that CBD effectively reduces PD-L1 expression in tumors, raising expectations for its application in tumor immunotherapy.

[0022] Paroxetine hydrochloride (PAR) is an antidepressant. This invention, based on experimental results, discovered 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 the use of CBD or PAR in combination with a T-cell enhancer. The T-cell enhancer includes one or more of the following actions: (1) promoting T-cell proliferation and differentiation, increasing the proportion or absolute number of T-cells; and (2) enhancing T-cell activity, improving the immune function of T-cells. The concept of such a 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, and the tumor cell-killing effect of T-cells after immunosuppression is lifted can be significantly enhanced. To understand this, 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, and the effect of the T-cell enhancer will be greatly reduced. Therefore, the present invention aims to improve the antitumor activity of a drug by having a pharmaceutical composition exhibit a synergistic effect when CBD or PAR is used in combination with a T-cell enhancer.

[0024] In some embodiments, the T-cell enhancer comprises at least one of vitamin E and thymosin. Vitamin E is an important antioxidant, but also an effective immunomodulator, promoting the development of immune organs and the differentiation of immune cells, and enhancing the function of cellular and humoral immunity. Thymosin is a thymic tissue extract derived from calves, pigs, or sheep, and is a soluble polypeptide that can enhance T-cell immune function and is used in the treatment of congenital or acquired T-cell immunodeficiency disorders, autoimmune diseases, and tumors.

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

[0026] In some embodiments, the antitumor drug composition comprises cannabidiol, paroxetine hydrochloride, vitamin E, and thymosin. Here, the ratio 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, excipients, wetting agents, emulsifiers, and pH buffers.

[0028] Appropriate pharmaceutically acceptable carriers are well known to those skilled in the art. A comprehensive 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, equilibrium salt solution, glycerin, and sorbitol. These carriers may also contain auxiliary substances such as lubricants, fluidizers, wetting agents or emulsifiers, pH buffers, and stabilizers such as albumin. At the time of use, a safe and effective dose of the antitumor drug described in the present invention is administered to a mammal (e.g., human). Of course, the specific dose should take into account factors such as the route of administration and the patient's health condition, all of which are within the scope of the skills of a skilled physician. The exact effective dose for a particular subject depends on the subject's body type and health condition, the nature and severity of the disease, and the selection of the therapeutic agent and / or combination of therapeutic agents to be administered. For a given situation, the effective dose can be determined by routine experimentation, and a clinician can make that determination.

[0029] In some embodiments, the dosage form of the pharmaceutical composition includes oils, granules, tablets, powders, capsules, pills, powders, oral solutions, sols, sprays, atomizers, and the like.

[0030] Another aspect of the present invention further provides applications of the previously described immune checkpoint blockade-based antitumor drug composition in the manufacture of antitumor drugs.

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

[0032] In the following, by linking the attached drawings and examples, the experimental process and results of the present invention will be described in detail, explaining in detail how the antitumor drug composition of the present invention can suppress 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 by T cells.

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

[0034] Unless otherwise specified, the materials and reagents used in the following examples can all be obtained through commercial channels. In Examples 1 to 4, cannabidiol was purchased from Yuxi Hongbao Biotechnology Co., Ltd. Paroxetine hydrochloride was purchased from MCE, product number BRL29060A. Thymosin enteric-coated tablets were purchased from Heilongjiang Dilong Pharmaceutical Co., Ltd. (National Pharmaceutical Standard Code H20058365). Vitamin E was purchased from Dingrui Chemical (Shanghai) Co., Ltd. (product number A04GS156945). Anti-PD-1 was purchased from Bioxcell (Ultra-LEAF). TM Purified anti-mouse CD279 (PD-1, BE0146) was purchased. Corn oil was purchased from Jin Taiyang Oil Co., Ltd. Curcumin was purchased from MCE, product number HY-N0005. Capsaicin was purchased from MCE, product number HY-10448. Artemisinin was purchased from MCE, product number HY-B0094. In Examples 5 and 6, cannabidiol was purchased from Yuxi Hongbao Biotechnology Co., Ltd. Paroxetine hydrochloride active pharmaceutical ingredient was purchased from Zhejiang Huahai Pharmaceutical Co., Ltd. Vitamin E active pharmaceutical ingredient was purchased from Zhejiang Xinhecheng Co., Ltd. Anti-PD-1 was purchased from Bioxcell (Ultra-LEAF). TMPurified anti-mouse CD279 (PD-1, BE0146) was purchased. Corn oil was purchased from Kin Taiyo Oil Co., Ltd.

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

[0036] Example 1: Effects of CBD on tumor cells in in vitro culture (1) Experimental process 1. Proliferative effect of CBD on tumor cells in in vitro culture. Different types of tumor cells in the logarithmic growth phase (rectal cancer cells RKO, lung cancer cells H1975, and lung cancer cells A549) were inoculated into 96-well plates, with an inoculation density of 5 × 10⁶ per well. 3 Individual cells were then incubated for 24 hours in a 37°C, 5% CO2 incubator. CBD mother liquor (10 mM) was diluted to 5, 10, and 20 μM with culture medium, respectively. Old medium was aspirated from a 96-well plate, and 100 μL of medium containing different concentrations of CBD was added to each well to create a control well (0 μM CBD), and incubation was continued for 24 hours. After the culture period, the number of proliferating tumor cells was detected using an EDU kit (Biyuntian, C0085S). Finally, images were taken using a high-content imaging analysis system, and the data were statistically analyzed.

[0037] 2. Effects 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 and density of 80% or more were laid in 6-well plates, resulting in a density of 5 × 10⁶ 4 Cells were cultured in a well at a concentration of 2 mL per well, and then incubated in a 37°C incubator. After 12 hours of incubation, when the cells had fully adhered to the cell wall, different concentrations of CBD (0, 10, 20, 30, 40 μM) were administered. After continuing the culture for 24 hours, the culture was terminated and subsequent tests were performed. After culturing was complete, the cells were collected, washed twice with cold PBS to remove any PBS residue, lysed with an appropriate amount of cell lysate, incubated on ice for 15 minutes, then centrifuged at 12,000 rpm for 15 minutes in a 4°C centrifuge, the supernatant was carefully aspirated, the protein concentration was measured by BCA, then a corresponding amount of loading buffer was added and shaken evenly, incubated in a 100°C water bath for 10 minutes, and finally a Western blot experiment was performed on the sample.

[0038] 3. Effects of CBD on PD-L1 expression on tumor cell membranes (1) Different types of tumor cells in the logarithmic growth phase (colon cancer cells RKO, HCT116, HT29 and lung cancer cells A549, H1975, H460) were inoculated into 6-well plates, with an inoculation density of 2.5 × 10⁶ per well. 5 The cells were divided into cells / mL, with 2mL per well, and then incubated overnight in a cell incubator. The culture medium was aspirated, treated with different concentrations (0, 5, 10, 20 μM) of CBD for 24 hours, the supernatant was removed, washed twice with PBS, and the cells were digested with pancreatin. The cells were then collected by centrifugation at 300g and 4°C for 5 minutes. The cells were washed twice with pre-cooled PBS and centrifuged at 300g and 4°C for 5 minutes. The cells were washed twice with pre-cooled PBS, resuspended in 100 μL of PBS, and incubated at room temperature for 30 minutes with Anti-PD-L1-Alexa Fluor 647 antibody. The cells were then centrifuged at 300g and 4°C for 5 minutes, resuspended in 300 μL of PBS by flow cytometry, and the Alexa Fluor 647 signal (excitation / emission wavelengths 647 nm / 666 nm) of the sample was detected by flow cytometry.

[0039] (2) Logarithmic growth phase rectal cancer cells (RKO) were inoculated into a 6-well plate, with a density of 2.5 × 10⁻⁶. 5The cells were divided into 100 cells / well, with a suspension volume of 2 mL per well. The cells were placed in a cell incubator and incubated overnight. The culture medium was aspirated, 20 μM CBD was added and treated for 12 hours, the supernatant was removed, the cells were washed twice with PBS, then fixed with paraformaldehyde for 15 minutes, followed by fixation with 5% BSA for 1 hour, washed twice with PBST, PD-L1 antibody was added and incubated overnight, red fluorescent antibody was added and incubated at room temperature for 1 hour, then stained with DAPI for 10 minutes, and finally photographic analysis was performed using a laser confocal microscope.

[0040] 4. A study showed that CBD enhanced T-cell killing in tumor cells in vitro. Logarithmic growth phase RKO cells were inoculated into a 12-well plate, and the density was 5 × 10⁶. 4 Cells / mL were attached to the wall of a 5% CO2 incubator and grown overnight. The supernatant was removed, and different concentrations of CBD (0, 5, 10, 20 μM) were added to each cell, followed by co-incubation for 12 hours. NK-92 cells (T cells) were added at a 1:500 ratio and cultured for 24 hours, while a control group without NK-92 cells was simultaneously established. The supernatant was removed, washed twice with PBS, and then 4% paraformaldehyde-fixed cells were added. After washing twice more with PBS, crystal violet staining was added for 30 minutes, and finally, excess crystal violet was washed off with PBS. Photographs were taken to collect data.

[0041] (2) Experimental results 1. CBD showed no apparent toxicity to tumor cells in vitro. As shown in Figure 1, there was no significant difference in the fluorescence intensity of tumor cells after treating the same type of tumor cells with different concentrations of CBD. This indicates that CBD alone does not have any apparent toxicity to tumor cells when added to tumor cells cultured in vitro. Further research is needed to investigate the antitumor mechanism of CBD.

[0042] 2. CBD reduced the expression of PD-L1 in tumor cells. As shown in the A-C diagram in Figure 2, as a result of the Western blot experiment, CBD significantly inhibited the expression of PD-L1 in rectal cancer cells (RKO) and lung cancer cells (H1975 and A549), and showed concentration dependence at 5 μM, 10 μM, and 20 μM.

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

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

[0045] The results, as shown in Figure 5, the CBD+T cell co-culture treatment significantly decreased the survival rate of RKO cells compared to the control group and induced apoptosis of RKO cells. As a result, it was shown that by decreasing the expression of PD-L1 in RKO cells, CBD enhanced the cytotoxicity of T cells against RKO cells in a concentration-dependent manner at 5 μM, 10 μM, and 20 μM.

[0046] Example 2 In vivo antitumor effects of different pharmaceutical compositions of CBD and paroxetine hydrochloride (I) Experimental procedure 1. MC38 rectal cancer cells were inoculated into 6-week-old female C57BL / 6 mice (1×10 6 cells / mouse) to construct a subcutaneous tumor model. When the tumor volume reached 50 mm 3After reaching a certain threshold, mice were grouped and administered the following drug combinations: (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) (12) PAR (10 mg / kg) + Vitamin E (50 mg / kg) + Thymosine (2 mg / kg) group, (13) CBD (100 mg / kg) + Vitamin E (50 mg / kg) + Thymosine (2 mg / kg) group, (14) PAR (10 mg / kg) + Vitamin E (50 mg / kg) + Thymosine (2 mg / kg) + anti-CTLA4 (100 μg / dose) group, (15) CBD (100 mg / kg) + PAR (10 mg / kg) + Vitamin E (50 mg / kg) + Thymosine (2 mg / kg) + anti-CTLA4 (100 μg / dose) group. Mouse tumor weight, volume, and body weight were monitored every two days. The formula for calculating tumor volume was: tumor volume (mm²) 3 )=0.5×(length)×(width) 2 The mice were executed 14 days after administration, and the tumors were dissected.

[0047] In this study, the pharmaceutical composition was dissolved by injecting 100 μL / mice of 10% DMSO + corn oil into the stomach, while the control group of mice was injected with the same volume of solvent.

[0048] 1. Colon cancer cells MC38 were introduced into 6-week-old female nude mice (1 × 10⁻¹⁶). 6 A subcutaneous tumor model was constructed by inoculating individual cells (or animals). The tumor volume was 50 mm². 3After reaching a certain stage, 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, (4) PAR (10 mg / kg) + CBD (100 mg / kg) + vitamin E (50 mg / kg) + thymosin (2 mg / kg) group. Mouse tumor weight, volume, and body weight were monitored every two days. The formula for calculating tumor volume was: tumor volume (mm²) 3 )=0.5×(length)×(width) 2 The mice were executed 14 days after administration, 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 Figure 6 and Table 1, the experimental results from comparison groups (1), (2), (4), and (6) showed that the therapeutic effects of CBD and PAR were equivalent, and that both were equivalent to the therapeutic effects of existing general anti-PD-1 antibodies. Furthermore, the experimental results from comparison groups (1), (4), (6), and (8) showed that the combination of CBD and PAR resulted in a higher tumor suppression effect and a synergistic effect compared to the use of CBD or PAR alone.

[0050] When T-cell enhancers (vitamin E + thymosin) were added to the treatment, the experimental results from the comparison groups (1), (4), (9), and (10) showed that while there was no clear therapeutic effect when vitamin E + thymosin was used alone, the combination of CBD and vitamin E + thymosin resulted in a higher therapeutic effect and a synergistic effect compared to when CBD or vitamin E + thymosin were used alone.

[0051] Similarly, experimental results from the comparison groups (1), (6), (9), and (11) showed that the combination of PAR and vitamin E + thymosin resulted in a higher therapeutic effect and a synergistic effect compared to the use of PAR or vitamin E + thymosin alone.

[0052] From the experimental results of the comparison groups (1), (8), (9), and (12), it was found that the combination of CBD + PAR + vitamin E + thymosin had a stronger tumor growth inhibitory effect and a synergistic effect compared to the use of CBD + PAR or vitamin E + thymosin alone.

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

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

[0055] [Table 1]

[0056] 2. The three compositions of CBD and paroxetine hydrochloride did not have antitumor effects in T cell-deficient mice. Based on the above research results, we injected nude mice with three compositions exhibiting excellent antitumor activity and explored their effects on tumors in immunodeficient mice.

[0057] As shown in Figure 7, the results showed no significant difference in tumor volume among the four groups of mice. In other words, none of the three combination drugs (CBD + vitamin E + thymosin, PAR + vitamin E + thymosin, PAR + CBD + vitamin E + thymosin) had any therapeutic effect on the tumors in nude mice.

[0058] These results indicate that the three compositions cannot exert a clear antitumor effect when T cells are not present in the body.

[0059] 3. The three compositions of CBD and paroxetine hydrochloride exhibited superior antitumor effects when used in combination with other immune checkpoint suppressors. The present invention also attempts to obtain a pharmaceutical composition with even better antitumor activity by adding other immune checkpoint inhibitors to three compositions that exhibit excellent antitumor activity. In this example, an anti-CTLA4 antibody was used.

[0060] As shown in AD in Figure 6, the experimental results from the comparison groups (10) to (15) showed that when anti-CTLA4 antibody was added to the three compositions, the drug compositions exhibited a synergistic effect, resulting in a more pronounced antitumor effect. In particular, the CBD + PAR + vitamin E + thymosin + anti-CTLA4 composition showed the optimal antitumor effect compared to the other compositions.

[0061] Example 3 Three compositions enhanced in vitro T-cell killing of different types of tumor cells. (1) Experimental process Based on the results of Example 2, three compositions exhibiting excellent antitumor activity were added to different types of tumor cells along with T cells for further study. The specific experimental procedure was as follows: Logarithmically growing 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 sampled in 5 × 10⁻¹⁰ 4 Cells were inoculated into 12-well plates at a density of individual cells / mL and grown overnight in a 37°C, 5% CO2 incubator with the cells attached to the wall. The supernatant was removed, and media containing either PAR + vitamin E + thymosin, CBD + vitamin E + thymosin, or PAR + CBD + vitamin E + thymosin were added, while a blank medium was used as a control group. Both media were incubated together for 12 hours. NK-92 cells were added at a 1:500 ratio and cultured for 24 hours. The supernatant was removed, washed twice with PBS, and then 4% paraformaldehyde-fixed cells were added. After washing twice more with PBS, crystal violet staining was added for 30 minutes, and finally, excess crystal violet was washed off with PBS. Photographs were taken to collect data.

[0062] (2) Experimental results As shown in Figure 8 (AD) and Figure 9 (EH), compared to the control group, co-culture treatment with the three pharmaceutical compositions and T cells significantly reduced the survival rates 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 also induced apoptosis in tumor cells. These results demonstrate that all three pharmaceutical compositions according to the present invention can enhance the cytotoxicity of T cells against tumor cells by reducing PD-L1 expression in tumor cells.

[0063] Example 4: In vivo antitumor effects of CBD and artemisinin composition, and CBD and curcumin composition (1) Experimental process In this invention, we also attempted to obtain different pharmaceutical compositions that exhibit synergistic effects by adding other substances that may have the effect of regulating the number or activity of T cells, or substances that have antitumor effects. In this example, artemisinin and curcumin + 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 procedure was as follows: Colon cancer cells MC38 were introduced into 6-week-old female C57BL / 6 mice (1 × 10⁻¹⁴). 6 A subcutaneous tumor model was constructed by inoculating individual cells (or animals). The tumor volume was 50 mm². 3 After reaching a certain stage, mice in the artemisinin-administered 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. Mice in the curcumin-administered group were grouped and administered the following drug combinations. (1) Control group, (2) CBD (100 mg / kg) group, (3) Curcumin (50 mg / kg) + Piperine (1 mg / kg) group, (4) CBD (100 mg / kg) + Curcumin (50 mg / kg) + Piperine combination (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 in mice was monitored every two days. The formula for calculating tumor volume was: tumor volume (mm²) 3 )=0.5×(length)×(width) 2 The mice were executed 14 days after administration, and the tumors were dissected. The dissolution method for the pharmaceutical composition was to inject 100 μL / mice of 10% DMSO + corn oil into the stomach, and the same volume of solvent was injected into the control group of mice.

[0066] (2) Experimental results As shown in Figure 10, compared to the control group, tumor growth was suppressed in mice in the CBD + vitamin E + thymosine group and the CBD + vitamin E + thymosine + artemisinin group. However, there was no significant difference in the antitumor effects of the two groups, indicating that the addition of artemisinin did not further enhance the antitumor effect of the CBD + vitamin E + thymosine composition.

[0067] As shown in Figure 11, tumor growth in mice in the CBD group was suppressed to some extent compared to the control group, but tumor growth in mice in the curcumin + piperine group was not significantly suppressed. Furthermore, the antitumor effects of several pharmaceutical compositions, including 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 clear advantage over the effect of CBD alone, indicating that not all combinations synergistically enhance the antitumor effect.

[0068] In this invention, the following experiments were conducted to further verify the therapeutic effect of a pharmaceutical composition using only vitamin E as a T-cell enhancing agent.

[0069] Example 5: In vivo antitumor activity of different pharmaceutical compositions of CBD and paroxetine hydrochloride (1) Experimental process 1. Rectal cancer cells MC38 were introduced into 6-week-old female C57BL / 6 mice (1 × 10⁻¹⁴). 6 A subcutaneous tumor model was constructed by inoculating individual cells (or animals). The tumor volume was 50 mm². 3 After reaching a certain stage, mice were grouped and administered the following drug combinations: (1) Control group (no administration), (2) anti-PD-1 (100 μg / dose) group, (3) Vitamin E (50 mg / kg) group, (4) CBD (100 mg / kg) group, (5) CBD (100 mg / kg) + Vitamin E (50 mg / kg) group, (6) PAR (5 mg / kg) group, (7) PAR (5 mg / kg) + Vitamin E (50 mg / kg) group, (8) CBD (100 mg / kg) + PAR (5 mg / kg) group, (9) PAR (5 mg / kg) + CBD (100 mg / kg) + Vitamin E (50 mg / kg) group. Mouse tumor weight, volume, and body weight were monitored every two days. The formula for calculating tumor volume was: tumor volume (mm²) 3 )=0.5×(length)×(width) 2 The mice were executed 14 days after administration, and the tumors were dissected.

[0070] In this study, the pharmaceutical composition was dissolved by injecting 100 μL / mice of 10% DMSO + corn oil into the stomach, while the control group of mice 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 Figure 12 and Table 2, the experimental results from comparison groups (1), (2), (4), and (6) showed that the therapeutic effects of CBD and PAR were equivalent, and both were equivalent to the therapeutic effects of existing general anti-PD-1 antibodies. Furthermore, the experimental results from comparison groups (4), (4), and (8) showed that the combination of CBD and PAR resulted in a higher tumor suppression effect and a synergistic effect compared to using CBD or PAR alone.

[0072] When vitamin E was added during treatment, the experimental results from the comparison groups (1), (3), (4), and (5) showed that while vitamin E alone did not have a clear therapeutic effect, the combination of CBD and vitamin E had a higher therapeutic effect and a synergistic effect compared to the use of CBD or vitamin E individually.

[0073] Similarly, the experimental results from the comparison groups (1), (3), (6), and (7) showed that the combined use of PAR and vitamin E resulted in a higher therapeutic effect and a synergistic effect compared to the use of PAR or vitamin E alone.

[0074] From the experimental results of the comparison groups (1), (3), (8), and (9), it was found that the combination of CBD + PAR + vitamin E had a stronger tumor growth inhibitory effect and a synergistic effect compared to the use of vitamin E and CBD + PAR alone.

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

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

[0077] [Table 2]

[0078] Example 6: The PAR+CBD+Vitamin E composition enhanced T-cell killing in vitro in different types of tumor cells.

[0079] (1) Experimental process We conducted research by adding PAR + CBD + vitamin E, which have good antitumor effects, to T cells along with different types of tumor cells. The specific experimental procedure was as follows:

[0080] Logarithmically growing 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 sampled in 5 × 10⁻¹⁰ 4 Cells were inoculated into 12-well plates at a density of cells / mL and grown overnight in a 37°C, 5% CO2 incubator, attached to the wall. The supernatant was removed, and CBD + vitamin E, PAR + vitamin E, CBD + PAR, and PAR + CBD + vitamin E were added to each, while a blank medium was used as a control group. Both groups were incubated together for 12 hours. Jurkat cells (T cells) overexpressing PD-1 were added at a 1:500 ratio and cultured for 24 hours. The supernatant was removed, washed twice with PBS, and then 4% paraformaldehyde-fixed cells were added. After washing twice more with PBS, crystal violet staining was added for 30 minutes, and finally, excess crystal violet was washed off with PBS. Photographs were taken to collect data.

[0081] (2) Experimental results As shown in the AD diagrams in Figure 13 and Figure 14, compared to the control group, CBD + vitamin E, PAR + vitamin E, CBD + PAR, and PAR + CBD + vitamin E all enhanced the T-cell killing ability against 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 were able to induce apoptosis in tumor cells. The PAR + CBD + vitamin E compositions showed stronger tumor-killing ability than when CBD + vitamin E, PAR + vitamin E, or CBD + PAR were used alone. These results indicate that the CBD+vitamin E, PAR+vitamin E, CBD+PAR, and PAR+CBD+vitamin E compositions according to the present invention can all enhance the cytotoxicity of T cells against tumor cells by reducing PD-L1 expression in tumor cells (rectal cancer cells, lung cancer cells, liver cancer cells, gastric cancer cells, bladder cancer cells, esophageal cancer cells, breast cancer cells, and melanoma cells). The PAR+CBD+vitamin E composition was shown to enhance the cytotoxic effect of T cells against tumor cells and exhibit a stronger synergistic effect compared to 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. Through a series of in vitro experiments, the effects of the pharmaceutical composition on PD-L1 expression on tumor cell membranes and tumor cell killing activity were studied. As a result, it was discovered 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 the tumor cell killing activity by T cells. Furthermore, experiments demonstrated that the pharmaceutical components according to the present invention, cannabidiol, paroxetine hydrochloride, and vitamin E + thymosin, exhibit synergistic effects when used in different combinations, improving the antitumor effect.

[0083] Although the contents of the present invention have been described in detail by the preferred embodiments above, it should be recognized that the above description should not be considered as limiting the present invention. Those skilled in the art will find several modifications and substitutions of the present invention obvious after reviewing the above description. Therefore, the scope of protection of the present invention should be limited by the appended claims.

Claims

1. It contains paroxetine hydrochloride and T-cell enhancers such as vitamin E and thymosin. The aforementioned vitamin E is α-tocopherol. An antitumor drug composition characterized by immune checkpoint blockade.

2. The aforementioned antitumor drug composition further comprises cannabidiol. The antitumor drug composition based on immune checkpoint blockade as described in claim 1.

3. The aforementioned antitumor drug composition consists of cannabidiol, paroxetine hydrochloride, vitamin E, and thymosin. The antitumor drug composition based on immune checkpoint blockade as described in feature 2.

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

5. The pharmaceutical composition further comprises at least one of pharmaceutically acceptable carriers, excipients, wetting agents, emulsifiers, and pH buffers. The antitumor drug composition based on immune checkpoint blockade as described in claim 1.

6. The dosage form of the pharmaceutical composition includes at least one of the following: oil, granules, tablets, powder, capsule, microcapsule, pill, powder, oral solution, sol, spray, and atomizer. The antitumor drug composition based on immune checkpoint blockade as described in claim 1.

7. comprising paroxetine hydrochloride, cannabidiol, and vitamin E, The aforementioned vitamin E is α-tocopherol. An antitumor drug composition characterized by immune checkpoint blockade.

8. Use of an antitumor pharmaceutical composition based on immune checkpoint blocking according to any one of claims 1 to 7 for the manufacture of an antitumor drug.

9. The aforementioned tumor includes one of the following: rectal cancer, lung cancer, liver cancer, stomach cancer, bladder cancer, esophageal cancer, breast cancer, or melanoma. The use described in feature 8.

10. In the aforementioned drug combination, cannabidiol and paroxetine hydrochloride can suppress 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. The use described in feature 9.

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