A pharmaceutical composition and its application

Abstract

The present invention provides a pharmaceutical composition and its application, belonging to the technical field of anti-tumor drugs. The pharmaceutical composition includes intestinal probiotics and αPD-1 inhibitors; the intestinal probiotics are obtained by inducing changes in the intestinal microecology through mushroom polysaccharides. The present invention adopts mushroom polysaccharides to enhance the anti-tumor efficacy of αPD-1 by regulating the intestinal microbial ecological functional groups. After mushroom polysaccharides change the intestinal flora environment, intestinal probiotics can be obtained by enrichment, which can reduce Foxp3 + CD4 + Infiltration of regulatory T cells (Treg) into tumors enhances CD8 + The effector function of T cells is stimulated, thereby producing a synergistic effect with αPD-1 therapy; compared with monotherapy, the combined treatment of intestinal probiotic substances obtained by inducing changes in the intestinal microecology through mushroom polysaccharides and αPD-1 can significantly delay tumor growth.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor drugs, and in particular relates to a drug composition and application thereof. Background Art

[0002] Cancer is a type of malignant tumor, which refers to a disease caused by the uncontrolled proliferation and spread of abnormal cells. These abnormal cells are called cancer cells, which can invade surrounding tissues and spread to other parts of the body through the blood or lymphatic system. Cancer can occur in various organs and tissues of the human body, such as breast, lung, colon, liver, stomach, uterus, prostate, etc. The development of cancer can go through different stages, from primary tumor to local invasion and then to distant metastasis.

[0003] The causes of cancer are complex and varied, and may be related to genetic factors, environmental factors (such as smoking, exposure to carcinogens), poor diet, chronic infection, etc. Early detection and treatment of cancer are very important, because early diagnosis can provide more effective treatment options and improve cure and survival rates.

[0004] Common cancer treatments include surgical removal of tumors, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. At the same time, cancer prevention is also very important, including maintaining a healthy lifestyle, avoiding exposure to carcinogens, vaccination, and regular physical examinations to reduce the risk of cancer. Among the treatment methods, drug-based targeted therapy has become the preferred treatment for cancer due to its good efficacy, few side effects to the body, and rapid results.

[0005] αPD-1 belongs to a class of immunomodulators. PD-1 is a receptor in the immune system that regulates the function and activity of immune cells.

[0006] The anti-tumor mechanism of αPD-1 drugs mainly works through two aspects: relieving immunosuppression and activating immune responses. (1) Relieving immunosuppression: Cancer cells can use the immune checkpoint signaling pathway, especially the signal of the PD-1 receptor, to inhibit the activity of immune cells. αPD-1 drugs can bind to the PD-1 receptor and block its binding to ligands (such as PD-L1), thereby preventing the transmission of immunosuppressive signals. This enables activated immune cells to attack cancer cells more effectively and enhance the immune system's ability to respond to tumors. (2) Activating immune responses: The use of αPD-1 drugs can stimulate and enhance the function of tumor-associated immune cells. Under normal circumstances, immune cells control the amplitude of immune responses and prevent excessive inflammatory responses through the PD-1 signaling pathway. However, cancer cells deceive immune cells by expressing ligands such as PD-L1, causing them to lose their ability to attack tumors. The use of αPD-1 drugs can restore the ability of immune cells to recognize and attack tumors and enhance immune responses. By relieving immunosuppression and activating immune responses, αPD-1 drugs help restore or enhance the immune system's control over cancer, thereby inhibiting tumor growth and spread.

[0007] By using αPD-1 drugs, the signaling of PD-1 receptors can be blocked, breaking the cancer cells' escape mechanism from immune response, thereby activating the immune system's attack on cancer cells. This treatment method is called immune checkpoint inhibitor therapy, which has achieved remarkable results in the treatment of malignant tumors.

[0008] However, αPD-1 drugs are not suitable for all types of cancer and may cause some side effects. In addition, the single administration of αPD-1 inhibitors has limited initiation of T cell-mediated anti-tumor immune responses, and its efficacy is not ideal. Summary of the invention

[0009] In order to solve the above problems, the present invention provides a pharmaceutical composition comprising:

[0010] Intestinal probiotic substances and αPD1 inhibitors;

[0011] Among them, the intestinal probiotic flora substance is the intestinal probiotic flora substance obtained by inducing changes in the intestinal microecology through mushroom polysaccharides.

[0012] Preferably, the intestinal probiotic flora substances include intestinal bacteria;

[0013] Alternatively, the intestinal probiotic flora substances include the intestinal bacteria and metabolites of the intestinal bacteria.

[0014] Preferably, the intestinal flora includes any one or more of the following bacteria:

[0015] OTU483, OTU601, OTU435, OTU829, OTU419, OTU638, OTU440, OTU1041, OTU424, OTU471, OTU481, OTU506, OTU603, OTU546 and OTU582.

[0016] Preferably, the metabolite is selected from any one or more of the following metabolites:

[0017] L-tryptophan, L-tyrosine, 2-hydroxycinnamic acid, phenylacetylglutamine, 5-methoxyindole acetate, 3-hydroxycinnamic acid, p-coumaric acid, 10-hydroxydecanoic acid, phenylbutyric acid, 2-anthraquinonesulfonic acid, 3-oxalocholic acid, tetradecanedioic acid, cholic acid, deoxycholic acid, cytosine, cytidine, azacitidine, 1-naphthylamine, daidzein, dehydroglycol, fribasstat, nicotinamide maleate, 3-hydroxybenzaldehyde, 4-hydroxybenzaldehyde, PC(18:1(9Z) / P- 18:1(9Z)), PS(22:0 / 15:0), racemic-1,8-epoxy-p-menthane-2,6-diol, 2,4(1H,3H)-pyrimidinedione-5-fluoro-1-(tetrahydrofuran-2-yl)-, 1-(4-O-beta-D-glucopyranyl-3-methoxyphenyl)-3,5-dihydroxydecane, 3,5,7,8-tetrahydro-2-(4-trifluoromethylphenyl)-4H-thiofuro[4,3-d]pyrimidin-4-one.

[0018] Preferably, the mushroom polysaccharide includes one or more of Tremella fuciformis polysaccharide, Tremella fuciformis polysaccharide, Lycium barbarum polysaccharide, Ganoderma lucidum polysaccharide, Cordyceps sinensis polysaccharide, Hericium erinaceus polysaccharide, Agrocybe edulis polysaccharide, Lentinan polysaccharide, Boletus edulis polysaccharide, Agaricus edodes polysaccharide and Lactarius polysaccharide.

[0019] Preferably, the components of the Lactarius ruber polysaccharide include glucose, mannose, galactose and fucose.

[0020] Preferably, the pharmaceutical composition is administered by:

[0021] The intestinal probiotic flora substance is obtained by inducing the intestinal microecological changes in the body after oral administration of mushroom polysaccharides, and the αPD1 inhibitor is administered by injection;

[0022] Alternatively, the intestinal probiotic substance is administered orally, and the αPD1 inhibitor is administered by injection.

[0023] Preferably, the mushroom polysaccharide is prepared by the following method:

[0024] The mushroom fruiting bodies are taken and the mushroom polysaccharides are extracted by water extraction and alcohol precipitation method.

[0025] Preferably, the method of extracting mushroom fruiting bodies and obtaining mushroom polysaccharides by water extraction and alcohol precipitation comprises:

[0026] Taking the mushroom fruiting body, and obtaining a polysaccharide extract after defatting and water extraction;

[0027] After the polysaccharide extract is diluted, ethanol is added to perform alcohol precipitation treatment to obtain the mushroom polysaccharide.

[0028] Preferably, the step of obtaining the mushroom fruiting bodies, defatting and extracting with water to obtain a polysaccharide extract further includes freeze-drying and pulverizing the mushroom fruiting bodies.

[0029] In addition, to solve the above problems, the present invention also provides a use of the above-mentioned pharmaceutical composition in the preparation of an anti-tumor product, wherein the anti-tumor product includes a drug, a detection reagent and a kit.

[0030] The present invention provides a pharmaceutical composition and its application, wherein the pharmaceutical composition comprises an intestinal probiotic substance and an αPD1 inhibitor; the intestinal probiotic substance is an intestinal probiotic substance obtained by inducing and changing the intestinal microecology through mushroom polysaccharide. The present invention adopts mushroom polysaccharide to enhance the anti-tumor efficacy of αPD-1 by regulating the intestinal microbial ecological functional group. After mushroom polysaccharide changes the intestinal flora environment, intestinal probiotic substances can be obtained by enrichment, which can reduce Foxp3 + CD4 + Infiltration of regulatory T cells (Treg) into tumors enhances CD8 + The effector function of T cells is enhanced, thereby producing a synergistic effect with αPD-1 therapy; animal experiments have shown that the intestinal probiotic substances enriched by mushroom polysaccharides regulating the intestinal microecology are the key to combined synergy. Compared with any single therapy, the intestinal probiotic substances obtained by inducing changes in the intestinal microecology by mushroom polysaccharides and αPD-1 combined treatment can significantly delay tumor growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a comparison chart of the weight changes of mice in each group in Example 1 of the present invention;

[0032] Figure 2 This is a comparison chart of the changes in tumor volume of mice in each group in Example 1 of the present invention;

[0033] Figure 3 This is a comparison of the appearance of tumor tissues of mice in each group in Example 1 of the present invention;

[0034] Figure 4 This is a comparison chart of the tumor masses of mice in each group in Example 1 of the present invention;

[0035] Figure 5This is a comparison of the changes in lymphocyte T cells in tumor tissues of mice in each group in Example 1 of the present invention;

[0036] Figure 6 This is a comparison chart of the weight changes of mice in each group in Example 2 of the present invention;

[0037] Figure 7 This is a comparison chart of the changes in tumor volume of mice in each group in Example 2 of the present invention;

[0038] Figure 8 This is a comparison of the appearance of tumor tissues of mice in each group in Example 2 of the present invention;

[0039] Fig. 9 This is a comparison chart of the tumor masses of mice in each group in Example 2 of the present invention;

[0040] Fig.10 This is an alpha diversity analysis diagram in Example 3 of the present invention;

[0041] Fig.11 This is a β diversity analysis diagram in Example 3 of the present invention;

[0042] Fig.12 This is a heat map of the relative abundance of intestinal microorganisms at different classification levels in Example 3 of the present invention;

[0043] Fig.13 A bar graph showing relative abundance of different species in Example 3 of the present invention;

[0044] Fig.14 This is a heat map of the correlation analysis between intestinal flora and host indicators in Example 3 of the present invention;

[0045] Fig.15 This is the volcano plot of differential metabolites in Example 3 of the present invention;

[0046] Fig.16 This is a heat map of the correlation analysis between serum metabolites and tumor volume and tumor immunity in Example 3 of the present invention.

[0047] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0048] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] The present invention provides a pharmaceutical composition comprising:

[0050] Intestinal probiotic substances and αPD1 inhibitors;

[0051] Among them, the intestinal probiotic flora substance is the intestinal probiotic flora substance obtained by inducing changes in the intestinal microecology through mushroom polysaccharides.

[0052] As mentioned above, mushroom polysaccharides refer to polysaccharide substances extracted from fungi (such as ganoderma lucidum, shiitake mushrooms, winter mushrooms, etc.). Mushroom polysaccharides are mainly composed of a variety of simple sugar molecules, such as glucose, xylose, mannose, etc.

[0053] Mushroom polysaccharides have the following characteristics and functions:

[0054] (1) Immune regulation: Mushroom polysaccharides can enhance the function of the immune system, promote the activity of immune cells such as macrophages and NK cells, and enhance the body's disease resistance.

[0055] (2) Anti-tumor effect: Some studies have shown that mushroom polysaccharides can inhibit the growth and spread of tumor cells and promote apoptosis of tumor cells. In addition, mushroom polysaccharides can enhance the efficacy of chemotherapy and radiotherapy and reduce their toxic side effects on normal cells.

[0056] (3) Antioxidant: Mushroom polysaccharides have strong antioxidant activity, which can scavenge free radicals, reduce the damage of oxidative stress to the body, and help improve the body's antioxidant capacity.

[0057] (4) Anti-inflammatory activity: Mushroom polysaccharides can inhibit the production of inflammatory factors, regulate immune responses, and alleviate inflammatory responses, thus having a certain inhibitory effect on inflammatory diseases.

[0058] (5) Regulating blood sugar and blood lipids: Some mushroom polysaccharides have the function of regulating blood sugar and blood lipids, which helps prevent and treat metabolic diseases such as diabetes and hyperlipidemia.

[0059] Mushroom polysaccharides can regulate the gastrointestinal flora environment. The gastrointestinal flora is a community of large microorganisms in the human body, which has an important impact on health. Mushroom polysaccharides can induce adjustments to the flora environment after entering the gastrointestinal tract, thereby obtaining intestinal probiotic flora substances.

[0060] The present invention adopts mushroom polysaccharide to enhance the anti-tumor effect of αPD-1 by regulating the ecological functional group of intestinal microorganisms. After mushroom polysaccharide changes the intestinal flora environment, intestinal probiotic flora substances can be obtained by enrichment, which can reduce Foxp3 + CD4 + Infiltration of regulatory T cells (Treg) into tumors enhances CD8 +The effector function of T cells is enhanced, thereby producing a synergistic effect with αPD-1 therapy; animal experiments have shown that the intestinal probiotic substances enriched by mushroom polysaccharides regulating the intestinal microecology are the key to combined synergy. Compared with any single therapy, the intestinal probiotic substances obtained by inducing changes in the intestinal microecology by mushroom polysaccharides and αPD-1 combined treatment can significantly delay tumor growth.

[0061] Furthermore, the intestinal probiotic bacteria substance includes:

[0062] (1) Intestinal bacteria;

[0063] (2) Intestinal bacteria and their metabolites.

[0064] Furthermore, the intestinal flora includes any one or more of the following bacteria:

[0065] OTU483, OTU601, OTU435, OTU829, OTU419, OTU638, OTU440, OTU1041, OTU424, OTU471, OTU481, OTU506, OTU603, OTU546 and OTU582.

[0066] As mentioned above, the intestinal flora includes any one or a combination of Enterorhabdus (OTU483), Bacteroides_uniformis (OTU601), Alistipes (OTU435), Eubacterium (OTU829), Muribaculaceae (OTU419), Muribaculaceae (OTU638), Muribaculaceae (OTU440), Robinsoniella_peoriensis (OTU1041), Lachnospiraceae_UCG-006 (OTU424), Roseburia (OTU471), Lachnospiraceae (OTU481), Lachnospiraceae (OTU506), Lachnospiraceae_NK4A136_group (OTU603), Ruminococcaceae (OTU546), and Ruminococcaceae (OTU582).

[0067] Furthermore, the metabolite is selected from any one or more of the following metabolites:

[0068] L-Tryptophan(L-tryptophan), L-Tyrosine(L-tyrosine), 2-Hydroxycinnamicacid(2-hydroxycinnamic acid), Phenylacetylglutamine(phenylacetylglutamine), 5-Methoxyindoleacetate(5-methoxyindoleacetate), M-Coumaric acid(3-hydroxycinnamic acid), P-Coumaric acid(p-coumaric acid), 10-Hydroxydecanoic Acid(10-hydroxydecanoic acid), Benzenebutanoic acid(phenylbutyric acid), 2-Anthraquinonesulfonic acid(2-anthraquinonesulfonic acid), 3-Oxocholic acid(3-oxocholic acid), Tetradecanedioic acid(tetradecanedioic acid), Cholic Acid(cholic acid), Deoxycholic acid(deoxycholic acid), Cytosine(cytosine), Cytidine(cytidine), Azacitidine(azacitidine), 1-Naphthylamine(1-naphthylamine), Daidzein(daidzein), Anhydroglycinol(anhydroglycinol), Firibastat(fluribast), Nicousamide Pyrotinib Maleate(Nicotinamide Maleate), 3-Hydroxybenzaldehyde(3-Hydroxybenzaldehyde), 4-Hydroxybenzaldehyde(4-Hydroxybenzaldehyde), PC(18:1(9Z) / P-18:1(9Z)), PS(22:0 / 15:0), Exo,exo-1,8-Epoxy-p-menthane-2,6-diol, 2,4(1H,3H)-Pyrimidinedione,5-fluoro-1-(tetrahydro-2-furanyl)-,(R)-,1-(4-O-beta-D-glucopyranosyl-3-methoxyphenyl)-3,5-dihydroxydecane, 3,5,7,8-Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one.

[0069] Furthermore, the mushroom polysaccharide includes one or more of Tremella fuciformis polysaccharide, Tremella fuciformis polysaccharide, Lycium barbarum polysaccharide, Ganoderma lucidum polysaccharide, Cordyceps sinensis polysaccharide, Hericium erinaceus polysaccharide, Agrocybe edulis polysaccharide, Lentinus edodes polysaccharide, Boletus edulis polysaccharide, Agaricus edodes polysaccharide and Lactarius polysaccharide.

[0070] In a preferred embodiment, the mushroom polysaccharide is Lactarius ruber polysaccharide.

[0071] It should be noted that red milk mushroom (scientific name: Lactifluus volemus) is an edible fungus, also known as agaric, big milk mushroom, small milk mushroom, etc. It belongs to the genus Milk Mushroom under the Boletaceae family and is a truffle-type fungus.

[0072] Lactarius volemus polysaccharide (LHP) refers to a polysaccharide extracted from Lactifluus volemus.

[0073] Furthermore, the components of the red juice lactarius polysaccharide include glucose, mannose, galactose and fucose.

[0074] Furthermore, the pharmaceutical composition is administered in the following manner:

[0075] Method (1): administering mushroom polysaccharides orally to induce changes in the intestinal microecology in the body to obtain the intestinal probiotic flora substance, and administering the αPD1 inhibitor by injection;

[0076] In the above method, after oral administration of mushroom polysaccharides, the autologous intestinal flora is changed, and intestinal probiotic substances are directly generated, which play a synergistic role with the injected αPD1 inhibitor.

[0077] Therefore, the substances administered in method (1) are mushroom polysaccharides and αPD1 inhibitors, wherein the intestinal probiotics are produced by the body after taking mushroom polysaccharides.

[0078] Method (2): administering the intestinal probiotic substance orally and administering the αPD1 inhibitor by injection.

[0079] In the above method, the prepared intestinal probiotic substances are directly orally ingested, which works synergistically with the injected αPD1 inhibitor.

[0080] Therefore, the substances administered in method (2) are intestinal probiotic substances and αPD1 inhibitors, wherein the intestinal probiotic substances are finished products that have been prepared and are produced by mushroom polysaccharides after administration under other conditions.

[0081] Furthermore, the mushroom polysaccharide is prepared by the following method:

[0082] The mushroom fruiting bodies are taken and the mushroom polysaccharides are extracted by water extraction and alcohol precipitation method.

[0083] As mentioned above, water extraction and alcohol precipitation is a method for extracting mushroom polysaccharides, mainly based on the following reasons:

[0084] (1) Efficient extraction: Water is a relatively mild solvent that can effectively extract mushroom polysaccharides. Mushroom polysaccharides have good solubility in water, and the polysaccharides can be fully extracted by controlling the appropriate temperature and time.

[0085] (2) Protecting active ingredients: The water extraction and alcohol precipitation method does not involve high temperature treatment and the use of organic solvents. It is relatively mild and is conducive to protecting the active ingredients and biological activity of mushroom polysaccharides.

[0086] (3) High purity of polysaccharides: Polysaccharides can be precipitated from aqueous solutions by alcohol precipitation (usually using ethanol or isopropanol), removing most impurities and non-polysaccharide components such as proteins. This can improve the purity of polysaccharides and facilitate subsequent separation, purification and further research.

[0087] (4) Easy operation: The water extraction and alcohol precipitation method is relatively simple to operate and has relatively low equipment requirements, making it suitable for small-scale laboratories or industrial production.

[0088] Furthermore, the method of extracting mushroom fruiting bodies and obtaining mushroom polysaccharides by water extraction and alcohol precipitation includes:

[0089] Taking the mushroom fruiting body, and obtaining a polysaccharide extract after defatting and water extraction;

[0090] After the polysaccharide extract is diluted, ethanol is added to perform alcohol precipitation treatment to obtain the mushroom polysaccharide.

[0091] Furthermore, the step of obtaining the mushroom fruiting bodies, defatting and extracting with water to obtain a polysaccharide extract further includes freeze-drying and pulverizing the mushroom fruiting bodies.

[0092] In the method of extracting mushroom polysaccharides, the fruiting bodies are freeze-dried, crushed and defatted in advance for the following reasons:

[0093] (1) Protecting the activity of polysaccharides: Freeze drying is a commonly used sample processing method that can preserve plant tissues or fungi intact and maintain the integrity of active ingredients such as polysaccharides. Freeze drying can prevent polysaccharides from being decomposed or losing their activity under high temperature and humidity conditions.

[0094] (2) Improving dissolution efficiency: Grinding the freeze-dried fruiting bodies helps to increase their surface area, making it easier for the extraction solvent to contact the sample, thereby improving the dissolution efficiency of polysaccharides. Grinding can also destroy the cell wall structure and release more polysaccharides.

[0095] (3) Removal of non-polysaccharide components: The purpose of defatting is to remove non-polysaccharide components such as fat and oil in the fungus to avoid interference of these substances in the polysaccharide extraction and further separation and purification process. Non-polysaccharide components may reduce the purity of polysaccharides and affect the results of subsequent experiments and applications.

[0096] In summary, pretreatment methods such as freeze-drying, crushing and defatting can protect the activity of mushroom polysaccharides, improve the extraction efficiency, and remove non-polysaccharide components that are not related to the polysaccharide separation and purification process. These steps are of great significance for the subsequent polysaccharide separation, purification and research.

[0097] In addition, the present invention also provides a use of the above-mentioned pharmaceutical composition in the preparation of an anti-tumor product, wherein the anti-tumor product includes a drug, a detection reagent and a kit.

[0098] In the above applications, the tumors targeted by the anti-tumor products can be one or more of respiratory system tumors, digestive system tumors, reproductive and urinary system tumors, lymphatic system tumors, and skin tumors.

[0099] In a preferred embodiment, the tumor targeted by the anti-tumor product is lung cancer among respiratory system tumors.

[0100] The present invention is further described below by means of specific examples, but it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0101] Embodiment 1:

[0102] This example investigates the anti-tumor effect of Lactarius edulis polysaccharide (LHP) combined with αPD1 inhibitors through tumor-bearing mouse experiments.

[0103] Experimental methods:

[0104] (1) Take Lewis tumor cell lines in logarithmic growth phase and the concentration is 1×10 7 / mL, 0.2mL / mouse was inoculated into the right axilla of male C57 mice;

[0105] (2) When the mouse tumor volume reaches 20-35mm 3 The mice were divided into 4 groups (n=8) according to tumor volume and body weight:

[0106] ① Model group: After grouping, the mice were gavaged with 500 mg / kg sterile water every day;

[0107] ② Polysaccharide intervention group (LHP): after grouping, 500 mg / kg LHP was intragastrically administered daily;

[0108] ③ Monoclonal antibody intervention (αPD-1) group: after grouping, 500 mg / kg of sterile water was administered intragastrically every day;

[0109] ④ The combined intervention group (LHP+αPD-1) was intragastrically administered with 500 mg / kg LHP daily after grouping;

[0110] (3) When the tumor volume is about 50 mm 3 The ③αPD-1 group and ④LHP+αPD-1 group were intraperitoneally injected with αPD1 at a dose of 250μg / mouse, while ①Model and ②LHP were given an equal amount of prepared solvent for intraperitoneal injection, and then the injection was given once every three days for a total of 5 times.

[0111] After grouping, the weight changes of mice were monitored and recorded. Figure 1 The comparison chart of weight changes of mice in each group shows that there is no significant statistical difference in weight among the four groups of mice.

[0112] After grouping, the tumor volume of each group of mice was measured and recorded. Figure 2 The comparison chart of tumor volume changes in each group of mice shows that the tumor volume of the combined intervention group was significantly reduced compared with that of the model control group (P<0.001); in addition, the tumor volume of the combined intervention group was significantly reduced compared with that of the single intervention group (P<0.05).

[0113] The mice were sacrificed, and the tumor tissue was removed and photographed. Figure 3 Comparison photos of tumor tissue appearance of mice in each group are shown; tumor mass was measured and obtained Figure 4 The comparison chart of tumor mass of mice in each group is shown; the results showed that the overall tumor mass of mice in all groups was significantly smaller than that in the model control group (P < 0.05), and the tumor volume of mice in the LHP combined with αPD1 inhibitor group was the smallest, which was statistically different from other groups (P < 0.05).

[0114] Flow cytometry analysis of CD4 in tumor tissue + 、CD8 + and FoxP3 + CD4 + For immune cell changes, single cell suspensions were stained with the following antibodies: APC anti-mouse CD3, FITC anti-mouse CD4, and PE / Cy7 anti-mouse CD8 (Biolegend, clone 17A2, clone RM4-5, and clone 53-6.7, respectively, USA). Flow cytometric analysis was performed using a FACSArial III flow cytometer (BD Biosciences, USA). Figure 5The comparison of the changes in lymphocyte T cells in tumor tissues of mice in each group is shown in the figure. Compared with the model group, the LHP group and the combined intervention group can significantly increase the CD8 + CD3 + / CD4 + CD3 + In addition, compared with the model group and the αPD1 intervention group, the combined treatment group could significantly inhibit the expression of Tregs cells in tumor tissue (FoxP3 + CD4 + T)(P<0.05).

[0115] LHP intervention alone and combined with αPD-1 intervention can significantly promote the immune effector T cells (CD8 + and CD4 + ), inhibiting immunosuppressive Tregs cells (FoxP3 + CD4 + T) infiltrated tumor tissue (P < 0.05), while αPD1 intervention alone did not show such effect.

[0116] The experimental results of this example show that LHP combined with αPD-1 can significantly promote immune effector T cells (CD8 + and CD4 + ), inhibiting immunosuppressive Tregs cells (FoxP3 + CD4 + T) infiltrates tumor tissue and initiates T cell-mediated anti-tumor immune response, thus showing that the combined intervention has a better effect on inhibiting lung cancer growth than αPD-1 inhibitor or LHP intervention alone.

[0117] Embodiment 2:

[0118] This example investigates the anti-tumor effect of the combination of LHP-modified intestinal flora and αPD-1 inhibitors through a flora transplantation experiment.

[0119] Experimental methods:

[0120] (1) Take Lewis tumor cell lines in logarithmic growth phase and the concentration is 1×10 7 / mL, 0.2mL / mouse was inoculated into the right axilla of male C57 mice;

[0121] (2) When the mouse tumor volume reaches 20-35mm 3 The mice were divided into 5 groups (n=8) according to tumor volume and body weight:

[0122] ① Model group: After grouping, the mice were gavaged with 500 mg / kg sterile water every day;

[0123] ②Intestinal flora donor group: After grouping, 500 mg / kg of LHP was gavaged daily;

[0124] ③ Monoclonal antibody intervention (αPD-1) group: after grouping, 500 mg / kg of sterile water was administered intragastrically every day;

[0125] ④Factory transplantation group (FMT-LHP);

[0126] ⑤Intestinal flora combined with αPD-1 treatment group (FMT-LHP+αPD-1).

[0127] (3) When the tumor volume is about 50 mm 3 The ③αPD-1 group and ⑤FMT-LHP+αPD-1 group were intraperitoneally injected with αPD 1 at a dose of 250μg / mouse, while the ①Model group and ②intestinal flora donor group were intraperitoneally injected with an equal amount of prepared solvent, and then injected once every three days for a total of 5 times.

[0128] For the ④ microbiota transplantation group (FMT-LHP) and the ⑤ intestinal microbiota combined with αPD-1 treatment group (FMT-LHP+αPD-1), they were divided into groups and received sterile drinking water containing ATB for 3 days, and then the fresh feces of the ② intestinal microbiota donor group were collected to prepare fecal microbiota liquid, and 200 μL was gavaged daily to the recipient mice in groups ④ and ⑤. Microbiota preparation and transplantation method: fresh feces of mice in group ② were collected (gently massage the abdomen of the mice and immediately collected using a sterile tube), the feces were weighed and soaked in PBS buffer with a volume of 10 times the weight of the feces, vortexed on a vortex instrument for about 1 minute until all fecal particles were disintegrated, centrifuged at 800g for 3 minutes, and the supernatant was taken to obtain fresh fecal microbiota liquid. The bacterial suspension collection and gavage were completed within 10 minutes.

[0129] Experimental methods:

[0130] After grouping, the weight changes of mice were monitored and recorded. Figure 6 The comparison chart of weight changes of mice in each group shows that there is no significant statistical difference in weight among the four groups of mice.

[0131] After grouping, the tumor volume of each group of mice was measured and recorded. Figure 7 The comparison of tumor volume changes in each group of mice is shown. The tumor volume of the microbiota combined with αPD-1 intervention group was significantly reduced compared with the model control group (P<0.001); in addition, the tumor volume of the combined intervention group was significantly reduced compared with the αPD-1 intervention group alone (P<0.05).

[0132] The mice were sacrificed, and the tumor tissue was removed and photographed. Figure 8 Comparison photos of tumor tissue appearance of mice in each group are shown; tumor mass was measured and obtained Fig. 9The comparison chart of tumor mass of mice in each group is shown; the results showed that the overall tumor mass of mice in all intervention groups was significantly smaller than that in the model control group (P < 0.05), and the tumor volume of mice in the microbiota combined with αPD1 inhibitor group was the smallest, and there was a statistical difference compared with the αPD1 intervention group alone (P < 0.05).

[0133] The experimental results of this example show that intestinal flora is the decisive factor in LHP enhancing the efficacy of αPD-1. Identifying the intestinal bacteria that play a role can broaden the development of anti-tumor active substances and provide more options for improving αPD-1 immunotherapy.

[0134] Embodiment 3:

[0135] In this example, 16S rDNA sequencing analysis was performed on the cecal contents of each group of mice collected in Example 1, and non-targeted metabolomics analysis was performed on the serum; candidate intestinal bacteria and intestinal flora metabolites that may alleviate the tumor burden of mice and promote the efficacy of αPD-1 were determined through multivariate statistics and whole microbiome association analysis.

[0136] The cecal contents of each group of mice were collected and sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for sequencing using the Miseq PE300 platform of Illumina, and the data were analyzed using the Meiji Cloud platform. Fig.10 Although all intervention groups showed similar richness and diversity of intestinal microbiota in terms of operational taxonomic units (OTUs) and Shannon diversity index, Fig.11 The analysis of the differences in the intestinal flora structure of each group showed that LHP and the combination of α-PD-1 caused obvious clustering of the microbial community structure. The PCoA analysis based on Bray-Curtis distance and the permutational multifactorial analysis of variance (permutational MANOVA) were used to test the significance of the differences in the flora structure between different groups.

[0137] To explore which groups were changed by LHP and α-PD-1, the Wilcox rank sum test was used to analyze the species (OTU) with different abundances in the microbial communities between the two groups. At the OUT classification level, 52 species in the LHP group changed significantly compared with the Model group, and 86 species in the LHP combined with αPD-1 group changed significantly compared with the α-PD-1 group. Many different groups reflected the strong effect of LHP on the intestinal flora of tumor-bearing mice. We focused on those groups that were significantly increased by both LHP and LHP combined with αPD-1, such as Fig.12As shown in the heat map of relative abundance of intestinal microorganisms at different classification levels, the relative abundance of 19 OUTs in the LHP combined with αPD-1 group was significantly increased compared with the α-PD-1 group, of which 7 OUTs were continuously enriched by LHP (OTUs that were significantly increased in LHP compared with Model, LHP combined with αPD-1 compared with αPD-1, and LHP combined with αPD-1 compared with Model), such as Fig.13 The relative abundance of differentially expressed species is shown in the bar graph, which shows OTU582 from the Ruminococcaceae family, OTU419 from the Muribaculaceae family, OTU424 from the Lachnospiraceae_UCG-006 genus, OTU1041 from the Robinsoniella genus, OTU435 from the Alistipes genus, OTU601 from the Bacteroides genus, and OTU829 from the Eubacterium genus. OTU603 of the genus Oup, OTU440 of the family Muribaculaceae, OTU483 of the genus Enterorhabdus, OTU481 and OTU506 of the family Lachnospiraceae, OTU471 of the genus Roseburia, OTU638 of the family Muribaculaceae, and OTU546 of the family Ruminococcaceae. These eight OTUs were the species that increased significantly in the combination of LHP and αPD-1 compared with αPD-1 and in the combination of LHP and αPD-1 compared with Model.

[0138] In the Firmicutes phylum, most members of the Lachnospiraceae and Ruminococcaceae families have considerable ability to hydrolyze starch and other dietary polysaccharides to produce butyrate and other short-chain fatty acids, but there is great variation between species and strains. It has been reported that most members of the Lachnoclostridium and Ruminococcaceae families can affect the occurrence and development of tumors by regulating the intestinal immune system, promoting CD8 + T cell recruitment and activation, thereby enhancing anti-tumor immunity, high abundance of Lachnoclostridium spp. is associated with intratumoral CD8 + T cell infiltration, intratumoral bacteria and patient survival are related; in addition, it has been reported that members of the genus Alistipes have been shown to play a beneficial role in cancer immunotherapy by regulating the tumor microenvironment, and members of the genus Eubacterium can ferment dietary fiber to produce short-chain fatty acids, which are related to anti-colon cancer.

[0139] The correlation analysis of the 19 continuously enriched intestinal bacteria with tumor volume and tumor immune cells was obtained. Fig.14 Heat map of correlation analysis between intestinal flora and host indicators. Most species are negatively correlated with tumor volume and tumor-infiltrating regulatory T cells. + / CD4 + T is positively correlated.

[0140] These consistently enriched species can serve as candidate intestinal bacteria for alleviating tumor burden in mice and promoting the efficacy of αPD-1.

[0141] The serum of mice in each group was collected and sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for identification of serum metabolites using AB SCIEX's ultra-high performance liquid chromatography tandem time-of-flight mass spectrometry UPLC-TripleTOF system, and the Meiji cloud platform was used for data analysis. The selection of significantly different metabolites was based on the variable weight value (VIP) obtained by the OPLS-DA model and the student's t-test p value. Metabolites with VIP>1 and p<0.05 were significantly different metabolites. Fig.15The volcano plot of differential metabolites showed that a total of 90 differential metabolites were screened in the combined treatment group compared with the αPD-1 single treatment group, of which 30 were significantly upregulated and 60 were significantly downregulated. The metabolites significantly upregulated in the combined treatment group were L-Tryptophan, L-Tyrosine, 2-Hydroxycinnamic acid, Phenylacetylglutamine, 5-Methoxyindoleacetate, M-Coumaric acid, P-Coumaric acid, 10-Hydroxydecanoic Acid, Benzenebutanoic acid, 2-Anthraquinonesulfonic acid, 3-Oxocholic acid, Tetradecanedioic acid, Cholic Acid, and 10-Hydroxydecanoic Acid. Acid, Deoxycholic acid, Cytosine, Cytidine, Azacitidine, 1-Naphthylamine, Daidzein, Anhydroglycinol, Firibastat, Nicousamide Pyrotinib Maleate(Nicotinamide Maleate), 3-Hydroxybenzaldehyde(3-Hydroxybenzaldehyde), 4-Hydroxybenzaldehyde(4-Hydroxybenzaldehyde), PC(18:1(9Z) / P-18:1(9Z)), PS(22:0 / 15:0), Exo,exo-1,8-Epoxy-p-menthane-2,6-diol, 2,4(1H,3H)-Pyrimidinedione,5-fluoro-1-(tetrahydro-2-furanyl)-,(R)-,1-(4-O-beta-D-glucopyranosyl-3-methoxyphenyl)-3,5-dihydroxydecane, 3,5,7,8-Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one.

[0142] The correlation analysis of 30 significantly upregulated serum metabolites with tumor volume and tumor immune cells was obtained. Fig.16 Heat map of correlation analysis between serum metabolites and tumor volume and tumor immunity shows that most metabolites are negatively correlated with tumor volume and tumor-infiltrating regulatory T cells and positively correlated with CD8+ / CD4+ T cells. These significantly enriched metabolites can be used as candidate compounds to alleviate tumor burden in mice and promote the efficacy of αPD-1.

[0143] In summary, the present invention adopts mushroom polysaccharides to enhance the anti-tumor efficacy of αPD-1 by regulating the intestinal microbial system. After mushroom polysaccharides change the intestinal flora environment, intestinal probiotic flora substances can be obtained by enrichment, which can synergistically activate cytotoxic T cells with αPD-1 to kill tumor cells; animal experiments show that the intestinal probiotic flora substances enriched by mushroom polysaccharides regulating the intestinal microecology are the key to combined synergy. Compared with any single therapy, the intestinal probiotic flora substances obtained by inducing changes in the intestinal microecology by mushroom polysaccharides and αPD-1 combined treatment can significantly delay tumor growth.

[0144] The above are preferred embodiments and corresponding examples of the present invention. It should be noted that, for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, including but not limited to the adjustment of the proportion, process, dosage and reaction vessel, such as the use of a continuous flow reactor, which all fall within the scope of protection of the present invention. The above are preferred embodiments and corresponding examples of the present invention. It should be noted that, for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, including but not limited to the adjustment of the proportion, process, dosage, which all fall within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition, characterized in that include: Intestinal probiotic substances and αPD-1 inhibitors; Wherein, the intestinal probiotic bacteria substance is the intestinal probiotic bacteria substance obtained by inducing and changing the intestinal microecology through red juice lactucin polysaccharide; The red juice lactarius polysaccharide is prepared by the following method: Taking mushroom fruiting bodies, degreasing and extracting with water to obtain a polysaccharide extract; diluting the polysaccharide extract, adding ethanol for alcohol precipitation to obtain the mushroom polysaccharide; The intestinal probiotic flora substances include intestinal bacteria.

2. The pharmaceutical composition according to claim 1, characterized in that The components of the red juice lactarius polysaccharide include glucose, mannose, galactose and fucose.

3. The pharmaceutical composition according to claim 1, characterized in that The step of obtaining the mushroom fruiting bodies, defatting and extracting with water to obtain a polysaccharide extract further includes freeze-drying and pulverizing the mushroom fruiting bodies.

4. Use of the pharmaceutical composition according to any one of claims 1 to 3 in the preparation of an anti-tumor product, characterized in that: The anti-tumor products include drugs, detection reagents and kits; The tumor targeted by the anti-tumor product is lung cancer among respiratory system tumors.

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