Sensitization camptothecin drug anti-tumor drug combination composition and application thereof

Abstract

The invention provides a sensitizing camptothecin drug anti-tumor combined drug composition and application thereof, and particularly relates to application of an apigenin and camptothecin drug composition in preparation of anti-tumor drugs. A natural dietary supplement and an anti-tumor chemotherapeutic drug are creatively combined, a colorectal cancer in-vitro cell model is utilized to prove that the apigenin has the anti-tumor effect of sensitizing camptothecin drugs, the apigenin and the low-dose camptothecin drugs can activate a PI3K-AKT pathway and a dsDNA-cGAS-STING pathway, tumor immunogenic death is induced, and the apigenin has the anti-tumor effect. And the anti-tumor immune response is activated. The invention not only has the effects of reducing toxicity and enhancing efficacy on clinically applied camptothecin medicines, but also provides a new strategy for sensitizing colorectal cancer immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a sensitized camptothecin drug anti-tumor combined drug composition and application thereof. Background Art

[0002] Cancer statistics in 2023 show that colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide and is common in men. Currently, the treatments for CRC are mainly chemotherapy, radiotherapy, surgery and immunotherapy. Among them, chemotherapy is still the main treatment for patients with advanced CRC. Irinotecan, a semi-synthetic derivative of camptothecin, exerts cytotoxic effects by specifically targeting DNA topoisomerase and inhibiting the reconnection of DNA chains. It has been approved by the FDA as a standard treatment for advanced CRC. Although irinotecan has significant anti-tumor efficacy, its clinical application is severely hindered by the existence of insensitive patient subgroups and strong adverse reactions. Summary of the invention

[0003] In order to overcome at least one of the defects of the prior art, the present invention provides a sensitized camptothecin anti-tumor drug combination composition and its application, which can solve the serious obstacles to clinical application due to the existence of insensitive patient subgroups and strong adverse reactions.

[0004] The technical solution adopted by the present invention to solve the problem is:

[0005] A combined drug composition for sensitizing camptothecin drugs to fight tumors comprises camptothecin drugs and apigenin.

[0006] By adopting the above scheme, the combined administration of the camptothecin drug irinotecan and apigenin has the effect of synergistically inhibiting the proliferation of colorectal cancer cells and promoting the apoptosis of colorectal cancer cells.

[0007] Furthermore, the molar ratio of the camptothecin drug to the apigenin is 1:30-1:3.

[0008] By adopting the above scheme, the best synergistic anti-tumor effect can be achieved by setting the molar ratio of camptothecin drugs and apigenin between 1:30 and 1:3. In different types of colorectal cancer cells, the combination of drugs within this molar ratio range can significantly inhibit cell proliferation and promote cell apoptosis. In particular, for cells that are insensitive to both irinotecan and apigenin, the best effect is achieved when the molar ratio is 3:1; while for cells that are more sensitive to irinotecan, the best effect is achieved when the molar ratio is 30:1.

[0009] Within this molar ratio range, the ideal anti-tumor effect can be achieved with a lower dose of camptothecin drugs, thereby significantly reducing the toxic side effects of the drugs. This not only improves the treatment safety of patients, but also improves their tolerance and quality of life.

[0010] Furthermore, the pharmaceutical composition is a clinically used chemotherapy drug containing a camptothecin core structure in the drug chemical structure.

[0011] Furthermore, the chemotherapy drug is one or a combination of irinotecan and topotecan.

[0012] The present invention also provides the use of the above-mentioned sensitizing camptothecin drug combination composition in the preparation of anti-tumor drug therapy.

[0013] Furthermore, the tumor is colorectal cancer.

[0014] Furthermore, the combination drug composition is in any pharmaceutically acceptable dosage form.

[0015] Furthermore, the dosage form includes any one of liposomes, microemulsions, and nanoparticles.

[0016] Furthermore, the administration forms of the combination composition include but are not limited to intravenous injection, intraperitoneal injection, oral administration or rectal administration.

[0017] Furthermore, the application is to induce tumor immunogenic death and activate anti-tumor immune response by activating the PI3K-AKT pathway and the dsDNA-cGAS-STING pathway.

[0018] By adopting the above scheme, the combination of apigenin and camptothecin drugs can significantly enhance the anti-tumor effect by activating the PI3K-AKT pathway and the dsDNA-cGAS-STING pathway. Specifically, activation of the PI3K-AKT pathway can promote cell apoptosis, while activation of the dsDNA-cGAS-STING pathway induces immunogenic cell death, making tumor cells more easily recognized and cleared by the immune system, thereby improving the tumor immune microenvironment. In addition, this combination drug strategy can achieve the ideal therapeutic effect at a lower dose, significantly reduce the toxic side effects of camptothecin drugs, and improve the safety and tolerability of patient treatment.

[0019] In summary, the anti-tumor combined drug composition for sensitizing camptothecin drugs provided by the present invention and its application have the following technical effects:

[0020] The present invention has demonstrated for the first time in three human colorectal cancer in vitro cell models that the combined administration of the camptothecin drug irinotecan and apigenin has the effect of synergistically inhibiting the proliferation of colorectal cancer cells and promoting the apoptosis of colorectal cancer cells. By examining the sensitivity of colorectal cancer cells to irinotecan and apigenin respectively, it was determined that when the molar ratio of the combined drug was 3:1, the growth inhibition effect of cells insensitive to both was the best, while for cells sensitive to irinotecan, the growth inhibition effect was the best when the molar ratio of the combined drug was 30:1.

[0021] The present invention discloses for the first time the mechanism of action of apigenin in sensitizing irinotecan against colorectal cancer. Apigenin combined with irinotecan can activate the downstream PI3K-AKT pathway and the signal transduction of the dsDNA-cGAS-STING pathway, leading to tumor immunogenic death. The two have a synergistic anti-tumor effect. In addition, the activated endogenous immune response of the tumor also improves the tumor immune microenvironment, enhances the killing effect of immune cells on tumor cells, and has the effect of sensitizing colorectal cancer immunotherapy. The two can be used in combination directly, or they can be made into a combined preparation for the treatment of colorectal cancer and sensitization of colorectal cancer immunotherapy, which has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Apigenin has the effect of sensitizing irinotecan against colorectal cancer. A is the synergistic inhibition growth index of apigenin and irinotecan or apigenin and SN38 (active metabolite of irinotecan) on three colorectal cancer cell lines (LOVO, HCT8 and HCT15 cells); b and c are the cell survival curves and IC50 values ​​of LOVO, HCT8 and HCT15 cells treated with different concentrations of apigenin and irinotecan for 72 hours; d is the cell survival rate analysis of LOVO, HCT8 and HCT15 cells treated with apigenin (3μM), irinotecan (1μM), irinotecan (0.1μM) and their combination for 72 hours. Data are expressed as mean ± standard deviation, n = 3; e is the effect of different concentrations of apigenin, irinotecan and their combination on the colony formation ability of LOVO, HCT8 and HCT15 cells; f is a representative picture of cell apoptosis detected by Annexin V apoptosis detection kit after different concentrations of apigenin, irinotecan and their combination acted on LOVO, HCT8 and HCT15 cells for 72 hours. Red fluorescence indicates apoptotic cells; g is the quantitative calculation of cell apoptosis rate by the red fluorescence intensity of stained apoptotic cells and the percentage of bright field cell coverage. Data are expressed as mean ± standard.

[0023] Figure 2Apigenin combined with irinotecan can activate the PI3K-AKT pathway. a is a screening flow chart for the top 30 Hub genes for apigenin, irinotecan and colorectal cancer determined by network pharmacology. b is a compound-disease-target gene network reflecting the relationship between apigenin, irinotecan and potential targets of colorectal cancer. The specific targets of irinotecan are indicated in yellow, the specific targets of apigenin are indicated in red, and the shared targets of irinotecan and apigenin are indicated in orange. c is the compound-disease-target gene network of irinotecan-specific targets, apigenin-specific targets and common targets of the two. The node size represents the gene enrichment degree, and the node color represents the betweenness centrality between each gene; d is the Gene Ontology (GO) enrichment analysis results of the predicted targets of irinotecan alone and apigenin combined with irinotecan; e is the RNA-seq data of apigenin and irinotecan combined treatment and irinotecan alone treatment using Hallmark gene enrichment analysis, which confirms that the PI3K-AKT signaling pathway is significantly activated in the combined treatment group compared with irinotecan alone treatment. The one-tailed Fisher's exact test was used to evaluate the statistical differences; f is the expression levels of GAPDH, total Akt and phosphorylated Akt after apigenin, irinotecan and apigenin and irinotecan combined treatment detected by Western blotting technology.

[0024] Figure 3Figure 1: Apigenin combined with irinotecan activates type I interferon response through the dsDNA-cGAS-STING pathway. a: Hallmark gene enrichment analysis was used to compare the differences in enriched pathways between apigenin and irinotecan combination therapy and irinotecan alone. The inflammatory response, IFNα response pathway, and IFNγ response pathway were significantly activated after combined therapy and are indicated in red; b: GSEA barcode diagram showing that the Hallmark gene sets related to the inflammatory response, IFNα response pathway, and IFNγ response pathway were significantly activated after apigenin and irinotecan combination therapy compared with irinotecan alone in the LOVO cell line, and the statistical differences were evaluated using the one-tailed Fisher's exact test; c: qPCR technology was used to confirm that apigenin and irinotecan combination therapy can induce increased mRNA expression of IFNα and IFNγ compared with irinotecan alone in LOVO and HCT15 cell lines, and Oneway ANOVA was used for significant statistical analysis. Data are expressed as mean ± SD, n = 3; d is the downregulation of DNA repair pathways in LOVO cell line after irinotecan combined treatment compared with irinotecan alone, and the statistical difference was evaluated by one-tailed Fisher's exact test; e is a representative image of dsDNA detection using immunofluorescence technology with DNA damage-specific antibody p-H2AX, with a scale of 50 μm in LOVO and 25 μm in HCT15; f is a quantitative analysis of dsDNA immunofluorescence data, and statistical difference analysis was performed using Oneway-ANOVA; g is a representative image of dsDNA detection using immunofluorescence technology with p-H2AX, with a scale of 50 μm in LOVO and 25 μm in HCT15; Representative immunoblot images of the expression levels of GAPDH and p-H2AX in OVO and HCT15 cell lines after treatment with apigenin, irinotecan, and the combination of apigenin and irinotecan; h is the quantitative data of g, and the statistical difference was analyzed by Oneway-ANOVA, n=3; i is a representative immunoblot image of the expression levels of GAPDH, c-GAS, STING, p-STING, TBK1, p-TBK1, STAT1, p-STAT1, STAT3, p-STAT3 and PD-L1 after treatment with apigenin, irinotecan, and the combination of apigenin and irinotecan.

[0025] Figure 4Apigenin combined with irinotecan enhances anti-tumor immune response. a is the immunofluorescence detection of dsDNA in CT26 and MC38 cell lines treated with apigenin, irinotecan, and apigenin and irinotecan combined using the DNA damage-specific antibody p-H2AX, with a scale of 25 μM; b is the quantitative analysis of immunofluorescence data, with Oneway-ANOVA used for statistical difference analysis; c is a representative protein immunoblot of the expression levels of GAPDH and p-H2AX in mouse cell lines treated with apigenin, irinotecan, and apigenin and irinotecan combined; d is the qPCR technology confirming that the expression levels of GAPDH and p-H2AX were significantly higher than those of irinotecan alone. Compared with the treatment, the combined treatment of apigenin and irinotecan induced the expression of IFNα and IFNγ in mouse cell lines. Oneway-ANOVA was used for statistical analysis, and the data were expressed as mean ± standard deviation, n = 3; e is a schematic diagram of the experimental process of co-culture of mouse tumor cells and spleen cells; f is the enhanced killing effect of spleen cells on MC38 mouse tumor cells after combined treatment of apigenin and irinotecan, and the killing status of MC38 cells was represented by MC38 cell apoptosis. Oneway-ANOVA was used for statistical difference analysis, and the data were expressed as mean ± standard deviation, n = 6. DETAILED DESCRIPTION

[0026] For better understanding and implementation, the technical solutions in the embodiments of the present invention will be clearly and completely described and discussed below in conjunction with the accompanying drawings of the present invention. Obviously, what is described here is only a part of the examples of the present invention, not all the examples. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0027] In order to facilitate the understanding of the embodiments of the present invention, the following will be further explained by taking specific embodiments as examples in conjunction with the drawings, and each embodiment does not constitute a limitation on the embodiments of the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0030] See also Figure 1-Figure 4 As shown, the effect of the combined pharmaceutical composition of apigenin and irinotecan in Example 1 on colorectal cancer cell lines HCT8, HCT15, and LOVO.

[0031] Human colorectal cancer cell lines LOVO, HCT8, and HCT15 were kindly provided by Zhejiang Cancer Hospital, China. Cells were cultured at 37°C in a 5% CO2 incubator in RPMI-1640 medium (Gibco) supplemented with 10% v / v fetal bovine serum (FBS).

[0032] The effect of combined drug use on cell proliferation was evaluated by cell proliferation experiment: cells in logarithmic growth phase were taken and inoculated in 96-well plates at a density of 3000 cells per well. After they adhered to the wall, apigenin (Aladding, A106676) and irinotecan (Aladding, I102602) or SN38 (Selleck, S4908) were prepared in culture medium in a three-fold dilution manner to prepare 49 different combination concentrations of drug-containing culture medium ranging from 30μM to 0μM, and added to the well plate. After incubation for 72 hours at 37°C and 5% CO2, CCK8 reagent (Biosharp, BS350B) was used to detect cell survival. The optical density (OD) value was determined using an enzyme reader (Tecan, Switzerland). The cell survival rate was determined using the following formula:

[0033] Cell survival rate (%) = (OD 处理 -OD 本底 ) / (OD 未处理 -OD 本底 )

[0034] The Bliss synergy score was calculated based on the above cell viability and visualized using the Bliss score package in RStudio software (version 4.2.0, Boston, USA) to evaluate the degree of drug synergy.

[0035] The long-term inhibitory effect of the combined drug on tumor cell proliferation was evaluated by cell colony analysis: 10,000 cells were seeded in 6-well plates per well. After adherence, the cells were cultured in drug-containing medium containing apigenin (3 μM), irinotecan (1 μM), irinotecan (0.1 μM), apigenin (3 μM) combined with irinotecan (1 μM), and apigenin (3 μM) combined with irinotecan (0.1 μM) for 13 days, with 0.1% DMSO as a control. After 13 days, the cells were fixed with 4% paraformaldehyde (Biosharp, BL539A), stained with crystal violet solution (Beyotime, C0121) for 1 hour, and photographed.

[0036] The effect of combined drug therapy on tumor cell apoptosis was evaluated by cell apoptosis experiment: cells in logarithmic growth phase were taken and inoculated into 96-well plates at 3000 cells per well, and cultured in drug dilution medium of apigenin (3μM), irinotecan (1μM), irinotecan (0.1μM), apigenin (3μM) combined with irinotecan (1μM), and apigenin (3μM) combined with irinotecan (0.1μM) for 72 hours, with 0.1% DMSO as control. Incucyte Annexin VRedDye reagent (Incucyte, 4641) was added to the cells to mark apoptotic cells, and the cell apoptosis process was continuously monitored every 6 hours using IncucyteSX5 live cell imaging equipment (Sartorius, Germany).

[0037] Experimental results: The results are as follows Figure 1 As shown. Three human colorectal cancer cells (LOVO, HCT8, and HCT15) were treated with different concentrations of apigenin (0-30 μM, three-fold dilution) and irinotecan (0-30 μM, three-fold dilution) in 49 different combinations, which proved that apigenin and irinotecan or apigenin and SN38 (the active metabolite of irinotecan) had a synergistic inhibitory effect on the above cell lines. However, due to the different sensitivities of different cell lines to these drugs, the optimal synergistic concentrations were also different. The optimal synergistic concentrations in LOVO cells were 3 μM apigenin and 1 μM irinotecan, and the optimal synergistic concentrations in HCT8 and HCT15 cells were 3 μM apigenin and 0.1 μM irinotecan. This shows that the combination of irinotecan and apigenin can enhance the tumor killing effect of irinotecan at a lower dose.

[0038] Example 2 The mechanism of action of apigenin in sensitizing irinotecan to inhibit colorectal cancer.

[0039] The potential mechanism of apigenin sensitizing irinotecan to inhibit colorectal cancer was predicted by network pharmacology. The potential targets of apigenin and irinotecan were predicted by SwissTargetPrediction server (http: / / www.swisstargetprediction.ch / ), and the research subjects were selected as human. Target genes related to colorectal cancer were downloaded from CTD (https: / / ctdbase.org / ) and GeneCards (https: / / www.genecards.org / ) databases. Subsequently, the collected target information was integrated using Cytoscape3.7.1 software to construct a compound-disease-target gene network. The Cytohubba plug-in in Cytoscape3.7.1 software was used to screen the hub genes affected by apigenin, irinotecan and their combination, and the top 30 hub genes were selected. The ggplot2 R software package was used to perform GO pathway enrichment analysis and visualization of the top 30 hub genes when irinotecan was used alone and in combination with apigenin.

[0040] The effect of combined drug treatment on the PI3K-AKT signaling pathway was verified by Western Blot: Human colorectal cancer cells (LOVO, HCT15) were treated with 0.1% DMSO, apigenin (3μM), irinotecan (1μM), and apigenin (3μM) combined with irinotecan for 72 hours, and then the cells were collected on ice and lysed at 4°C for 10 minutes using a lysis buffer (20mM Tris-HCl pH8.0, 150mMNaCl, 10mM MgCl2, 1mM EDTA, 0.5% Triton X-100) containing 1mM phosphatase inhibitor and 1x protease inhibitor (BeyotimeBiotechnology). Protein quantification was performed using a BCA protein assay kit (BeyotimeBiotechnology). After adding the loading buffer and denaturing at 95°C for 5 minutes, 20μg of lysed protein was separated on a 10% gradient gel and then transferred to a PVDF (Millipore, USA) membrane. After the PVDF membrane was blocked with 5% skim milk powder at room temperature for 1 hour, the primary antibody was incubated overnight at 4°C, including AKT1 antibody (HUABIO, ET1609-47; 1:800), p-AKT1 antibody (HUABIO, ET1607-73; 1:8005483t; 1:2000), and GAPDH antibody (CST, 5174S; 1:2000). The membrane was then incubated with HRP-labeled goat anti-rabbit IgG antibody (Wanlei Bio, WLA023, 1:5000) at room temperature for 1 hour. Immunoreactive bands were detected using an ECL luminescence kit (Biosharp, BL520B), and the membrane was imaged using the Amersham ImageQuant 800 Western blot imaging system (Cytiva, Japan).

[0041] RNA-sequencing was used to analyze the effect of combined drug treatment on the gene expression level of colorectal cancer cells. Library construction and sequencing were performed by GENEWIZ (GENEWIZ Biotechnology Co., Ltd., Suzhou, China) according to the manufacturer's instructions, based on the Illumina Hi Seq platform, in a 2×150 paired-end (PE) configuration. The reference genome sequence was prepared and indexed using Hisat2 (v2.0.1) for subsequent analysis. The data obtained by sequencing were aligned to the reference genome using Hisat2 software (v2.0.1). The known gff annotation files were converted to fasta format transcripts and appropriately indexed for further analysis. HTSeq (v0.6.1) used reference gene files to evaluate the expression levels of genes and isoforms from paired-end data. Gene expression analysis was performed using the DESeq2 package and sorted according to statistical values ​​(https: / / github.com / mikelove / DESeq2). The Hallmark gene set and GO gene set were derived from MsigDB (https: / / www.gsea-msigdb.org / gsea / msigdb / ). The clusterProfiler R package was used for gene set enrichment analysis (GSEA), and the enrichmentplot R package was used to generate pathway enrichment maps.

[0042] Experimental results: The results are as follows Figure 2 As shown. The results of network pharmacology, KEGG pathway enrichment analysis, and RNA-Seq sequencing analysis all showed that the PI3K-AKT pathway was significantly enriched after the combined administration of apigenin and irinotecan, indicating the key role of the PI3K-AKT pathway in mediating the synergistic anti-tumor effect of combined drug administration. And compared with the administration of irinotecan alone, the PI3K-AKT signaling pathway was significantly upregulated after the combined use of apigenin and irinotecan. This observation was verified at the protein level, and Western blot experiments proved that AKT phosphorylation was involved in the synergistic anti-tumor effect of apigenin and irinotecan.

[0043] Example 3 Effect of apigenin-irinotecan combination on immune response

[0044] First, gene set enrichment analysis (GSEA) was used to compare the differences in enriched pathways after apigenin combined with irinotecan and irinotecan alone in the LOVO cell line.

[0045] Real-time quantitative polymerase chain reaction (qRT-PCR) was used to verify the mRNA expression of IFN-α and IFN-γ after combined drug treatment: LOVO cells were treated with 0.1% DMSO, apigenin (3 μM), irinotecan (1 μM), and apigenin (3 μM) combined with irinotecan (1 μM), and HCT15 cells were treated with 0.1% DMSO, apigenin (3 μM), irinotecan (0.1 μM), and apigenin (3 μM) combined with irinotecan (0.1 μM). After 72 hours of drug treatment, cells were collected and total RNA was extracted using a total RNA extraction kit (Vazyme, RC112-01). Then, it was reverse transcribed into cDNA using a cDNA synthesis kit (Vazyme, R223-01). Real-time quantitative PCR was performed on a LightCycler96 real-time PCR system (BioRad) using SYBR Green (Vazyme, Q712-02) as a detection reagent. Specific primers are used to detect the expression of IFN-α and IFN-β. The primer sequences are shown in Table 1:

[0046] Table 1. Primer sequence list

[0047]

[0048] Using DNA damage-specific antibody p-H2AX as an indicator, immunofluorescence technology was used to detect cell DNA damage after combined drug use. Cells were fixed with 4% paraformaldehyde and then blocked with 3% bovine serum albumin (Biosharp, 9048-46-8). p-H2AX antibody (CST, 9718S; 1:100) and TRITC-labeled rabbit IgG antibody (Beyotime, A0516; 1:500) were used for staining, and DAPI (Beyotime, C1005) was used to stain the cell nucleus. The stained cells were imaged using a Sigle Photon confocal microscope (NIKON, A1HD25), and the images were analyzed using ImageJ software (v1.8.0).

[0049] Experimental results: Figure 3As shown. HALLMARK pathway analysis showed that when apigenin was combined with irinotecan, the inflammatory response, interferon-γ response and interferon-α response pathways were significantly enriched. The above results indicate that the combined treatment of apigenin and irinotecan can stimulate the activation of endogenous immune response. Subsequently, qPCR verified that apigenin combined with irinotecan could significantly upregulate the mRNA levels of IFNα and IFNβ, further confirming that apigenin combined with irinotecan treatment has the ability to upregulate the interferon pathway and activate endogenous immune response. GESA GO functional enrichment analysis confirmed that the combined use of apigenin and irinotecan would inhibit the DNA repair pathway. Immunofluorescence and western blot analysis confirmed that apigenin combined with irinotecan treatment could significantly induce DNA damage and promote the production of dsDNA, while increasing the expression level of p-H2AX protein in LOVO cells. Further testing of the changes in the cGAS-STING pathway, a classic downstream pathway of dsDNA, showed that the downstream cGAS-STING pathway signal was significantly activated after treatment with apigenin combined with irinotecan, and the protein levels of c-GAS, STING, and p-STING were significantly enhanced. Subsequently, TBK1 was phosphorylated and activated, and this activation ultimately induced the production of type I interferon. The above results further reveal the potential immunomodulatory effect of apigenin combined with irinotecan on colorectal cancer.

[0050] Example 4 used CT26 and MC38 mouse cell models to co-culture with spleen cells to explore the effect of combined drug use on anti-tumor immune response.

[0051] Mouse colorectal cancer cell lines CT26 and MC38 were kindly provided by Zhejiang Cancer Hospital, China. Cells were cultured at 37°C in an incubator containing 5% CO2 in RPMI-1640 medium (Gibco) supplemented with 10% v / v fetal bovine serum (FBS).

[0052] The effect of combined drug therapy on immunomodulatory function was evaluated by in vitro co-culture experiments of tumor cells and spleen cells. Cells in the logarithmic growth phase were taken and inoculated in 6-well plates at a density of 2000 cells per well, and incubated at 37°C for 72 hours in a drug dilution medium containing 0.1% DMSO, apigenin (3μM), irinotecan (1μM), and apigenin (3μM) combined with irinotecan (0.1μM). Subsequently, the pre-treated tumor cells were co-cultured with splenocytes isolated from mouse spleens. Splenocyte activation was stimulated with activation factors such as CD3 (Biolegend, 100301), CD28 (Biolegend, 102115), and IL-2 (Solarbio, P0019), and the apoptosis reagent Annexin V (Vazyme, A211-01) was added to mark the apoptosis of tumor cells, and the co-culture continued for 48 hours. During the entire co-culture process, the interaction between cells was monitored and the changes in the fluorescence intensity of tumor cell apoptosis were recorded every 2 hours using the Incucyte SX5 live cell imaging device (Sartorius, Germany). The acquired images were analyzed using the Incucyte software based on the ratio of apoptotic cell green fluorescence and bright field cell coverage.

[0053] Experimental results: The results are as follows Figure 4 As shown. In order to explore the effect of combined drug treatment on immunomodulatory function in mouse cells, the effect of combined drug treatment on endogenous immunity was first verified on CT26 and MC38 cells. The results showed that after MC38 cells were treated with apigenin combined with irinotecan, dsDNA accumulation, p-H2AX expression, and IFN-α and IFN-β mRNA production were more obvious than those in CT26 cells, making them more suitable as an in vitro co-culture model. Subsequently, MC38 cells were used as a model to further evaluate the immunomodulatory function of apigenin combined with irinotecan. The co-culture results showed that when apigenin and irinotecan were treated in combination, the apoptosis of tumor cells under the action of spleen cells increased significantly, confirming that combined drug treatment would enhance the cytotoxic effect of spleen cells on tumor cells, while apigenin or irinotecan alone had no significant effect. These results show that the combination of apigenin and irinotecan has a significant effect on anti-tumor immunomodulation.

[0054] Data processing: Data were statistically analyzed using GraphPad Prism7 software. Significant differences were evaluated by one-way analysis of variance (ANOVA), with P < 0.05 as the standard of significant difference. All experiments were repeated and the results were expressed as mean ± standard deviation.

[0055] The technical means disclosed in the scheme of the present invention are not limited to the technical means disclosed in the above-mentioned implementation mode, but also include technical schemes composed of any combination of the above-mentioned technical features. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications are also regarded as the protection scope of the present invention.

Claims

1. A combined drug composition for sensitizing camptothecin drugs against tumors, characterized in that: Including camptothecins and apigenin.

2. A sensitizing camptothecin anti-tumor drug combination composition according to claim 1, characterized in that: The molar ratio of the camptothecin drug to the apigenin is 1:30-1:

3.

3. A sensitizing camptothecin anti-tumor drug combination composition according to claim 2, characterized in that: The pharmaceutical composition is a clinically used chemotherapy drug containing a camptothecin mother nucleus structure in the drug chemical structure.

4. A sensitizing camptothecin anti-tumor drug combination composition according to claim 3, characterized in that: The chemotherapy drug is one or a combination of irinotecan and topotecan.

5. Use of the combined drug composition of sensitizing camptothecin drugs according to any one of claims 1 to 4 in the preparation of anti-tumor drugs.

6. The use according to claim 5, characterized in that: The tumor is colorectal cancer.

7. The use according to claim 5, characterized in that: The combined pharmaceutical composition is in any pharmaceutically acceptable dosage form.

8. The use according to claim 7, characterized in that: The dosage form includes any one of liposomes, microemulsions, and nanoparticles.

9. The use according to claim 5, characterized in that: The administration forms of the combined pharmaceutical composition include, but are not limited to, intravenous injection, intraperitoneal injection, oral administration or rectal administration.

10. The use according to claim 5, characterized in that: The application is to induce tumor immunogenic death and activate anti-tumor immune response by activating the PI 3K-AKT pathway and the dsDNA-cGAS-STING pathway.

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