Application of pachymaran combined with immune checkpoint inhibitors and combined drugs

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of pachyman triterpenes and immune checkpoint inhibitors and a combined drug. The pachyman triterpenes and the immune checkpoint inhibitors are combined to enhance the body's anti-tumor immune response through a synergistic mechanism: on the one hand, the immune checkpoint inhibitors remove the immune suppression of T cells by the tumor microenvironment by blocking the immune inhibitory checkpoint signal; on the other hand, the pachyman triterpenes enhance the activation and proliferation of CD4 + T cells, CD8 + T cells and NK cells, enhance the immune surveillance function of the peripheral immune system, promote the recruitment of immune effector cells to the tumor microenvironment, and thus produce a synergistic anti-tumor effect with the immune checkpoint inhibitors. The immune checkpoint inhibitors remove the immune suppression "brake" of T cells, and the pachyman triterpenes enhance the activation and proliferation capacity of multiple immune cells. The two work synergistically through different but complementary mechanisms in the same immune response chain to achieve an overall anti-tumor effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of the combined use of Poria triterpenes and immune checkpoint inhibitors, and the combined drugs. Background Technology

[0002] Malignant tumors are a major disease that seriously threatens human life and health. Their global incidence and mortality rates are rising year by year, and they have become one of the leading causes of death. Most malignant tumors have no specific clinical symptoms in their early stages, and most patients are diagnosed at a locally advanced stage or with distant metastasis, directly leading to a significant decrease in clinical cure rates and 5-year survival rates. Among them, colorectal cancer is a highly prevalent malignant tumor of the digestive tract, originating from the colonic mucosal epithelium. Due to its insidious early symptoms and lack of specific manifestations, the vast majority of patients are diagnosed at a locally advanced stage or with distant metastasis, resulting in a poor clinical prognosis.

[0003] Chemotherapy is a fundamental treatment for advanced malignant tumors, but its clinical application has significant drawbacks. Most patients cannot tolerate the various toxic side effects of chemotherapy, resulting in limited overall clinical efficacy. Targeted therapy exerts its anti-tumor effect by inhibiting angiogenesis in the tumor microenvironment and has achieved some clinical efficacy. However, tumor cells readily develop drug resistance, significantly limiting the long-term therapeutic effect of targeted drugs.

[0004] Tumor immunotherapy is an emerging non-invasive anti-tumor treatment technology. Its core mechanism of action is to activate the body's own immune cells and enhance the body's specific killing ability against tumor cells, which has significant advantages over traditional treatment methods. Immune checkpoints, represented by PD-1 / PD-L1 and CTLA-4, are key targets for regulating tumor immune escape. Among them, immune checkpoint inhibitors that block the PD-1 / PD-L1 signaling axis have become the core strategy of tumor immunotherapy and are widely used in the clinical treatment of various solid tumors. Currently, anti-PD-1 / PD-L1 monoclonal antibodies such as pembrolizumab, nivolumab, and atezolizumab have been approved for the treatment of various advanced solid tumors, including melanoma, non-small cell lung cancer, and renal cell carcinoma.

[0005] However, existing monotherapy with immune checkpoint inhibitors has significant limitations: the overall objective response rate is only 15%–30%, indicating a low efficacy rate; it has virtually no therapeutic effect on "cold tumors" with low immunogenicity, such as MSS-type colorectal cancer; and primary and acquired tumor resistance are frequent problems. Current clinical studies have confirmed that peripheral blood CD4... +High expression of PD-1 on the surface of T cells is an independent risk factor for the failure of immunotherapy in colorectal cancer patients. After PD-1 binds to PD-L1, it can activate tyrosine groups, inhibiting the downstream PI3K / Akt signaling pathway, causing T cell dysfunction and loss of anti-tumor immune capacity, thereby mediating tumor immune escape and ultimately leading to immunotherapy failure. Simultaneously, clinical characteristics such as poor / low-grade tumor differentiation, clinical stage III-IV, lymph node metastasis, distant metastasis, NLR ≥ 5, and PLR ≥ 135 further reduce the effectiveness of immune checkpoint inhibitors. Simply blocking the PD-1 / PD-L1 signaling axis cannot completely rebuild an effective anti-tumor immune response in the body.

[0006] To overcome the limitations of monotherapy for immunotherapy, existing technologies are gradually exploring combined treatment options such as the use of natural products and regulation of gut microbiota, in order to improve the efficacy of immunotherapy.

[0007] In the field of combination therapy with natural products, CN122398784A discloses a technical solution for treating B-cell lymphoma using taxine combined with immune checkpoint inhibitors. This solution works by anchoring the METTL1 catalytic core with taxine and blocking the m7G methylation cascade reaction, thereby synergistically enhancing the anti-tumor effect. However, this technical solution has a limited applicability, targeting only B-cell lymphoma and failing to cover clinically prevalent solid tumors such as colon cancer and lung cancer. Furthermore, taxine suffers from poor water solubility, low bioavailability, and limitations in oral administration, making it unable to achieve CD4+ target efficacy. + The synergistic activation of T and NK cells has significant shortcomings in terms of its mechanism of action and scope of application.

[0008] In the field of combined therapy based on gut microbiota regulation, CN122351490A discloses a technical solution that uses vancomycin to reshape the gut microbiota and reduce L-asparagine levels, thereby enhancing the antigen presentation capacity of dendritic cells and improving the therapeutic sensitivity of MSS-type colorectal cancer to PD-1 / PD-L1 inhibitors. However, this technical solution relies on antibiotics to indirectly regulate the immune microenvironment, and long-term use is prone to causing gut microbiota dysbiosis, bacterial resistance, and other safety risks, making it difficult to guarantee clinical safety; moreover, this technical solution only targets and regulates CD4. + The T cell initiation process cannot directly activate CD4. + Key immune cells such as T cells and NK cells have a single dimension of immune activation, resulting in limited synergistic therapeutic effects.

[0009] Poria cocos triterpenes are triterpenoid compounds extracted from the dried sclerotia of the fungus Poria cocos (Polyporaceae family). Existing studies have confirmed that they possess various pharmacological activities, including anti-inflammatory, diuretic, hepatoprotective, and hypoglycemic effects. Currently, in publicly available patents and literature, Poria cocos triterpenes are only used in routine health care fields such as hair care and diuretic regulation; there are no studies or reports on their combined use with immune checkpoint inhibitors for immunotherapy of solid tumors. Summary of the Invention

[0010] 1. The problem to be solved To address the shortcomings of existing anti-tumor immunotherapies such as monotherapy with immune checkpoint inhibitors, combination therapy with natural products and immune checkpoint inhibitors, and combination therapy with antibiotics and immune checkpoint inhibitors, this invention aims to provide the application of Poria cocos triterpenes in combination with immune checkpoint inhibitors in the preparation of drugs for the prevention and / or treatment of tumors, as well as the combination drugs. This invention, by combining Poria cocos triterpenes with immune checkpoint inhibitors, can significantly improve the therapeutic effect on tumors.

[0011] 2. Technical Solution To achieve the above objectives, the present invention provides the following technical solution.

[0012] This invention provides the application of the combination of Poria triterpenes and immune checkpoint inhibitors in the preparation of drugs for the prevention and / or treatment of tumors.

[0013] Furthermore, the tumor is a solid tumor.

[0014] Furthermore, the solid tumors include colon cancer, lung cancer, and ovarian cancer.

[0015] The poria triterpenes are obtained by alcohol extraction of poria. Further, the preparation method of the poria triterpenes includes the following steps: pulverizing poria and adding alcohol for extraction; filtering while hot after extraction and collecting the filtrate; recovering the solvent from the filtrate and freeze-drying the resulting concentrate to obtain an alcohol extract; dispersing the alcohol extract in water, adding an organic solvent for extraction, and collecting the extract; recovering the organic solvent from the extract and freeze-drying the resulting concentrate to obtain poria triterpenes.

[0016] Furthermore, the alcohol is C 1-6 Fatty alcohols.

[0017] Furthermore, the alcohol is C 1-3 Fatty alcohols.

[0018] Furthermore, the alcohol is at least one of methanol, ethanol, and propanol.

[0019] Furthermore, the alcohol is ethanol.

[0020] Furthermore, the mass of the alcohol is 4 to 10 times the mass of Poria cocos.

[0021] Furthermore, the mass of the alcohol is 6 to 8 times the mass of Poria cocos.

[0022] Furthermore, the extraction is a reflux extraction.

[0023] Furthermore, the extraction is performed 2 to 3 times.

[0024] Furthermore, the extraction time for each extraction is 1 to 3 hours.

[0025] Furthermore, the organic solvent is ethyl acetate.

[0026] Furthermore, the volume ratio of the organic solvent to the water is 0.5 to 1:1.

[0027] Furthermore, the extraction is performed 2 to 5 times.

[0028] Furthermore, the immune checkpoint inhibitor is selected from at least one of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, and LAG-3 inhibitors.

[0029] Furthermore, the immune checkpoint inhibitor is a PD-1 inhibitor.

[0030] Furthermore, the PD-1 inhibitor is a PD-1 antibody.

[0031] Furthermore, prevention and / or treatment of tumors includes at least one of the following: Slowing down tumor growth; Inhibit tumor growth; Reduce terminal tumor burden; Improve survival rate; Extend the lifespan; Increase the sensitivity of tumors to immune checkpoint inhibitors; Increase CD4 in the tumor environment + T cells, CD8 + The ratio of T cells to NK cells.

[0032] Furthermore, the drug also includes pharmaceutically acceptable carriers and / or excipients. These pharmaceutically acceptable carriers and / or excipients include, but are not limited to, diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc., the amounts of which can be determined by those skilled in the art according to actual needs.

[0033] Furthermore, the drug can be prepared into a pharmaceutically acceptable dosage form according to conventional methods in the art, including but not limited to pills, tablets, capsules, granules, powders, granules, lozenges, suspensions, syrups, oral solutions, or injections.

[0034] The present invention also provides a combination drug for the prevention and / or treatment of tumors, comprising poria triterpenoids and immune checkpoint inhibitors.

[0035] Furthermore, the tumor is a solid tumor.

[0036] Furthermore, the solid tumors include colon cancer, lung cancer, and ovarian cancer.

[0037] The poria triterpenes are obtained by alcohol extraction of poria. Further, the preparation method of the poria triterpenes includes the following steps: pulverizing poria, adding alcohol for extraction; filtering while hot after extraction, collecting and combining the filtrates; recovering the solvent from the filtrate, freeze-drying the obtained concentrate to obtain an alcohol extract; dispersing the alcohol extract in water, adding an organic solvent for extraction, collecting the extract; recovering the organic solvent from the extract, freeze-drying the obtained concentrate to obtain poria triterpenes.

[0038] Furthermore, the alcohol is C 1-6 Fatty alcohols.

[0039] Furthermore, the alcohol is C 1-3 Fatty alcohols.

[0040] Furthermore, the alcohol is at least one of methanol, ethanol, and propanol.

[0041] Furthermore, the alcohol is ethanol.

[0042] Furthermore, the mass of the alcohol is 4 to 10 times the mass of Poria cocos.

[0043] Furthermore, the mass of the alcohol is 6 to 8 times the mass of Poria cocos.

[0044] Furthermore, the extraction is a reflux extraction.

[0045] Furthermore, the extraction is performed 2 to 3 times.

[0046] Furthermore, the extraction time for each extraction is 1 to 3 hours.

[0047] Furthermore, the organic solvent is ethyl acetate.

[0048] Furthermore, the volume ratio of the organic solvent to the water is 0.5 to 1:1.

[0049] Furthermore, the extraction is performed 2 to 5 times.

[0050] Furthermore, the immune checkpoint inhibitor is selected from at least one of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, and LAG-3 inhibitors.

[0051] Furthermore, the immune checkpoint inhibitor is a PD-1 inhibitor.

[0052] Furthermore, the PD-1 inhibitor is a PD-1 antibody.

[0053] Furthermore, the immune checkpoint inhibitor and Poria triterpenoids are administered simultaneously or separately.

[0054] Further, the mass ratio of the immune checkpoint inhibitor to Poria triterpenes is 1:(5-100). Further, the mass ratio of the immune checkpoint inhibitor to Poria triterpenes is 1:5, 1:8, 1:10, 1:12, 1:15, 1:17, 1:20, 1:22, 1:25, 1:27, 1:30, 1:32, 1:35, 1:37, 1:40, 1:42, 1:45, 1:47, 1:50, 1:52, 1:55, 1:57, 1:60, 1:62, 1:65, 1:67, 1:70, 1:72, 1:75, 1:77, 1:80, 1:82, 1:85, 1:87, 1:90, 1:92, 1:95, 1:97, 1:100, or a range of any two values.

[0055] For the combined drug of the present invention, there are no particular restrictions on the order of administration of the immune checkpoint inhibitor and the poria triterpenoid.

[0056] Furthermore, immune checkpoint inhibitors are administered via intravenous injection, while poria triterpenes are administered orally.

[0057] The present invention also relates to a method for preventing and / or treating tumors, comprising administering the combination drugs as described above to a subject in need.

[0058] 3. Beneficial effects This invention combines the naturally derived active ingredient, Poria cocos triterpenes, with an immune checkpoint inhibitor, significantly enhancing the therapeutic effect against tumors. The combination of Poria cocos triterpenes and immune checkpoint inhibitors enhances the body's anti-tumor immune response through a synergistic mechanism: on the one hand, the immune checkpoint inhibitors relieve the immunosuppression of T cells by the tumor microenvironment by blocking immunosuppressive checkpoint signals; on the other hand, Poria cocos triterpenes promote CD4+... + T cells, CD8 +The activation and proliferation of T cells and NK cells enhance the immune surveillance function of the peripheral immune system and promote the recruitment of immune effector cells to the tumor microenvironment, thereby producing a synergistic anti-tumor effect with immune checkpoint inhibitors. Immune checkpoint inhibitors release the immunosuppressive "brake" on T cells, while Poria triterpenes enhance the activation and proliferation of various immune cells. Through different but complementary mechanisms, they work synergistically on the same immune response chain to achieve an overall anti-tumor effect. Furthermore, the combined use of Poria triterpenes and immune checkpoint inhibitors can enhance the blocking effect of immunosuppressive pathways such as PD-1 / PD-L1, improve the sensitivity of tumors to immunotherapy, reverse the "cold tumor" immunosuppressive state, thereby overcoming primary and acquired immunotherapy resistance and further expanding the applicable population for immunotherapy.

[0059] Furthermore, the compositions provided by this invention are not limited to the treatment of colon cancer and lung cancer, but have the potential to be extended to other solid tumors, and have broad clinical application prospects. Attached Figure Description

[0060] The present invention will be further described below with reference to the accompanying drawings. The drawings are merely illustrative, and the scope of protection of the present invention is not limited to these drawings. Obviously, those skilled in the art can derive other forms of drawings based on the principles of the present invention and the following drawings without any inventive effort.

[0061] Figure 1 The growth curves of subcutaneous colon cancer xenografts in MC38 mice after treatment with solvent control group, Poria triterpenoid treatment group, PD-1 inhibitor treatment group, and combined treatment group are shown.

[0062] Figure 2 Survival curves of MC38 mouse colon cancer models after treatment with solvent control group, Poria triterpenoid treatment group, PD-1 inhibitor treatment group and combination treatment group.

[0063] Figure 3 Gross images of subcutaneous xenografts in MC38 mice on day 13 after treatment of the solvent control group, the Poria triterpenoid treatment group, the PD-1 inhibitor treatment group, and the combined treatment group.

[0064] Figure 4 The growth curves of subcutaneous xenografts in Lewis lung cancer (LLC, 3LL subline) mice after treatment with solvent control group, Poria triterpenoid treatment group, PD-1 inhibitor treatment group and combination treatment group are shown.

[0065] Figure 5 Survival curves of Lewis lung cancer (LLC, 3LL subspecies) mouse models after treatment with solvent control group, Poria triterpenoid treatment group, PD-1 inhibitor treatment group and combination treatment group.

[0066] Figure 6Gross images of subcutaneous xenografts in Lewis lung cancer (LLC, 3LL subline) mice on day 13 after treatment with solvent control group, Poria triterpenoid treatment group, PD-1 inhibitor treatment group, and combination treatment group.

[0067] In the figure, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001. Detailed Implementation

[0068] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0069] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0070] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0071] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0072] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0073] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0074] The IgG isotype control antibody used was IgG1-D265A, purchased from Syd Labs, catalog number PA007135.

[0075] The PD-1 inhibitor used was RMP1-14, purchased from Syd Labs, catalog number PA007162.

[0076] Example 1: Extraction of triterpenes from Poria cocos The coarse pieces of Poria cocos were crushed and extracted by reflux with 85% ethanol for a total of 3 extractions, each extraction lasting 1.5 hours, with the amount of 85% ethanol used each time being 6 times the mass of Poria cocos. After hot filtration, the filtrates were collected and combined. The ethanol in the filtrate was removed by vacuum distillation, and the resulting concentrate was freeze-dried to obtain the ethanol extract. The ethanol extract was dispersed in water, and extracted with an equal volume of ethyl acetate as water for a total of 4 extractions. The extracts were combined, and the ethyl acetate was removed by vacuum distillation. The resulting concentrate was freeze-dried to obtain the ethyl acetate extract, i.e., Poria cocos triterpenes.

[0077] Example 2: Anti-colon cancer effect of the combination of Poria triterpenes and PD-1 inhibitors 1. Laboratory animals Female C57BL / 6J mice, aged 6-8 weeks and weighing 16-20 g, were provided by the Changzhou Cavens Laboratory. The animals were housed in an SPF-grade barrier environment with free access to water at a temperature of 22±2℃ and a relative humidity of 60±5%. All animal experiments were conducted strictly in accordance with the regulations and procedures of the Ethics Committee of Changzhou First People's Hospital and international animal welfare and health standards.

[0078] 2. Modeling and Grouping MC38 cells in logarithmic growth phase were digested with trypsin, washed twice with PBS, resuspended in sterile PBS, and the cell concentration was adjusted to 2 × 10⁻⁶ cells / year. 7 Cell viability was >95% as measured by trypan blue staining, yielding MC38 tumor cell suspension. Each mouse was inoculated with 50 μL of MC38 tumor cell suspension. Prior to inoculation, the fur on the abdomen and sides of the trunk of the mice was shaved, and the injection was administered subcutaneously on the left side of the trunk. Six days after modeling, the mice were randomly divided into four groups of five: a solvent control group, a Poria cocos triterpenoid treatment group, a PD-1 inhibitor treatment group, and a combination therapy group.

[0079] 3. Experimental Methods: Solvent control group: Mice were given the drug starting on day 6 after inoculation with MC38 subcutaneous xenografts. They were also given an intraperitoneal injection of IgG isotype control antibody (100 μg / mouse, dissolved in 100 μL PBS) once every 4 days for a total of 4 times. At the same time, they were given 200 μL of corn oil by gavage daily until day 30 after inoculation.

[0080] Poria triterpenoid treatment group: Mice were given medication starting on day 6 after inoculation with MC38 subcutaneous xenografts. IgG isotype control antibody (100 μg / mouse, dissolved in 100 μL PBS) was injected intraperitoneally every 4 days for a total of 4 doses. Simultaneously, Poria triterpenoid (1000 μg / mouse, dispersed in 200 μL corn oil) was administered by gavage daily until day 30 after inoculation.

[0081] PD-1 inhibitor treatment group: Mice were given PD-1 inhibitor (100 μg / mouse, dissolved in 100 μL PBS) intraperitoneally starting on day 6 after inoculation with MC38 subcutaneous xenografts. The administration was repeated every 4 days for a total of 4 times. At the same time, 200 μL of corn oil was administered by gavage daily until day 30 after inoculation.

[0082] Combined treatment group: Mice were given PD-1 inhibitor (100 μg / mouse, dissolved in 100 μL PBS) intraperitoneally starting on day 6 after MC38 subcutaneous xenograft inoculation, once every 4 days for a total of 4 times; at the same time, Poria triterpenes (1000 μg / mouse, dispersed in 200 μL corn oil) were administered by gavage daily until day 30 after inoculation.

[0083] Peripheral blood was collected from mice on day 14 after tumor inoculation, single-cell suspensions were prepared, and CD4 levels in the peripheral blood were detected by flow cytometry. + T cells, CD8 + Changes in the proportions of T cells and NK cells. Starting from day 6 after tumor inoculation, the long and short diameters of the subcutaneous tumors in mice were measured every two days, and the results were calculated using the formula V = long diameter × short diameter. 2 / 2 Calculate tumor volume. Assume a tumor volume of 2000 mm². 3 Humanitarian endpoints were defined as mice having a maximum tumor length exceeding 20 mm or exhibiting significant weight loss and general deterioration. Survival time was recorded for each group of mice, and survival analysis was performed using the Kaplan-Meier method.

[0084] 4. Experimental Results Table 1. CD4 levels in peripheral blood during colon cancer experiments + T cells, CD8 + The ratio of T cells to NK cells

[0085] Note: Compared with the solvent control group, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0086] Flow cytometry detection of CD4 in peripheral blood + T cells, CD8 +The ratios of T cells and NK cells are shown in Table 1. Compared with the solvent control group, the peripheral blood CD4 counts in the Poria cocos triterpenoid treatment group, the PD-1 inhibitor treatment group, and the combination therapy group were significantly higher. + T cells, CD8 + The proportions of T cells and NK cells were increased to varying degrees, especially in the combination therapy group, where the level of CD4+ in peripheral blood was higher. + T cells, CD8 + The proportions of T cells and NK cells increased significantly (P < 0.01 or P < 0.001).

[0087] Combination Figures 1 to 3 The growth curves, survival curves, and gross images of the xenografts showed that, compared with the solvent control group, the treatment groups of Poria triterpenoids and PD-1 inhibitors significantly slowed the growth rate of MC38 subcutaneous xenografts and prolonged the survival time of tumor-bearing mice to varying degrees. However, the single-drug interventions in both groups only achieved moderate anti-tumor effects and could not effectively control the continuous growth of tumors. The combined treatment group significantly enhanced the in vivo anti-tumor effect, not only significantly inhibiting tumor volume growth (P < 0.001) and reducing terminal tumor burden, but also significantly improving the survival benefit of tumor-bearing mice (P < 0.01). The survival advantage was significantly better than that of either single-drug treatment group, indicating that the combination of Poria triterpenoids and PD-1 inhibitors has a synergistic anti-colon cancer effect.

[0088] Example 3: Anti-lung cancer effect of the combination of Poria triterpenes and PD-1 inhibitors 1. Laboratory animals Female C57BL / 6J mice, aged 6-8 weeks and weighing 16-20 g, were provided by the Changzhou Cavens Laboratory. The animals were housed in an SPF-grade barrier environment with free access to water at a temperature of 22±2℃ and a relative humidity of 60±5%. All animal experiments were conducted strictly in accordance with the regulations and procedures of the Ethics Committee of Changzhou First People's Hospital and international animal welfare and health standards.

[0089] 2. Modeling and Grouping MC38 cells in logarithmic growth phase were digested with trypsin, washed twice with PBS, resuspended in sterile PBS, and the cell concentration was adjusted to 2 × 10⁻⁶ cells / year. 7 Cell viability was >95% as measured by trypan blue staining, yielding MC38 tumor cell suspension. Each mouse was inoculated with 50 μL of MC38 tumor cell suspension. Prior to inoculation, the fur on the abdomen and sides of the trunk of the mice was shaved, and the injection was administered subcutaneously on the left side of the trunk. Six days after modeling, the mice were randomly divided into four groups of five: a solvent control group, a Poria cocos triterpenoid treatment group, a PD-1 inhibitor treatment group, and a combination therapy group.

[0090] 3. Experimental Methods: Solvent control group: Mice were given the drug starting on day 6 after inoculation with subcutaneous xenografts of Lewis lung cancer (LLC, 3LL subline) and intraperitoneally injected with IgG isotype control antibody (100 μg / mouse, dissolved in 100 μL PBS) every 4 days for a total of 4 times; at the same time, they were given 200 μL corn oil by gavage daily until day 30 after inoculation.

[0091] Poria triterpenoid treatment group: Mice were given medication starting on day 6 after inoculation with subcutaneous xenografts of Lewis lung cancer (LLC, 3LL subline). IgG isotype control antibody (100 μg / mouse, dissolved in 100 μL PBS) was injected intraperitoneally every 4 days for a total of 4 doses. Simultaneously, Poria triterpenoid (1000 μg / mouse, dispersed in 200 μL corn oil) was administered by gavage daily until day 30 after inoculation.

[0092] PD-1 inhibitor treatment group: Mice were given PD-1 inhibitor (100 μg / mouse, dissolved in 100 μL PBS) intraperitoneally starting on day 6 after inoculation with subcutaneous xenografts of Lewis lung cancer (LLC, 3LL subline) and administered once every 4 days for a total of 4 times; at the same time, they were given 200 μL corn oil by gavage daily until day 30 after inoculation.

[0093] Combined treatment group: Mice were given PD-1 inhibitors (100 μg / mouse, dissolved in 100 μL PBS) intraperitoneally starting on day 6 after inoculation with subcutaneous xenografts of Lewis lung cancer (LLC, 3LL subline) and administered once every 4 days for a total of 4 times; at the same time, they were given Poria triterpenes (1000 μg / mouse, dispersed in 200 μL corn oil) by gavage daily until day 30 after inoculation.

[0094] Peripheral blood was collected from mice on day 14 after tumor inoculation, single-cell suspensions were prepared, and CD4 levels in the peripheral blood were detected by flow cytometry. + T cells, CD8 + Changes in the proportions of T cells and NK cells. Starting from day 6 after tumor inoculation, the long and short diameters of the subcutaneous tumors in mice were measured every two days, and the values ​​were calculated using the formula V = long diameter × short diameter. 2 / 2 Calculate tumor volume. Assume a tumor volume of 2000 mm². 3 Humanitarian endpoints were defined as mice having a maximum tumor length exceeding 20 mm or exhibiting significant weight loss and general deterioration. Survival time was recorded for each group of mice, and survival analysis was performed using the Kaplan-Meier method.

[0095] 4. Experimental Results Table 2. CD4 levels in peripheral blood during lung cancer experiments + T cells, CD8 + The ratio of T cells to NK cells

[0096] Note: Compared with the solvent control group, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0097] Flow cytometry detection of CD4 in peripheral blood + T cells, CD8 + The ratios of T cells and NK cells are shown in Table 2. Compared with the solvent control group, the peripheral blood CD4 counts in the Poria cocos triterpenoid treatment group, the PD-1 inhibitor treatment group, and the combination therapy group were significantly higher. + T cells, CD8 + The proportions of T cells and NK cells were increased to varying degrees, especially in the combination therapy group, where the level of CD4+ in peripheral blood was higher. + T cells, CD8 + The proportions of T cells and NK cells increased significantly (P < 0.001), indicating that the combination of Poria triterpenes and PD-1 inhibitors can synergistically promote the proliferation and activation of peripheral anti-tumor immune cells and enhance the systemic anti-tumor immune response.

[0098] like Figures 4 to 6 As shown, using tumor growth level, tumor volume, and mouse survival time as evaluation indicators, the solvent control group showed rapid tumor proliferation and the shortest mouse survival time. Compared with the solvent control group, both the Poria triterpenoid treatment group and the PD-1 inhibitor treatment group could inhibit tumor growth and prolong the survival time of tumor-bearing mice to a certain extent, but their anti-tumor effects were limited. The combined treatment group showed significantly better results in reducing tumor volume and improving mouse survival rate than each single-drug group, indicating that the combination of Poria triterpenoids and PD-1 inhibitors has a synergistic anti-lung cancer ability.

[0099] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. Application of Poria triterpenes in combination with immune checkpoint inhibitors in the preparation of drugs for the prevention and / or treatment of tumors.

2. The application according to claim 1, characterized in that, The tumor is a solid tumor.

3. The application according to claim 1 or 2, characterized in that, The preparation method of Poria triterpenes includes the following steps: after crushing Poria, add alcohol for extraction; filter while hot after extraction and collect the filtrate; recover the solvent in the filtrate and freeze-dry the obtained concentrate to obtain the alcohol extract; disperse the alcohol extract in water, add organic solvent for extraction and collect the extract; recover the organic solvent in the extract and freeze-dry the obtained concentrate to obtain Poria triterpenes.

4. The application according to claim 3, characterized in that, The alcohol is C 1-6 fatty alcohols; and / or The mass of the alcohol is 4 to 10 times the mass of Poria cocos; and / or The extraction is a reflux extraction; and / or The number of extractions is 2 to 3; and / or Each extraction takes 1–3 hours; and / or The organic solvent is ethyl acetate; and / or The volume ratio of the organic solvent to water is 0.5 to 1:1; and / or The extraction is performed 2 to 5 times.

5. The application according to claim 1 or 2, characterized in that, The immune checkpoint inhibitor is selected from at least one of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, and LAG-3 inhibitors.

6. The application according to claim 1 or 2, characterized in that, Prevention and / or treatment of tumors includes at least one of the following: Slowing down tumor growth; Inhibit tumor growth; Reduce terminal tumor burden; Improve survival rate; Extend the lifespan; Increase the sensitivity of tumors to immune checkpoint inhibitors; Increase CD4 in the tumor environment + T cells, CD8 + The ratio of T cells to NK cells.

7. The application according to claim 1 or 2, characterized in that, The drug also includes pharmaceutically acceptable carriers and / or excipients.

8. A combination drug for the prevention and / or treatment of tumors, characterized in that, Including poria triterpenes and immune checkpoint inhibitors.

9. The combination drug for the prevention and / or treatment of tumors according to claim 8, characterized in that, The mass ratio of the immune checkpoint inhibitor to the poria triterpenoid is 1:(5-100).

10. The combination drug for the prevention and / or treatment of tumors according to claim 8 or 9, characterized in that, The immune checkpoint inhibitors are formulated as injectable dosage forms, and the poria triterpenes are formulated as oral dosage forms.

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