Application of dihydroartemisinin in preparation of drugs for treating colorectal cancer metastasis

Abstract

The application belongs to the field of colorectal cancer treatment, and particularly relates to application of dihydroartemisinin in preparation of colorectal cancer liver metastasis drugs. The application research finds that dihydroartemisinin (DHA) can inhibit expression of mutant KRAS, reshape tumor immune microenvironment, thereby enhancing therapeutic effect of regorafenib combined with PD-1 inhibitor. These findings provide new insights for overcoming drug resistance in KRAS mutant colorectal cancer liver metastasis (CRCLM), and support further development of DHA as an adjuvant drug for combination therapy to improve patient prognosis.

Description

Technical Field

[0001] This invention belongs to the field of colorectal cancer treatment, specifically relating to the application of dihydroartemisinin in the preparation of drugs for colorectal cancer metastasis. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors worldwide, and metastasis is the leading cause of death among patients, with the liver being the most common secondary site of involvement. Patients with colorectal cancer liver metastases (CRCLM) primarily receive chemotherapy, targeted therapy, and immunotherapy. CRCLM exhibits high genetic heterogeneity and a highly suppressive tumor immune microenvironment (TIME), thus complicating its treatment due to multiple gene mutations and the highly immunosuppressive tumor microenvironment, limiting the effectiveness of the aforementioned treatment methods.

[0003] In recent years, numerous clinical trials on the combination of targeted therapy and immunotherapy have been conducted globally. Previous studies have reported that microsatellite stable tumors, conventionally considered insensitive to immune checkpoint inhibitors (ICBs), can be effectively suppressed through combination therapy with targeted therapy and immunotherapy, such as the use of regorafenib combined with nivolumab. However, another trial, IMBlaze370, found that the efficacy of the two-drug combination was not superior to monotherapy. In actual clinical practice, the combination of regorafenib and a PD-1 monoclonal antibody (mAb) has been used to treat CRCLM patients, but only a portion of these patients achieved disease remission.

[0004] Approximately 50% of CRC patients have KRAS mutations, in which KRAS G12D It is the most common mutation type, followed by KRAS. G12V and KRAS G13D Mutated KRAS continuously activates its downstream signaling pathways, such as p-Erk1 / 2 and p-Akt, which may lead to multidrug resistance through mechanisms such as mediating bypass signaling pathways, inhibiting anti-tumor immune responses, and inducing secondary mutations.

[0005] KRAS was initially considered an "undrugable" target due to its smooth protein surface. However, since the first target for KRAS was developed in 2013... G12C Since the discovery of small molecules that induce apoptosis, the exploration of mutant KRAS has become one of the main areas of cancer research. Currently, clinically approved inhibitors are limited to KRAS. G12C Primarily used for lung cancer, but also for KRAS. G12D KRAS G12V and KRAS G13D There are currently no approved drugs for CRC mutations. On the other hand, in the treatment of CRC, targeting KRAS is crucial. G12C sotorasib and KRASG12D MRTX1133 may develop resistance due to compensatory activation of the EGFR signaling pathway, which may also be the reason for the resistance of KRAS-mutant CRC to regorafenib. Therefore, there is an urgent need to develop new alternative KRAS-mutant targeted drugs. Summary of the Invention

[0006] The treatment of colorectal cancer liver metastases (CRCLM) is complicated by multiple gene mutations and a highly immunosuppressive tumor microenvironment, which limits the effectiveness of traditional treatments such as chemotherapy, targeted drugs and immune checkpoint inhibitors. In order to overcome the above-mentioned technical problems, this invention provides the application of dihydroartemisinin in the preparation of colorectal cancer drugs.

[0007] The application of dihydroartemisinin according to specific embodiments of the present invention in the preparation of KRAS inhibitors.

[0008] Preferably, the inhibitor inhibits the expression of mutant KRAS.

[0009] The application of dihydroartemisinin according to specific embodiments of the present invention in the preparation of colorectal cancer drugs.

[0010] Preferably, the drug also includes a pharmaceutically acceptable carrier.

[0011] Preferably, the pharmaceutically acceptable carrier includes at least one of the following: filler, diluent, disintegrant, binder, lubricant, flow aid, surfactant, solvent, flavoring agent, stabilizer, colorant, and preservative.

[0012] Preferably, the drug is an oral or injectable drug, and the dosage form includes tablets, capsules, powders, granules, pills, or solutions.

[0013] The present invention provides the use of a combination of drugs in the preparation of a drug for the treatment of colorectal cancer, the combination of drugs comprising an effective amount of dihydroartemisinin, an effective amount of regorafenib and an effective amount of a PD-1 inhibitor.

[0014] Preferably, the dihydroartemisinin inhibits the expression of mutant KRAS.

[0015] The method of using the above-mentioned drugs includes administering an effective amount of the drug to the subject. The administration route may be oral, intravenous injection, or transdermal penetration, applied to the patient requiring treatment.

[0016] A pharmaceutically effective dose refers to the amount of medication sufficient to treat a disease with a reasonable benefit / risk ratio. The level of the effective dose can be determined based on several factors, including the patient's disease type and severity, the drug's activity, drug sensitivity, timing of administration, route of administration, excretion rate, treatment duration, concurrent medications, and other factors known in the pharmaceutical field. The drug of this invention can be administered as a standalone therapeutic agent or in combination with other therapeutic agents. It is important to consider all the above factors and administer the minimum dose that produces the maximum effect without side effects, which can be determined by the physician based on the patient's condition, age, etc.

[0017] The beneficial effects of this invention are:

[0018] This invention establishes that KRAS mutations weaken the efficacy of regorafenib combined with anti-PD-1 therapy in CRCLM. Furthermore, this invention discovers that dihydroartemisinin (DHA) is a potential KRAS mutation inhibitor that can effectively downregulate KRAS expression. Most importantly, DHA can enhance the efficacy of regorafenib combined with PD-1 monoclonal antibodies by blocking ERBB pathway activation and restoring IFN response activity.

[0019] DHA has demonstrated excellent efficacy in inhibiting mutant KRAS expression and enhancing the therapeutic effects of regorafenib and PD-1 monoclonal antibodies, with minimal adverse reactions such as weight loss, hepatotoxicity, and nephrotoxicity. Furthermore, DHA can serve as a broad-spectrum KRAS inhibitor to address secondary KRAS mutations that may occur during treatment.

[0020] DHA plays a crucial role in the remodeling of the tumor immune microenvironment (TIME) by inhibiting mutant KRAS expression, validating the impact of low IFN response induced by KRAS mutation on T cell function, and further supporting the potential mechanism by which weakened IFN response in cancer cells impairs T cell chemotaxis and cytotoxicity.

[0021] This invention provides a mechanism by which KRAS mutations mediate resistance to regorafenib combined with anti-PD-1 therapy in CRCLM. Furthermore, it demonstrates that DHA can act as a broad-spectrum KRAS inhibitor, suppressing the expression of KRAS mutant proteins, blocking the activation of the ERBB signaling pathway, restoring IFN response, and remodeling the tumor immune microenvironment (TIME).

[0022] DHA can enhance the efficacy of regorafenib combined with PD-1 inhibitors while exhibiting very few adverse reactions. This provides a strong theoretical basis for incorporating DHA into combination therapy for CRCLM and other KRAS-mutant malignancies, which will help the clinical translation of DHA-based treatment strategies and promote the development of precision medicine, thereby overcoming the problem of drug resistance in cancer treatment. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The study showed that KRAS mutations lead to resistance to combination therapy with regorafenib and PD-1 monoclonal antibodies in CRCLM patients.

[0025] (A) KRAS status of CRCLM responders and non-responders treated with regorafenib in combination with anti-PD-1 therapy;

[0026] (B) GSEA analysis based on clinical sample RNA-seq;

[0027] (C) Using mIF to detect TIME characteristics in patient tumor tissue;

[0028] (D) Experimental procedure of C57BL / 6J mouse CRCLM model;

[0029] Statistical analysis of liver and liver weight relative to body weight in (E–F) CRCLM mouse model;

[0030] (G) Western blot detection of downstream signaling pathways (p-Erk1 / 2 and p-Akt) in tumor tissue of mouse model.

[0031] (H) TIME characteristics of tumor tissue in mouse model.

[0032] Figure 2 The results show that DHA can inhibit mutant KRAS and its downstream signaling pathways, among which...

[0033] (A) Virtual drug screening for KRASG12D;

[0034] (B) Binding site of DHA on KRASG12D;

[0035] (C–D) Western blot verification of the inhibitory effect of DHA on KRAS-MUT or KRAS-WT and its downstream signaling pathways.

[0036] Figure 3 The in vitro and in vivo experiments showed that DHA and regorafenib synergistically inhibited CRC.

[0037] (A–B) Synergistic analysis of regorafenib and DHA in LS180 and SW480 cells;

[0038] (C) Synergistic antiproliferative effect of regorafenib and DHA;

[0039] (D) Synergistic pro-apoptotic effect of regorafenib and DHA;

[0040] (E) Western blot analysis confirmed that regorafenib and DHA synergistically inhibited p-Erk1 / 2 and p-Akt;

[0041] (F) Subcutaneous tumors in a BALB / c nude mouse model;

[0042] (G) Tumor weight at the time of sampling.

[0043] (H) Changes in tumor size during the experiment;

[0044] (I) HE, IHC and TUNEL staining of tumor tissue.

[0045] Figure 4 The results showed that DHA can enhance the efficacy of combination therapy of regorafenib and PD-1 monoclonal antibody in CRCLM.

[0046] (A) Experimental procedure of BALB / c mouse CRCLM model;

[0047] (B) Liver of BALB / c mouse model;

[0048] (C) Statistical analysis of liver weight relative to body weight in BALB / c mouse model;

[0049] (D–E) KEGG and GO enrichment analysis based on RNA-seq of tumor tissues from BALB / c mouse model;

[0050] (F) Western blot detection of downstream signaling pathways (p-Erk1 / 2 and p-Akt) in tumor tissue of mouse model.

[0051] (G) Heatmap showing the expression of immunomodulatory factors in tumor tissue (based on RNA-seq);

[0052] (H) Immune cell infiltration was assessed using the CIBERSORT algorithm based on RNA-seq.

[0053] Figure 5 The study showed adverse reactions in mice with subcutaneous colorectal cancer xenografts treated with DHA combined with regorafenib and PD-1 monoclonal antibody, and in mice with colorectal cancer liver metastases treated with DHA combined with regorafenib and PD-1 monoclonal antibody.

[0054] (A) Experimental procedure for the BALB / c nude mouse subcutaneous colorectal cancer model;

[0055] (B) Changes in body weight in the BALB / c nude mouse model;

[0056] (C) HE staining of liver and kidney of BALB / c nude mice;

[0057] (D) Changes in body weight in the BALB / c mouse CRCLM model;

[0058] (E) HE staining of kidneys in BALB / c mouse model.

[0059] Figure 6 This demonstrates the synergistic effect of regorafenib and DHA, among which...

[0060] (AE) Synergistic analysis of regorafenib and DHA;

[0061] (F) Synergistic antiproliferative effect of regorafenib and DHA in HCT116 cells;

[0062] (G) Synergistic pro-apoptotic effects of regorafenib and DHA in HCT116 cells;

[0063] (H) The synergistic inhibitory effect of regorafenib and DHA on p-Erk1 / 2 and p-Akt was verified by Western blotting. Detailed Implementation

[0064] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0065] Example 1

[0066] 1. Clinical Sample Collection

[0067] Sixteen cases of CRCLM were collected for molecular pathological characterization. Tumor tissue from six patients, including three patients who responded to regorafenib combined with anti-PD-1 therapy (KRAS-WT) and three non-responders (KRAS-MUT), was used for transcriptome sequencing (RNA-seq). Paraffin-embedded CRCLM tissue from the patients was used for immunohistochemical (IHC) staining or multiplex immunofluorescence (mIF) staining.

[0068] Table 1. Information on 16 patients included in the molecular pathology analysis.

[0069]

[0070] like Figure 1 As shown in A, more of the 16 cases collected were non-responders with detected KRAS mutations.

[0071] 2. Tissue RNA sequencing and data analysis

[0072] Total RNA was extracted from paraffin-embedded human tumor tissue using the RecoverAll™ FFPE Total Nucleic Acid Isolation Kit (AM1975, ThermoFisher). Total RNA was extracted from mouse colorectal cancer liver metastases using the Trizol assay. RNA sequencing was performed by BGI (Wuhan, China). Functional enrichment analyses were performed using GO, KEGG, and GSEA. Immune infiltration assessment was performed using the CIBERSORT algorithm.

[0073] Batch transcriptome sequencing was performed on 3 well-preserved case specimens from each of 8 treatment responders and 8 non-responders. Functional enrichment analysis showed differences in the enrichment of certain gene pathways between the two groups, including anti-tumor immune and cell proliferation pathways. Figure 1 B).

[0074] In situ detection of TIME (tumor immune microenvironment) using mIF also revealed that responders (KRAS) WT In tumor tissues of [specific type], there is greater infiltration of CD8+ T cells and less infiltration of CD206+ TAMs (tumor-associated macrophages). Figure 1 C).

[0075] 3. Cell lines and cell culture

[0076] According to the Cancer Cell Line Encyclopedia, LS180 cells carry KRAS. G12D Mutation, SW480 cells carry KRAS G12V Mutation, HCT116 cells carry KRAS G13D Mutations, while Caco-2 and HT29 cells are KRAS WT Cells. CT26 cells carry Kras G12D Mutation, while MC38 cells are Kras WT cell.

[0077] LS180, SW480, HCT116, HT29, CT26, and MC38 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin; Caco-2 cells were cultured in DMEM medium containing 10% FBS and 1% penicillin / streptomycin. All cells were cultured at 37°C, 5% CO2, and humidified, and passaged every two to three days.

[0078] 4. Plasmid and stable cell construction

[0079] MC38 cells were used to construct mouse Kras. G12D Overexpressing cells, pLV3-CMV-Kras G12D -Puro plasmid was purchased from Miaoling Plasmid Co., Ltd., China. Caco-2 and HT29 cells were used to construct human KRAS. G12D Overexpressing cells, pLVX-EF1α-KRAS G12D The IRES-Puro plasmid was purchased from Beijing Laike Biotechnology Co., Ltd. Lentiviral packaging plasmids pMD2.G (#12259) and psPAX2 (#12260) were purchased from Addgene. Lentiviral cells were prepared by transfecting HEK293T cells with the above plasmids (at a ratio of 7:2:5) using Neofect transfection reagent. Cell infection was promoted using 10 μg / mL polybrene (TR-1003, Sigma-Aldrich), and positive cells were selected using puromycin (6 μg / mL for MC38 cells, 2 μg / mL for Caco-2 and HT29 cells). KRAS G12D / Kras G12D The stability of the expression was verified by Sanger sequencing and Western blot.

[0080] 5. Mouse model construction and therapeutic drugs

[0081] Four-week-old female C57BL / 6J and BALB / c mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. The mice were treated by injecting 100 μL of 1×10⁻⁶ PBS suspension into their spleens. 6 MC38 or CT26 cells were used to construct a mouse model of colorectal cancer liver metastasis. Figure 1 D-1H, Figure 4 and Figure 5 D-5E).

[0082] Four-week-old female BALB / c nude mice were purchased from Charles River. 2 × 10⁻⁶ mice were subcutaneously injected into the left axilla. 6HCT116 cells (suspended in 100 μL PBS solution containing 50% Matrigel). Treatment was initiated when the tumor volume increased to approximately 50 mm³ (tumor volume = 1 / 2 × length × width²). Figure 3 H-3I, Figure 5 A-5C).

[0083] Regorafenib (HY-10331, MedChemExpress) and DHA (HY-N0176, MedChemExpress) were administered via corn oil gavage, while the PD-1 monoclonal antibody (A2122, Selleck) was administered via intraperitoneal injection.

[0084] 6. Western blot

[0085] Cells were lysed in RIPA lysis buffer (R0010, Solarbio) as previously described. Primary antibodies are listed in Table 2. After incubation with secondary antibodies at room temperature for one hour, the cells were washed with TBS-T, and the membranes were exposed on an ImageQuant 800 system to detect protein levels.

[0086] 7. Multiplex immunofluorescence (mIF) staining

[0087] mIF staining was performed using a seven-color mIF kit (based on tyrosine signal enhancement technology). Experiments were performed according to the manufacturer's instructions. Primary antibodies are listed in Table 2. Captured images were analyzed using ImageJ software to estimate mean fluorescence intensity (MFI) or the percentage of positive cells.

[0088] Table 2 Antibody List

[0089]

[0090] 8. Determination of half-maximal inhibitory concentration (IC50)

[0091] The smooth surface of KRAS proteins makes them difficult to target with small molecules. Through a scoring and screening of 225 natural active compounds derived from traditional Chinese medicine, the proposed target for KRAS is artemisinin derivative DHA. G12D compounds ( Figure 2 AB).

[0092] Caco-2 and HT29 cells are two wild-type KRAS cell lines used to construct KRAS. G12D Cells were used to investigate whether DHA can regulate KRAS expression.

[0093] Cells were spaced at 1 × 10⁶ cells per well. 4Cells were seeded at a density of [number] cells per well in 96-well plates. After cell adhesion, a gradient of drug concentrations was added (regorafenib: 0 μM, 0.39 μM, 0.78 μM, 1.56 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM; DHA: 0 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, and 100 μM). After 48 hours, the drugs were removed, and 100 μL of cell culture medium (CK04, DOJINDO) containing 10% CCK8 solution was added and incubated for 1.5 hours. The absorbance was measured at 450 nm using a Bio-Rad microplate reader. The IC50 value was calculated using GraphPad Prism 8.0.

[0094] CRC cell lines were treated with a series of DHA concentration gradients to further measure changes in KRAS protein. The results showed that KRAS... G12D The expression of KRAS decreased significantly, while that of KRAS decreased significantly. WT Unaffected, its downstream pathways such as p-Erk1 / 2 and p-Akt showed a similar trend ( Figure 2 C). It is evident that DHA can also effectively inhibit LS180 (KRAS). G12D ), SW480 (KRAS) G12V ) and HCT116 (KRAS G13D KRAS expression in cells ( Figure 2 D).

[0095] 9. Analysis of drug synergistic effects

[0096] Based on the estimated IC50 values, a series of concentration gradients for DHA and regorafenib were established to analyze their synergistic effects. Synergistic effect analysis was performed using the SynergyFinder platform and measured by an HSA score (threshold 10), with higher HSA scores indicating stronger synergistic effects.

[0097] In various KRAS mutant CRC cell lines (including LS180, SW480, HCT116, and KRAS) G12D In mutant Caco-2 and HT29 cells, regorafenib combined with DHA showed a synergistic antiproliferative effect. Figure 3 AB, Figure 6 AE).

[0098] LS180, SW480, and HCT116 cells were seeded in 6-well plates at densities of 1000 cells / well, 500 cells / well, and 500 cells / well, respectively, and cultured in conditioned media containing different drugs. After incubation for 10–15 days, cells were fixed with 4% paraformaldehyde (PFA) and stained with crystal violet. Cell counting was performed using ImageJ software. In vitro experiments, including clonogenic assays and apoptosis detection, showed that the combination of regorafenib and DHA significantly inhibited the proliferation of LS180, SW480, and HCT116 cells compared to monotherapy. Figure 3 C, Figure 6 F) and promotes its apoptosis (F) Figure 3 D, Figure 6 G). By detecting the levels of p-Erk1 / 2 and p-Akt in cell lines, it was found that regorafenib monotherapy had limited effect on downregulating these phosphorylated proteins, while combination therapy with DHA significantly reduced phosphorylation levels. Figure 3 E, Figure 6 H) indicates that the inhibitory effect on the downstream of the KRAS mutation is enhanced.

[0099] To verify the synergistic effect in vivo, an HCT116 mouse xenograft model was established. Mice were sacrificed at the end of the treatment regimen. Tumor size and weight both indicated that regorafenib combined with DHA had a good synergistic effect in inhibiting CRC. Figure 3 FH).

[0100] Furthermore, Ki-67 immunohistochemical staining and TUNEL assays evaluated the efficacy of the combination therapy in inhibiting CRC proliferation and promoting apoptosis, respectively. Simultaneously, angiogenesis, assessed by CD31 expression, was also inhibited. Figure 3 I). The above data indicate that DHA can synergistically combat CRC with regorafenib by simultaneously inhibiting proliferation and promoting apoptosis, and its main mechanism is the downregulation of the p-Erk1 / 2 and p-Akt signaling pathways.

[0101] 10. Treatment of a mouse model of colorectal cancer liver metastasis

[0102] Using MC38 cells (Kras) WT and Kras G12D A mouse model of CRCLM was constructed, and treatment was administered according to the planned protocol. Figure 1 D). Regorafenib in combination with PD-1 monoclonal antibody in patients carrying Kras WT The therapeutic effect is better in mouse models of tumors. Figure 1 EF). In KRAS G12D In tumors, downstream signaling pathways regulating cell growth (such as p-Erk1 / 2 and p-Akt) of KRAS were not significantly inhibited by treatment. Figure 1G). Furthermore, with KRAS G12D Compared to tumors, regorafenib combined with PD-1 monoclonal antibodies significantly increased KRAS. WT CD8+ T cell infiltration in tumors and reduced recruitment of CD206+ TAMs ( Figure 1 H). This reveals the important role of KRAS mutation in affecting the sensitivity of CRCLM to regorafenib combined with PD-1 monoclonal antibody therapy.

[0103] A CRCLM model was established by injecting CT26 cells (KrasG12D) into the spleen of BALB / c mice, and treatment was administered according to plan. Figure 4 A). At the experimental endpoint, after euthanasia and tumor resection in mice, it was found that treatment with regorafenib in combination with PD-1 monoclonal antibody or DHA alone had limited efficacy. However, when both were used in combination, tumor suppression was significantly enhanced, indicating a stronger therapeutic synergy. Figure 4 B–C). To preliminarily investigate how DHA enhances the efficacy of combination therapy with regorafenib and PD-1 monoclonal antibodies, we performed RNA sequencing (RNA-seq) on randomly selected samples. KEGG and GO enrichment analyses showed that DHA can regulate proliferation-related signaling pathways and modulate anti-tumor immune responses (B–C). Figure 4 D–E). Consistent with transcriptome data, western blot results confirmed that in DHA-containing combination therapy, the levels of Kras, p-Erk1 / 2, and p-Akt proteins were significantly reduced, while regorafenib monotherapy showed weak inhibitory effects on these pathways in vivo. Figure 4 F). Furthermore, DHA treatment reduced the expression of some immunosuppressive factors (such as Tgfb1–3 and Il-10) while increasing the expression of some immunostimulatory mediators (such as Cxcl9, Cxcl10, Gzmb, and Ifng). Figure 4 G). TIME (tumor immune microenvironment) atlases plotted using the CIBERSORT algorithm showed increased infiltration of CD8+ T cells and M1 macrophages, while decreased infiltration of M2 macrophages. Figure 4(H). This indicates that DHA can significantly enhance the anti-CRCLM efficacy of regorafenib combined with PD-1 monoclonal antibody by inhibiting the activity of proliferation-related signaling pathways and remodeling the tumor immune microenvironment. Single-cell transcriptome sequencing (scRNA-seq) further revealed that in KRAS-mutant CRC, the expression of ERBB ligands EREG, AREG, and HBEGF was upregulated after regorafenib treatment, which is a manifestation of the reactivation of Erk1 / 2 and Akt, ultimately leading to regorafenib resistance. In addition, KRAS mutations can induce immune escape by inhibiting the interferon response of cancer cells and reducing the chemotaxis and cytotoxicity of CD8+ T cells; while DHA can alleviate this immune escape phenomenon by potently inhibiting the expression of KRAS mutations.

[0104] This invention further validated through experiments that DHA has a significant regulatory effect on TIME, promoting cytotoxic and pro-inflammatory immune cell populations while inhibiting immunosuppressive subsets. Because DHA can inhibit the expression of mutant KRAS and remodel TIME, it enhances the efficacy of PD-1 monoclonal antibodies.

[0105] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The use of a combination of drugs in the preparation of a therapeutic drug for colorectal cancer liver metastases, characterized in that, The combination of drugs includes an effective amount of dihydroartemisinin, an effective amount of regorafenib, and an effective amount of a PD-1 inhibitor; The dihydroartemisinin inhibits the expression of mutant KRAS.

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