Application of dihydroartemisinin in preparation of medicine for colorectal cancer metastasis

Abstract

The invention belongs to the field of colorectal cancer treatment, and particularly relates to application of dihydroartemisinin in preparation of a medicine for treating colorectal cancer liver metastasis. Research finds that dihydroartemisinin (DHA) can inhibit expression of mutant KRAS and remodel a tumor immune microenvironment, so that the treatment effect of regorafenib combined with a PD-1 inhibitor is enhanced. These findings provide a new insight 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] The present invention belongs to the field of colorectal cancer treatment, and particularly relates to the application of dihydroartemisinin in the preparation of colorectal cancer metastasis drugs. Background Art

[0002] Colorectal cancer (CRC) is one of the most common malignancies worldwide. Metastasis is the leading cause of death, with the liver being the most common site of secondary involvement. Patients with colorectal cancer liver metastasis (CRCLM) primarily receive chemotherapy, targeted therapy, and immunotherapy. However, CRCLM has high genetic heterogeneity and a highly suppressive tumor immune microenvironment (TIME). Therefore, treatment is complicated by multiple genetic mutations and a highly immunosuppressive tumor microenvironment, which limits the effectiveness of these treatments.

[0003] In recent years, numerous clinical trials combining targeted therapy with immunotherapy have been conducted worldwide. Previous studies have reported that microsatellite-stable tumors, which are generally considered insensitive to immune checkpoint inhibitors (ICBs), can also be effectively suppressed by combined 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 no better than that of monotherapy. In actual clinical practice, regorafenib and PD-1 monoclonal antibody (mAb) have been used to treat CRCLM patients, but only a subset of patients achieved disease remission.

[0004] Approximately 50% of CRC patients have KRAS mutations, including G12D 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 by mediating bypass signaling pathways, inhibiting anti-tumor immune responses, and inducing secondary mutations.

[0005] KRAS was initially considered an “undruggable” target due to its smooth protein surface. However, since 2013, the first KRAS-targeted 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 , mainly used for lung cancer, but targeting KRAS G12D 、KRAS G12V and KRAS G13D There are no approved drugs for CRC with KRAS mutations. G12C sotorasib and targeting KRASG12D The study also suggests that MRTX1133 may induce resistance due to compensatory activation of the EGFR signaling pathway, which may also be the cause of regorafenib resistance in KRAS-mutant CRC. Therefore, the development of new alternative mutant KRAS-targeted drugs is urgently needed. Summary of the Invention

[0006] The treatment of colorectal cancer liver metastasis (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). To overcome the above technical problems, the present invention provides the use of dihydroartemisinin in the preparation of colorectal cancer drugs.

[0007] According to a specific embodiment of the present invention, dihydroartemisinin is used in the preparation of KRAS inhibitors.

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

[0009] According to a specific embodiment of the present invention, dihydroartemisinin is used in the preparation of colorectal cancer drugs.

[0010] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

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

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

[0013] The present invention provides use of a combined drug combination in preparing a drug for treating colorectal cancer, wherein the combined drug combination comprises 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 for using the above-mentioned drug comprises administering an effective amount of the above-mentioned drug to a subject. The administration method can be oral, intravenous injection or transdermal permeation, and the drug is applied to the patient in need of treatment.

[0016] A pharmaceutically effective amount refers to a quantity sufficient to treat a disease at a reasonable benefit / risk ratio that can be achieved with the use of a drug. The level of an effective dose can be determined based on factors including the patient's disease type, severity, activity of the drug, sensitivity to the drug, time of administration, route of administration, excretion rate, treatment cycle, concurrently used drugs, and other factors well known in the medical field. The drug of the present invention can be administered as an independent therapeutic agent or in combination with other therapeutic agents. It is important to consider all of the above factors and administer the drug at the minimum dose that produces the greatest effect without side effects, which can be determined by a physician based on the patient's condition, age, etc.

[0017] Beneficial effects of the present invention: This study confirms that KRAS mutations impair the efficacy of regorafenib combined with anti-PD-1 therapy in CRCLM. Furthermore, the study discovered that dihydroartemisinin (DHA) is a potential inhibitor of KRAS mutations, effectively downregulating its expression. Most importantly, DHA can enhance the efficacy of regorafenib combined with anti-PD-1 monoclonal antibody therapy by blocking activation of the ERBB pathway and restoring interferon (IFN) response activity.

[0018] DHA has shown excellent efficacy in inhibiting mutant KRAS expression and enhancing the therapeutic effects of regorafenib and PD-1 monoclonal antibodies, with few adverse reactions such as weight loss, hepatotoxicity, and nephrotoxicity. Furthermore, DHA can be used as a broad-spectrum KRAS inhibitor to address secondary KRAS mutations that emerge during treatment.

[0019] DHA is crucial for remodeling the tumor immune microenvironment (TIME) by inhibiting mutant KRAS expression, validating the impact of low IFN response caused by KRAS mutation on T cell function, further supporting the potential mechanism by which weakened IFN response in cancer cells impairs T cell chemotaxis and cytotoxicity.

[0020] This study 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, inhibiting the expression of KRAS mutant proteins, blocking activation of the ERBB signaling pathway, restoring IFN responses, and reshaping the tumor immune microenvironment (TIME).

[0021] DHA can enhance the efficacy of regorafenib combined with PD-1 inhibitors while showing very few adverse reactions, providing a strong theoretical basis for incorporating DHA into the combination treatment of CRCLM and other KRAS mutant malignant tumors, contributing to the clinical transformation of DHA-based treatment strategies and promoting the development of precision medicine, thereby overcoming the problem of drug resistance in cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 showed that KRAS mutations lead to resistance to the combination of regorafenib and PD-1 monoclonal antibody in CRCLM, among which, (A) KRAS status of CRCLM responders and non-responders to regorafenib combined with anti-PD-1 therapy; (B) GSEA analysis based on clinical sample RNA-seq; (C) Detection of TIME signatures in patient tumor tissues using mIF; (D) Experimental procedures of the C57BL / 6J mouse CRCLM model; (E–F) Statistical analysis of liver and liver weight relative to body weight in CRCLM mouse model; (G) Western blot analysis of downstream signaling pathways (p-Erk1 / 2 and p-Akt) in tumor tissues of mouse models; (H) TIME characteristics of tumor tissues in mouse models.

[0024] Figure 2 It shows that DHA can inhibit mutant KRAS and its downstream signaling pathways, (A) Virtual screening of drugs targeting KRASG12D; (B) DHA binding site on KRASG12D; (C–D) Western blot verification of the inhibitory effect of DHA on KRAS-MUT or KRAS-WT and its downstream signaling pathways.

[0025] Figure 3 In vitro and in vivo experiments showed that DHA and regorafenib synergistically inhibited CRC, (A–B) Synergistic analysis of regorafenib and DHA in LS180 and SW480 cells; (C) Synergistic antiproliferative effects of regorafenib and DHA; (D) Synergistic pro-apoptotic effect of regorafenib and DHA; (E) Western blot validation of regorafenib and DHA synergistically inhibiting p-Erk1 / 2 and p-Akt; (F) Subcutaneous tumor in BALB / c nude mouse model; (G) Tumor weight at the time of sampling.

[0026] (H) Changes in tumor size during the experiment; (I) HE, IHC, and TUNEL staining of tumor tissues.

[0027] Figure 4 It shows that DHA can enhance the efficacy of combined treatment with regorafenib and PD-1 monoclonal antibody in CRCLM. (A) Experimental procedures for the BALB / c mouse CRCLM model; (B) Liver of BALB / c mouse model; (C) Statistical analysis of liver weight relative to body weight in the BALB / c mouse model; (D–E) KEGG and GO enrichment analysis based on RNA-seq of tumor tissues from BALB / c mouse models; (F) Western blot analysis of downstream signaling pathways (p-Erk1 / 2 and p-Akt) in tumor tissues of mouse models; (G) Heat map showing the expression of immune regulatory factors in tumor tissues (based on RNA-seq); (H) Immune cell infiltration was assessed using the CIBERSORT algorithm based on RNA-seq.

[0028] Figure 5 The adverse reactions of DHA combined with Regorafenib in the treatment of mice with subcutaneous transplanted colorectal cancer and DHA combined with Regorafenib and PD-1 monoclonal antibody in the treatment of mice with liver metastasis of colorectal cancer were shown. (A) Experimental procedures for the BALB / c nude mouse subcutaneous colorectal cancer model; (B) Body weight changes in BALB / c nude mouse model; (C) HE staining of liver and kidney of BALB / c nude mice; (D) Body weight changes in the BALB / c mouse CRCLM model; (E) HE staining of the kidney of the BALB / c mouse model.

[0029] Figure 6 The synergistic effect of regorafenib and DHA was shown, among which, (AE) Synergistic analysis of regorafenib and DHA; (F) Synergistic antiproliferative effects of regorafenib and DHA in HCT116 cells; (G) Synergistic pro-apoptotic effect of regorafenib and DHA in HCT116 cells; (H) Western blotting confirmed the synergistic inhibitory effects of regorafenib and DHA on p-Erk1 / 2 and p-Akt. DETAILED DESCRIPTION

[0030] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without inventive effort are within the scope of protection of the present invention.

[0031] Example 1 1. Clinical Sample Collection Sixteen CRCLM cases 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 patients who did not respond (KRAS-MUT), was used for transcriptome sequencing (RNA-seq). Paraffin-embedded CRCLM tissue from the patients was used for immunohistochemistry (IHC) staining or multiplex immunofluorescence (mIF) staining.

[0032] Table 1 Information of 16 patients included in molecular pathology analysis

[0033] like Figure 1 As shown in A, more KRAS mutations were detected in non-responders among the 16 cases collected.

[0034] 2. Tissue RNA Sequencing and Data Analysis 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 metastasis tumor tissue using the Trizol method. RNA sequencing was performed by BGI (Wuhan, China). Functional enrichment analysis was performed using the GO, KEGG, and GSEA algorithms. Immune infiltration was assessed using the CIBERSORT algorithm.

[0035] Three well-preserved case specimens were selected from each of the eight treatment responders and eight non-responders for batch transcriptome sequencing. Functional enrichment analysis showed that there were differences in the enrichment of certain gene pathways between the two groups, including anti-tumor immunity and cell proliferation pathways ( Figure 1 B).

[0036] By detecting TIME (tumor immune microenvironment) in situ with mIF, it was also found that responders (KRASWT ) tumor tissues had more CD8+ T cell infiltration and less CD206+ TAMs (tumor-associated macrophages) infiltration ( Figure 1 C).

[0037] 3. Cell Lines and Cell Culture 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 CT26 cells carry Kras G12D mutation, while MC38 cells are Kras WT cell.

[0038] LS180, SW480, HCT116, HT29, CT26, and MC38 cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin; Caco-2 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. All cells were cultured at 37°C in a humidified atmosphere with 5% CO2 and passaged every two to three days.

[0039] 4. Plasmid and Stable Cell Construction MC38 cells were used to construct mouse Kras G12D Overexpressing cells, pLV3-CMV-Kras G12D Puro plasmid was purchased from Miaoling Plasmid Company, China. Caco-2 and HT29 cells were used to construct human KRAS G12D Overexpressing cells, pLVX-EF1α-KRAS G12D -IRES-Puro plasmid was purchased from Beijing Lake Biotechnology Co., Ltd. Lentiviral packaging plasmids pMD2.G (#12259) and psPAX2 (#12260) were purchased from Addgene. Lentivirus was prepared by transfecting HEK293T cells with the above plasmids (at a ratio of 7:2:5) using Neofect transfection reagent. Cell infection was facilitated by using 10 μg / mL polybrene (TR-1003, Sigma-Aldrich), and positive cells were selected using puromycin (6 μg / mL for MC38 cells and 2 μg / mL for Caco-2 and HT29 cells). KRAS G12D / Kras G12DThe expression stability was verified by Sanger sequencing and western blot.

[0040] 5. Mouse Model Construction and Therapeutic Drugs Four-week-old female C57BL / 6J and BALB / c mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. The spleens of mice were injected with 1×10 6 MC38 or CT26 cells were used to establish a mouse colorectal cancer liver metastasis model ( Figure 1 D-1H, Figure 4 and Figure 5 D-5E).

[0041] Four-week-old female BALB / c nude mice were purchased from Charles River. 2×10 6 HCT116 cells (suspended in 100 μL PBS solution containing 50% Matrigel). Treatment was started when the implanted tumor volume increased to approximately 50 mm³ (tumor volume = 1 / 2 × length × width²). Figure 3 H-3I, Figure 5 A-5C).

[0042] Regorafenib (HY-10331, MedChemExpress) and DHA (HY-N0176, MedChemExpress) were administered orally with corn oil, and PD-1 monoclonal antibody (A2122, Selleck) was administered by intraperitoneal injection.

[0043] 6. Western blot Cells were lysed in RIPA lysis buffer (R0010, Solarbio) and processed as previously described. The primary antibodies are listed in Table 2. After incubation with secondary antibodies for one hour at room temperature, the membranes were washed with TBS-T, and protein levels were detected using the ImageQuant 800 system.

[0044] 7. Multiplex Immunofluorescence (mIF) Staining mIF staining was performed using a seven-color mIF kit (based on tyrosine signal enhancement technology). The experiments were performed according to the manufacturer's instructions. The primary antibodies are listed in Table 2. The captured images were analyzed using ImageJ software, and the mean fluorescence intensity (MFI) or the proportion of positive cells was estimated.

[0045] Table 2 Antibody list

[0046] 8. Determination of half maximal inhibitory concentration (IC50) The smooth surface of KRAS protein makes it difficult to be targeted by small molecules. Through the scoring and screening of 225 natural active compounds from traditional Chinese medicine, the artemisinin derivative DHA is proposed as a target for KRAS. G12D Compounds ( Figure 2 AB).

[0047] Caco-2 cells and HT29 cells are two KRAS wild-type cell lines, and KRAS G12D cells to investigate whether DHA could regulate KRAS expression.

[0048] Cells were collected at 1×10 per well 4 Cells were seeded at a density of 100 μM in 96-well plates. After cell attachment, a concentration gradient of drugs (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) were added. 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. Absorbance was measured at 450 nm using a microplate reader (Bio-Rad). IC50 values ​​were calculated using GraphPad Prism 8.0.

[0049] CRC cell lines were treated with a series of DHA concentration gradients to further determine the changes in KRAS protein. G12D The expression of KRAS WT were not affected, and its downstream pathways such as p-Erk1 / 2 and p-Akt showed similar trends ( Figure 2 C). It can be seen that DHA can also effectively inhibit LS180 (KRAS G12D )、SW480(KRAS G12V ) and HCT116 (KRAS G13D ) KRAS expression in cells ( Figure 2 D).

[0050] 9. Drug synergy analysis Based on the estimated IC50 values, a series of DHA and regorafenib concentration gradients were set up to analyze their synergistic effect. Synergy analysis was completed using the SynergyFinder platform and measured by HSA score (threshold of 10), with higher HSA scores indicating stronger synergistic effects. In multiple KRAS mutant CRC cell lines (including LS180, SW480, HCT116 and KRASG12D In mutant Caco-2 and HT29 cells), combined treatment with regorafenib and DHA showed a synergistic antiproliferative effect ( Figure 3 AB, Figure 6 AE).

[0051] 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 medium containing different drugs. After 10-15 days of incubation, the cells were fixed with 4% paraformaldehyde (PFA) and stained with crystal violet. Cell counting was performed using ImageJ software. The results of in vitro experiments of clone formation assays and apoptosis detection showed that the combination of regorafenib and DHA significantly inhibited the proliferation of LS180, SW480, and HCT116 cells compared with monotherapy ( Figure 3 C, Figure 6 F) and promotes apoptosis ( 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 combined treatment with DHA significantly reduced the phosphorylation levels ( Figure 3 E, Figure 6 H), indicating enhanced inhibitory effects downstream of KRAS mutations.

[0052] To verify the synergistic effect in vivo, an HCT116 mouse xenograft model was established. At the end of the treatment regimen, the mice were sacrificed. Both tumor size and weight indicated that the combined treatment of regorafenib and DHA had a good synergistic effect on inhibiting CRC ( Figure 3 FH).

[0053] In addition, Ki-67 immunohistochemical staining and TUNEL assays were used to evaluate the high efficacy of the combined treatment in inhibiting CRC proliferation and promoting apoptosis, respectively. At the same time, angiogenesis, as assessed by CD31 expression, was also inhibited ( Figure 3 I). The above data indicate that DHA can synergize with regorafenib in the fight against CRC by simultaneously inhibiting proliferation and promoting apoptosis, and its main mechanism is to downregulate the p-Erk1 / 2 and p-Akt signaling pathways.

[0054] 10. Treatment of Colorectal Cancer Liver Metastasis Mouse Model Using MC38 cells (Kras WT and Kras G12D ), a CRCLM mouse model was constructed and treated according to the planned regimen ( Figure 1 D). Regorafenib combined with PD-1 monoclonal antibody in patients with Kras WTThe efficacy was better in mouse models of tumors ( Figure 1 EF). In KRAS G12D In tumors, KRAS downstream signaling pathways that regulate cell growth (such as p-Erk1 / 2 and p-Akt) were not significantly inhibited by treatment ( Figure 1 G). In addition, KRAS G12D Compared with the tumor, regorafenib combined with PD-1 monoclonal antibody significantly increased KRAS WT CD8+ T cell infiltration in tumors and reduced recruitment of CD206+ TAMs ( Figure 1 H). Thus, this revealed the important role of KRAS mutation in affecting the sensitivity of CRCLM to regorafenib combined with PD-1 monoclonal antibody treatment.

[0055] The CRCLM model was established by injecting CT26 cells (KrasG12D) into the spleen of BALB / c mice and treated as planned ( Figure 4 A). At the end of the experiment, mice were euthanized and tumors were removed. It was found that the treatment effects of regorafenib combined with PD-1 monoclonal antibody or DHA alone were limited. However, when the two were used together, tumor suppression was significantly enhanced, indicating a stronger therapeutic synergy ( Figure 4 To preliminarily explore how DHA enhances the efficacy of regorafenib and PD-1 monoclonal antibody combination therapy, we performed RNA sequencing (RNA-seq) on randomly selected samples. KEGG and GO enrichment analysis showed that DHA can regulate signaling pathways related to proliferation and modulate anti-tumor immune responses ( Figure 4 Consistent with the transcriptome data, western blot results confirmed that the levels of Kras, p-Erk1 / 2, and p-Akt proteins were significantly reduced in combination with DHA, while the inhibitory effect of regorafenib alone on these pathways in vivo was still weak ( Figure 4 F). In addition, DHA treatment reduced the expression of some immunosuppressive factors (such as Tgfb1–3 and Il-10) and increased the expression of some immunostimulatory mediators (such as Cxcl9, Cxcl10, Gzmb, and Ifng) ( Figure 4 G). The TIME (tumor immune microenvironment) map depicted by the CIBERSORT algorithm showed that the infiltration of CD8+ T cells and M1 macrophages increased, while the infiltration of M2 macrophages decreased ( Figure 4H). This study demonstrates that DHA can significantly enhance the efficacy of regorafenib combined with PD-1 monoclonal antibody against CRC-LM by inhibiting the activity of proliferation-related signaling pathways and reshaping the tumor immune microenvironment. Single-cell transcriptome sequencing (scRNA-seq) further revealed that in KRAS-mutant CRC, regorafenib treatment upregulates the expression of the ERBB ligands EREG, AREG, and HBEGF, a marker of reactivation of Erk1 / 2 and Akt, ultimately leading to regorafenib resistance. Furthermore, KRAS mutations can induce immune escape by suppressing the interferon response of cancer cells, reducing the chemotaxis and cytotoxicity of CD8+ T cells. DHA, by potently inhibiting the expression of KRAS mutations, can mitigate this immune escape.

[0056] Further experiments in this study confirmed that DHA significantly regulates TIME, promoting cytotoxic and pro-inflammatory immune cell populations while suppressing immunosuppressive subpopulations. Because DHA can inhibit the expression of mutant KRAS and reshape TIME, it can enhance the efficacy of PD-1 monoclonal antibodies.

[0057] 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 modifications or substitutions that can be easily conceived by a person 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 based on the scope of protection of the claims.

Claims

1. Application of dihydroartemisinin in the preparation of KRAS inhibitors.

2. The use according to claim 1, characterized in that The inhibitor suppresses the expression of mutant KRAS.

3. Application of dihydroartemisinin in the preparation of drugs for colorectal cancer metastasis.

4. The use according to claim 3, characterized in that The medicine also includes a pharmaceutically acceptable carrier.

5. The application according to claim 4, characterized in that: The pharmaceutically acceptable carrier includes at least one of a filler, a diluent, a disintegrant, a binder, a lubricant, a glidant, a surfactant, a solvent, a flavoring agent, a stabilizer, a colorant, and a preservative.

6. The application according to claim 3, characterized in that: The medicine is an oral medicine or an injectable medicine, and the dosage form includes tablets, capsules, powders, granules, pills or solutions.

7. Use of a combined drug combination in the preparation of a drug for treating colorectal cancer, characterized in that: The combined drug combination comprises an effective amount of dihydroartemisinin, an effective amount of regorafenib and an effective amount of a PD-1 inhibitor.

8. The use according to claim 7, characterized in that The dihydroartemisinin inhibits the expression of mutant KRAS.

9. The use according to claim 7, characterized in that Colorectal cancer is colorectal cancer liver metastasis.

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