Composition with synergistic effect on ICB therapy and application thereof

Abstract

The invention belongs to the technical field of food and medicine, and particularly discloses a composition with a synergistic effect on ICB therapy and application thereof.The composition is composed of curcumin, panoxadiol and 3-hydrogenated dehydropineoic acid, the curcumin, the panoxadiol and the 3-hydrogenated dehydropineoic acid are compounded and matched, and the composition has a synergistic effect on ICB therapy. The obtained composition has a remarkable synergistic effect on colorectal cancer immunotherapy, the effect of the composition is confirmed through animal model tests, and therefore the composition can be used for preparing products with auxiliary functions on colorectal cancer immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of food and medicine, and relates to the development of food and medicine with adjuvant effects on tumor immunotherapy. Specifically, it relates to a composition with a synergistic effect on ICB therapy and its application in the preparation of products with adjuvant functions for colorectal cancer immunotherapy. Background Art

[0002] Colorectal cancer (CRC) is a common malignant tumor of the digestive system clinically, and its incidence varies greatly in different regions of the world. In the past two decades or more, the incidence of colorectal cancer has shown an upward trend in most countries in the world.

[0003] Immune checkpoint blockade (ICB) therapy, as a new emerging immunotherapy, has achieved certain results in tumor treatment. Its principle is based on the activation mechanism of cytotoxic T lymphocytes (CTLs). At present, ICB therapy has become an important cancer treatment method after surgery, radiotherapy, chemotherapy and targeted therapy. For colorectal cancer, ICB therapy has become one of the important treatment methods currently applied. However, ICB therapy only has a certain effect on a small number of colorectal cancer patients with microsatellite instability-high (MSI-high), and the response speed is generally slow. How to improve the response speed and anti-tumor ability of ICB therapy so as to improve the patient's survival period and survival rate is an important problem that urgently needs to be solved in the current clinical treatment of colorectal cancer. It has been found that tumor microenvironment heterogeneity and its mediated immunosuppression are one of the key factors affecting the effect of colorectal cancer immunotherapy. The tumor microenvironment is composed of tumor cells, endothelial cells, immune cells, fibroblasts, signaling molecules and extracellular matrix. Similar to other solid tumors, colorectal tumors generally have an immunosuppressive microenvironment, with a high level of immunosuppressive cells and a low level of effector immune cells, and most of them are functionally exhausted, which will seriously hinder the response and efficacy of ICB therapy and lead to the occurrence of drug resistance. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a composition with a synergistic effect on ICB therapy. The components of the present invention include three ingredients: curcumin, ginsenoside diol and 3-hydroxydehydrotumulosic acid. This composition can activate anti-tumor immune responses inside colorectal tumors and reverse the immunosuppressive microenvironment, which is beneficial to the response and efficacy of ICB therapy, and achieves a significant synergistic effect on colorectal cancer immunotherapy.

[0005] The present invention is realized through the following technical solutions:

[0006] A composition with a synergistic effect on ICB therapy, which consists of curcumin, panaxadiol, and 3-hydro-dehydro-pinostrolic acid, and the mass ratio of curcumin, panaxadiol, and 3-hydro-dehydro-pinostrolic acid is 1-3:0.5-1.2:0.5-1.2.

[0007] Preferably, the mass ratio of curcumin, panaxadiol, and 3-hydro-dehydro-pinostrolic acid in the composition is 2:1:1.

[0008] In the above composition, after curcumin, panaxadiol, and 3-hydro-dehydro-pinostrolic acid are mixed, they are subsequently embedded with excipients, which can make the prepared product have better water solubility, facilitating intestinal absorption and injection administration.

[0009] A further improvement of the present invention is:

[0010] Use of the above composition in the preparation of a product with an auxiliary function for colorectal cancer immunotherapy.

[0011] The composition of the present invention has strong stability and is easy to process and form, and is suitable for the preparation of drugs, functional foods, and health products

[0012] Furthermore, the product is a drug, a functional food, or a health product.

[0013] Furthermore, the product also includes pharmaceutically, functionally, or nutraceutically acceptable formulation excipients.

[0014] Furthermore, the dosage form of the drug is injection, tablet, powder, capsule, oral liquid, or granule; the dosage form of the functional food includes - tablet, capsule, liquid dosage form, or granule; the dosage form of the health product includes soft capsule, hard capsule, oral liquid, granule, or tablet.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] The present invention combines three components, curcumin, panaxadiol, and 3-hydro-dehydro-pinostrolic acid, and the obtained composition has a significant synergistic effect on colorectal cancer immunotherapy. The present invention has confirmed the efficacy of the composition through animal model experiments. Therefore, the composition of the present invention can be used to prepare a product with an auxiliary function for colorectal cancer immunotherapy. Description of the Drawings

[0017] Figure 1 Results of the comparison of the proportions of different types of immune cells in the tumor tissues of tumor-bearing mice in each group;

[0018] Among them, (a) the proportion of white blood cells (CD45 + ); (b) the proportion of T cells (CD3 +); (c) proportion of helper T cells (CD4 + ); (d) proportion of cytotoxic T cells (CD8 + ); (e) proportion of regulatory T cells (Foxp3 + CD4 + ).

[0019] Figure 2 Effect diagram of the synergistic effect of the composition of the present invention in tumor immunotherapy for oral dosage form;

[0020] Among them, (a) comparison results of tumor masses of the blank control group, experimental group 1, experimental group 2 and experimental group 3 after 24 days of different treatments (Note: comparing the experimental groups with the blank control group respectively, *P < 0.05, **P < 0.01, ***P < 0.001; comparing experimental group 3 with experimental groups 1 and 2 simultaneously, #P < 0.05, ##P < 0.01, P < 0.001); (b) comparison results of tumor inhibition rates (tumor suppression rates) of mice in 3 experimental groups.

[0021] Figure 3 Effect diagram of the synergistic effect of the composition of the present invention in tumor immunotherapy for injection dosage form;

[0022] Among them, (a) comparison results of tumor masses of the blank control group, experimental group 1, experimental group 2 and experimental group 3 after 24 days of different treatments (Note: comparing the experimental groups with the blank control group respectively, ns no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001; comparing experimental group 3 with experimental groups 1 and 2 respectively, #P < 0.05, ##P < 0.01, P < 0.001); (b) comparison results of tumor inhibition rates (tumor suppression rates) of mice in 3 experimental groups. Detailed implementation mode

[0023] The present invention will be introduced in detail below in conjunction with specific embodiments.

[0024] Example 1

[0025] This example provides a composition with a synergistic effect on ICB therapy, specifically: 2 parts of curcumin, 1 part of ginsenoside diol and 1 part of 3-hydro-dehydrotumulosic acid.

[0026] Comparative Example 1

[0027] In this comparative example, 4 parts of curcumin were used, and ginsenoside diol and 3-hydro-dehydrotumulosic acid were not added.

[0028] Comparative Example 2

[0029] In this comparative example, 2 parts of ginsenoside diol and 2 parts of 3-hydro-dehydrotumulosic acid were mixed, and curcumin was not added.

[0030] Verification Example 1

[0031] To verify that the composition described in the present invention has a tumor immune microenvironment regulation effect, the product obtained in Example 1 was functionally verified as follows:

[0032] 1. Experimental animals

[0033] BALB / c mice were selected, SPF grade, male, 19 - 22 g.

[0034] 2. Experimental protocol

[0035] Cell line selection: The CT26 murine colorectal cancer cell line was used and cultured by normal passage.

[0036] In - situ tumor model construction: BALB / c male mice were selected as the model animals. After anesthetizing the mice, the abdomen was surgically incised to expose the cecum. A needle was inserted along the axial position of the cecum body into the cecal mesenteric triangle, and the cancer cell suspension (20 μL per mouse, containing 2×10 5 cells) was slowly injected. After withdrawing the needle, the injection site was gently pressed to ensure no liquid leakage. The peritoneal membrane and abdominal wall muscle layer of the mice were sutured, and finally the skin was sutured. After 20 days of feeding, when a palpable induration appeared in the abdomen of the mice, relevant experiments were carried out.

[0037] Preparation of oral dosage form: For the composition product or single - substance product obtained in the example or comparative example, 2 parts by weight of the product and 5 parts by weight of high - purity lecithin were added to a rotary evaporation flask. Absolute ethanol was added to the flask. The obtained mixture was heated at 60 °C for 20 minutes, and then ethanol was removed by reduced - pressure distillation and vacuum - dried. Finally, deionized water was added to the rotary evaporation flask and hydrated at 60 °C for 20 min, and then ultrasonically dispersed and homogenized at high speed to obtain an oral dosage form containing the composition.

[0038] Drug administration treatment: The tumor - bearing mice were randomly divided into 4 groups (20 mice in each group), including a blank control group, an experimental group, comparison group 1, and comparison group 2. The experimental group, comparison group 1, and comparison group 2 were respectively given the test composition by gavage to the mice in the group at a dose of 0.02 g / kg.bw. The test composition used in experimental group 1 was the product obtained in Example 1; the test compositions used in comparison group 1 and comparison group 2 were the products obtained in Comparative Example 1 and Comparative Example 2, respectively. The drug - administration frequency was 1 time every 2 days for 4 consecutive times. The blank control group was given the same volume of pure water. 12 hours after the 4th drug administration, the tumor tissues of the mice were dissected for immune cell detection.

[0039] Immune cell flow cytometry detection: Isolate tumor tissues, hold scissors to cut the tumor tissues into pieces (operate on ice), add digestive fluid to the fragmented tumor tissues, and digest at 37°C. After terminating the digestion, aspirate the tumor tissue suspension to filter through a cell strainer, and then centrifuge, remove the supernatant and resuspend. Finally, after blocking, antibody incubation, and washing, perform analysis on the machine. The detection items are: white blood cell ratio (CD45 + ), T cell ratio (CD3 + ), cytotoxic T cell ratio (CD8 + ), helper T cell ratio (CD4 + ), and regulatory T cell ratio (Foxp3 + CD4 + ). The detection results are Figure 1 shown as follows.

[0040] Figure 1 Among them, the experimental group and the comparison group were respectively compared with the blank control group, *P < 0.05, **P < 0.01, ***P < 0.001; the experimental group was compared with the comparison group, #P < 0.05, ##P < 0.01, P < 0.001.

[0041] The comparison results showed that the level of white blood cells (CD45 + ) in the tumor tissues of the experimental group mice increased the most, significantly higher than that of the blank control group and the two comparison groups; the level of T cells (CD3 + ) in the tumor tissues of the experimental group mice increased the most, significantly higher than that of the blank control group and the two comparison groups; the level of cytotoxic T cells (CD8 + ) in the tumor tissues of the experimental group mice increased the most, significantly higher than that of the blank control group and the two comparison groups; the level of helper T cells (CD4 + ) in the tumor tissues of the experimental group mice increased the most, significantly higher than that of the blank control group and the two comparison groups; the proportion of regulatory T cells (Foxp3 + CD4 + ) in the tumor tissues of the experimental group mice decreased the least, significantly lower than that of the blank control group and the two comparison groups. The above results indicate that the immune microenvironment of the tumor tissues of the experimental group (administered with the test composition provided in Example 1) mice was improved best.

[0042] Verification Example 2

[0043] In Verification Example 1, the test composition provided in Example 1 (the mass ratio of curcumin, ginsenoside diol, and 3-hydrodehydrotumulosic acid is 2:1:1) showed excellent tumor immune microenvironment regulation effect. To verify that the test composition provided in Example 1 has a synergistic effect on tumor immunotherapy, functional verification was carried out as follows:

[0044] 1. Experimental animals

[0045] BALB / c mice, SPF grade, male, weighing 19 - 22 g, were selected.

[0046] 2. Experimental protocol

[0047] Cell line selection: The CT26 mouse colorectal cancer cell line was used in the study and cultured by normal passage.

[0048] Establishment of orthotopic tumor model of colon cancer: BALB / c male mice were selected as model animals. After anesthetizing the mice, the abdomen was surgically incised to expose the cecum. A needle was inserted along the axial position of the cecum body into the cecal mesenteric triangle, and the cancer cell suspension (20 μL per mouse, containing 2×10 5 cells) was slowly injected. After withdrawing the needle, the injection site was gently pressed to ensure no liquid leakage. The peritoneal and abdominal wall muscle layers of the mice were sutured, and finally the skin was sutured. After 20 days of feeding, when a palpable induration appeared in the abdomen of the mice, relevant experiments were carried out.

[0049] Preparation of oral dosage form: 2 parts by weight of curcumin, 1 part by weight of ginsenoside Rb1, 1 part by weight of 3-hydroxydehydrotumulosic acid, and 10 parts by weight of high-purity lecithin were added to a rotary evaporation flask. Absolute ethanol was added to the flask. The obtained mixture was heated at 60 °C for 20 minutes, and then ethanol was removed by reduced pressure distillation and vacuum dried. Finally, deionized water was added to the rotary evaporation flask and hydrated at 60 °C for 20 min, and then ultrasonically dispersed and homogenized at high speed to obtain an oral dosage form containing the composition.

[0050] Drug administration treatment: The tumor-bearing mice were randomly divided into 4 groups (20 mice in each group), including a blank control group, experimental group 1 (intraperitoneally injected with 0.01 g / kg.bw PD-1 antibody), experimental group 2 (the test composition of Example 1 was given to the mice by gavage at 0.02 g / kg.bw), and experimental group 3 (the test composition of Example 1 was given to the mice by gavage at 0.02 g / kg.bw, and at the same time, 0.01 g / kg.bw PD-1 antibody was intraperitoneally injected). The administration frequency of the test composition of Example 1 was once every 2 days; the administration frequency of the PD-1 antibody was once every 3 days for 24 consecutive days. The blank control group was given the same volume of pure water. After 24 days, the tumors of the mice were dissected and weighed, and the tumor inhibition rate was calculated. The calculation formula was as follows: Tumor growth inhibition rate = (1 - average tumor weight of the treatment group / average tumor weight of the control group) × 100%. The obtained values were compared among groups, and the results are shown in Figure 2 .

[0051] Figure 2a shows that, compared with the blank control group, the tumor weights of the mice in experimental group 1 (intraperitoneal injection of PD-1 antibody) and experimental group 2 (intragastric administration of the test composition of Example 1 to mice) decreased significantly, while experimental group 3 (intragastric administration of the test composition of Example 1 to mice and simultaneous intraperitoneal injection of PD-1 antibody) showed a very significant decrease in tumor mass. Comparison among the experimental groups found that the degree of decrease in tumor weight of the mice in experimental group 3 was significantly higher than that in experimental group 1 and experimental group 2. Figure 2 b shows that the tumor inhibition rate of experimental group 3 is higher than that of experimental group 1 and experimental group 2.

[0052] Verification Example 3

[0053] In the present invention, Verification Example 3 further verifies that the test composition provided in Example 1 has a synergistic effect on tumor immunotherapy. The difference between Verification Example 3 and Verification Example 2 is that the tumor-bearing model used is a subcutaneous tumor model, and the method of administering the test composition and PD-1 antibody to the tumor-bearing mice in the experimental group is intravenous injection. Specifically as follows:

[0054] 1. Experimental animals

[0055] BALB / c mice were selected, SPF grade, male, 19 - 22 g.

[0056] 2. Experimental protocol

[0057] Cell line selection: The CT26 murine colorectal cancer cell line was used in the study and cultured by normal passage.

[0058] Inoculation of tumor: BALB / c male mice were selected as the model animals. The mice were fixed, and the cancer cell suspension (100 μL per mouse, containing 2×10 5 cells) was slowly injected subcutaneously on the left back. After withdrawing the needle, the injection site was gently pressed to ensure no liquid leakage. After 10 days of feeding, when a palpable induration appeared on the left back of the mice, relevant experiments were carried out.

[0059] Preparation of injection dosage form: 2 parts by weight of curcumin, 1 part by weight of panaxadiol, 1 part by weight of 3-hydro-dehydrotumulosic acid and 40 parts by weight of high-purity DSPE-PEG2000 were added to a rotary evaporation flask. Absolute ethanol was added to the flask. The obtained mixture was heated at 60 °C for 20 minutes, and then ethanol was removed by reduced pressure distillation and vacuum dried. Finally, deionized water was added to the rotary evaporation flask and hydrated at 60 °C for 20 min, and then ultrasonic dispersion was carried out to obtain an injection dosage form containing the composition.

[0060] Drug administration treatment: The tumor-bearing mice were randomly divided into 4 groups (20 mice in each group), including a blank control group, experimental group 1 (intravenously injected with 0.01 g / kg.bw PD-1 antibody), experimental group 2 (the test composition of Example 1 was intravenously injected into mice at a dose of 0.02 g / kg.bw), and experimental group 3 (the test composition of Example 1 was intravenously injected into mice at a dose of 0.02 g / kg.bw, and at the same time, 0.01 g / kg.bw PD-1 antibody was intravenously injected). The administration frequency of the test composition of Example 1 was once every 2 days; the administration frequency of PD-1 antibody was once every 3 days for 24 consecutive days. The blank control group was given the same volume of normal saline. After 24 days, the tumors of the mice were dissected and weighed, and the tumor inhibition rate was calculated. The calculation formula was as follows: Tumor growth inhibition rate = (1 - average tumor weight of the treatment group / average tumor weight of the control group) × 100%. The obtained values were compared among groups, and the results are shown in Figure 3 .

[0061] Figure 3 a shows that compared with the blank control group, the tumor weights of the mice in experimental group 1 (intravenously injected with PD-1 antibody) and experimental group 2 (the test composition of Example 1 was intravenously injected into mice) did not decrease significantly, while the mice in experimental group 3 (the test composition of Example 1 was intravenously injected into mice, and at the same time, PD-1 antibody was intravenously injected) showed a significant decrease in tumor mass. Comparison among experimental groups found that the degree of decrease in tumor weight of the mice in experimental group 3 was significantly higher than that in experimental groups 1 and 2. Figure 3 b shows that the tumor inhibition rate of experimental group 3 was higher than that of experimental groups 1 and 2.

[0062] The above embodiments are only for illustrating the technical concept and characteristics of the present invention, and the purpose is to enable those who are familiar with this technology to understand the content of the present invention and implement it accordingly. It should not be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A composition having a synergistic effect on ICB therapy, characterized in that, The composition consists of curcumin, panaxadiol, and 3-hydro-dehydrotumulosic acid, and the mass ratio of curcumin, panaxadiol, and 3-hydro-dehydrotumulosic acid is 1-3:0.5-1.2:0.5-1.

2.

2. The composition having a synergistic effect on ICB therapy according to claim 1, wherein: The mass ratio of curcumin, panaxadiol, and 3-hydro-dehydrotumulosic acid is 2:1:

1.

3. Use of the composition according to claim 1 or 2 in the preparation of a product having an auxiliary function for immunotherapy of colorectal cancer.

4. The application according to claim 3, characterized in that, The product is a drug, a functional food, or a health product.

5. The application according to claim 3 or 4, characterized in that: The product further includes pharmaceutically, functionally, or nutritionally acceptable formulation excipients.

6. The application according to claim 4, characterized in that: The dosage form of the drug is injection, tablet, powder, capsule, oral liquid, or granule; the dosage form of the functional food includes tablet, capsule, liquid dosage form, or granule; the dosage form of the health product includes soft capsule, hard capsule, oral liquid, granule, or tablet.

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