A combination for the treatment of braf v600e-mutated advanced colorectal cancer

Abstract

The application provides a combination drug for treating BRAF V600E mutant advanced colorectal cancer, and relates to the technical field of biological medicines.The effective component is composed of the following substances: Encorafenib, Cetuximab and a small molecule inhibitor of MOGAT3.The combination of the small molecule inhibitor of MOGAT3, Encorafenib and Cetuximab can effectively promote the targeted treatment effect of double-target drugs, and has synergistic effect, reverses drug resistance, and provides a reliable selection for drug resistance drugs and clinical drugs.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and more specifically, to a combination drug for treating BRAF V600E-mutant advanced colorectal cancer. Background Technology

[0002] Patients with advanced colorectal cancer who have BRAF V600E mutations have a survival time of one year. The combination therapy targeting BRAF (encoding RAF family serine / threonine protein kinases) and EGFR (Epidermal Growth Factor Receptor) is the latest standard treatment. However, the tumor develops resistance and progresses within 4.3 months after treatment. Currently, most drugs for treating these diseases are molecularly targeted drugs, which are expensive, have significant side effects, and develop resistance rapidly. There is an urgent need to further study the resistance mechanism and find strategies to reverse it and prolong patient survival. Summary of the Invention

[0003] The purpose of this application is to provide a combination drug for treating BRAF V600E-mutant advanced colorectal cancer, which has the characteristic of delaying the rapid resistance to molecularly targeted drugs.

[0004] The technical problem solved by this application is achieved by the following technical solution.

[0005] This application provides a combination drug for treating BRAF V600E-mutant advanced colorectal cancer, the active ingredients of which consist of the following substances: Encorafenib, Cetuximab and a small molecule inhibitor of MOGAT3.

[0006] In this study, researchers dynamically examined signaling pathway changes in a BRAF V600E-mutant colorectal cancer PDX model during the transition from BRAF and EGFR dual-targeted therapy to resistance. They found that MOGAT3-regulated lipid metabolism promotes resistance, and the small molecule inhibitor PF-06471553 can downregulate lipid metabolism and reverse resistance. Furthermore, the study revealed abnormal activation of lipid synthesis in dual-target resistant PDX tumors, with significantly increased expression of the monoacylglycerol acyltransferase MOGAT3 and reactivation of the MAPK signaling pathway. Knockout of MOGAT3 significantly reduced lipid accumulation in drug-resistant cells, and the cells regained sensitivity to both targets. The small molecule inhibitor PF-06471553 also reversed resistance in vivo. Therefore, the combination of small molecule inhibitors of MOGAT3 with encorafenib and cetuximab can effectively promote the targeted therapy effect of dual-target drugs, while synergistically enhancing the effect and reversing drug resistance. This provides a reliable new option for clinical use in the treatment of drug-resistant drugs.

[0007] Compared with the prior art, the embodiments of this application have at least the following advantages or beneficial effects:

[0008] This application provides a combination drug for treating BRAF V600E-mutant advanced colorectal cancer, which has the characteristic of delaying rapid resistance to molecularly targeted drugs. The combination of a small molecule inhibitor of MOGAT3 with encorafenib and cetuximab effectively promotes the targeted therapeutic effect of the dual-target drugs, while synergistically enhancing the efficacy and reversing drug resistance, providing a reliable option for addressing drug resistance and for clinical use. Attached Figure Description

[0009] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0010] Figure 1 This is a KEGG enrichment map from the screening experiment in Example 1 of this application;

[0011] Figure 2 This is a diagram showing the results of qPCR verification of metabolic pathway-related genes in the screening experiment of Example 1 of this application;

[0012] Figure 3 This is a diagram showing the immunohistochemical staining results from the screening experiment in Example 1 of this application;

[0013] Figure 4 This is a graph showing the immunohistochemical quantitative results from the screening experiment in Example 1 of this application;

[0014] Figure 5 This is a classification diagram of metabolic pathways in the screening experiment of Example 1 of this application;

[0015] Figure 6 This is a comparison chart of triglyceride content in the screening test of Example 1 of this application;

[0016] Figure 7 This is a diagram showing the staining results in the screening experiment of Example 1 of this application;

[0017] Figure 8 This is a graph showing the results of the WB test in the screening experiment of Example 1 of this application;

[0018] Figure 9 This is a graph showing the results of the metabolomics experiment in the screening test of Example 1 of this application;

[0019] Figure 10 This is a diagram showing the staining results from the in vitro experiment in Example 3 of this application;

[0020] Figure 11 This is a graph showing the cell viability test results in the in vitro experiment of Example 3 of this application;

[0021] Figure 12 This is a graph showing the level of apoptosis in the in vitro experiment of Example 3 of this application;

[0022] Figure 13 This is a graph showing the apoptosis rate in the in vitro experiment of Example 3 of this application;

[0023] Figure 14 This is a comparison chart of tumor volume in the in vivo experiment of Example 4 of this application;

[0024] Figure 15 This is a comparison chart of tumor weight in the in vivo experiment of Example 4 of this application;

[0025] Figure 16 This is a graph showing the detection of lipid levels in blood during an in vivo experiment in Example 4 of this application;

[0026] Figure 17 This is a graph showing tumor growth data in the in vivo experiment of Example 4 of this application;

[0027] Figure 18 This is a comparison chart of mouse body weight changes in the in vivo experiment of Example 4 of this application;

[0028] Figure 19 This is a diagram showing the staining results of tissue sections in the in vivo experiment of Example 4 of this application;

[0029] Figure 20 This is a graph showing the immunohistochemical quantitative results in the in vivo experiment of Example 4 of this application. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0031] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present application will now be described in detail with reference to specific embodiments.

[0032] This application provides a combination drug for treating BRAF V600E-mutant advanced colorectal cancer, which consists of the following components: Encorafenib, Cetuximab, and a small molecule inhibitor of MOGAT3.

[0033] This drug has the characteristic of delaying the rapid development of resistance to molecularly targeted drugs. Combining a small molecule inhibitor of MOGAT3 with encorafenib and cetuximab can effectively promote the targeted therapeutic effect of dual-target drugs, while synergistically reversing drug resistance, providing a reliable option for addressing drug resistance and for clinical use.

[0034] In some embodiments of this application, the small molecule inhibitor of MOGAT3 is PF-06471553.

[0035] In this study, PF-06471553 was specifically used for experimental verification. The combination of PF-06471553 with Encorafenib and Cetuximab significantly inhibited the increase of lipids in drug-resistant cells, reduced lipid droplets in drug-resistant cells, and downregulated lipid metabolism. Simultaneously, in the assessment of cell proliferation activity, it was found that the combination of dual targets with PF-06471553 could significantly inhibit the proliferation of drug-resistant cells, restore the sensitivity of drug-resistant cells, and reverse drug resistance. Furthermore, flow cytometry was used to assess the level of cell apoptosis, and it was found that the combination of dual targets with PF-06471553 (Pf) could significantly promote the apoptosis of drug-resistant cells, achieving a synergistic effect.

[0036] In some embodiments of this application, the molar concentration ratio of Encorafenib, Cetuximab, and PF-06471553 in the above-mentioned combination drug is 1:1:10.

[0037] Drugs combined in the above proportions exhibit excellent drug resistance characteristics and synergistic effects in promoting targeted therapy with dual-target drugs.

[0038] The features and performance of this application will be further described in detail below with reference to the embodiments.

[0039] Example 1

[0040] This embodiment screened for metabolites related to tumor regulation of dual-target drug-resistant advanced colorectal cancer with BRAF V600E mutation.

[0041] 1. Test materials and test methods (the same applies below)

[0042] Human specimen materials: Tumor specimens were collected from patients who underwent colorectal cancer surgery at Sir Run Run Shaw Hospital, affiliated with Zhejiang University. The excised tumor tissue specimens were rapidly frozen in liquid nitrogen and then stored at -80°C.

[0043] Lipid content in dual-target resistant and sensitive cells was determined using lipid metabolomics assays, while non-targeted metabolomics assays were performed by Megi Biotech (Shanghai, China) using LC-MS.

[0044] Cell viability and apoptosis assays: Cell viability was assessed using a CCK8 assay kit (Dojindo, Japan). Cells in the logarithmic growth phase were trypsinized, centrifuged, resuspended, and accurately counted using a cell counter. Cells were seeded at 5 × 10⁶ cells per well in 96-well plates. 3 Cells were cultured at 200 μL of complete medium per well. After 24 hours, once the cells had fully adhered, the medium was discarded, and 100 μL of complete medium containing the specified reagents was added to each well. The cells were then cultured for another 72 hours. After the culture, the medium was discarded, and freshly prepared 10% v / v water-soluble WST-8 dye was added to each well. The cells were incubated for 0.5–2 hours. The absorbance at 450 nm (OD value) was measured using a microplate reader, and cell viability was calculated. Apoptosis detection was performed using a BD (USA) kit, and the assay was performed using a BDFACSARIALL flow cytometer. Gating was used to exclude cell debris and adherent cell clusters, and 10,000 cellular events were recorded. Results were analyzed using Flowjo (Version X) software.

[0045] Nile Red, IHC, TUNEL staining, and triglyceride detection: Nile Red Staining Solution kit, TUNEL apoptosis detection kit (APPLYGEN, China); Ki67 antibody Cat No. 28074-1-AP (PROTEINTECH, China), MOGAT3 antibody DF9099 (Affnity, China). Immunohistochemical analysis was performed using the Avidin Biotin Comlex (ABC) immunohistochemical kit (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.). Peripheral blood samples were centrifuged at 3000-4000 rpm for 10 min, and the supernatant was collected for triglyceride (TG) detection using an automated biochemical analyzer (LW C400, Shenzhen Lanyun Medical Instruments). Tissue embedding and section preparation were completed in collaboration with the Department of Pathology, Sir Run Run Shaw Hospital.

[0046] 2. Test Results

[0047] 1) Transcriptomics sequencing analysis

[0048] Transcriptomic sequencing analysis was performed on BRAF V600E-mutant advanced colorectal cancer tumors that were resistant to dual-target drugs and sensitive to them, such as... Figure 1 As shown, KEGG enrichment indicates significant changes in metabolic pathways in BRAF V600E-mutant advanced colorectal cancer tumors that are resistant to dual-target drugs and sensitive tumors.

[0049] 2) qPCR validation of metabolic pathway-related genes

[0050] Genes related to metabolic pathways were validated using qPCR, such as Figure 2 As shown, the upregulation of MOGAT3 RNA levels was most pronounced in drug-resistant tissues.

[0051] 3) Immunohistochemistry

[0052] Immunohistochemical analysis was performed on BRAF V600E-mutant advanced colorectal cancer tumors that were resistant to and sensitive to dual-target drugs, such as... Figure 3 and Figure 4 As shown, the level of MOGAT3 protein in drug-resistant tissues is significantly increased.

[0053] 4) Metabolic pathway analysis

[0054] like Figure 5 As shown, changes in lipid metabolism pathways are the most pronounced among metabolic alterations. Figure 6 As shown, triglycerides were significantly higher in the drug-resistant group compared to the sensitive group.

[0055] 5) Staining of sections

[0056] like Figure 7 As shown, Nile Red staining indicates increased lipid content in drug-resistant tumor tissue.

[0057] 6) Western blot test

[0058] like Figure 8 As shown, the WB results indicated a significant increase in MOGAT3 protein in RKOEC-R cells, which are BRAF V600E mutant Encorafenib+Cetuximab resistant.

[0059] 7) Metabolomics experiments

[0060] like Figure 9 As shown, metabolomics assays revealed that the triglyceride content in dual-target resistant cells (RKO EC-R) was significantly higher than that in sensitive cells (RKO).

[0061] Based on the above experimental results, researchers found that lipid metabolism plays a crucial role in tumorigenesis and progression, but further research is needed on how lipids contribute to tumor drug resistance. Therefore, to gain a deeper understanding of the molecular mechanisms by which lipid remodeling plays a role in tumor drug resistance, this study further investigated the abnormally elevated metabolite MOGAT3 in dual-target resistant PDX tumors.

[0062] Example 2

[0063] This embodiment provides a combination drug for treating BRAF V600E-mutant advanced colorectal cancer, which contains the following components: 2 μM of Encorafenib, 2 μM of Cetuximab and 20 μM of MOGAT3 small molecule inhibitor PF-06471553, with the remainder being physiological saline.

[0064] Example 3

[0065] This embodiment verifies the combination drug provided in Example 2 through in vitro experiments.

[0066] Cell culture: The human colon cancer cell line RKO was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Cells were cultured in DMEM medium (RKO) containing 10% fetal bovine serum.

[0067] 1) Nile Red staining assay after cell treatment

[0068] Dual-targeted drug-resistant cell lines were seeded into 12-well plates and randomly divided into a control group (Vehicle), a PF-06471553(Pf) group (20 μM), an Encorafenib + Cetuximab group (2 μM Encorafenib and 2 μM Cetuximab), and a drug treatment group as described in Example 2. Each group was treated with the drugs for 48 hours. Lipid synthesis was assessed by Nile Red staining. Figure 10As shown, the dual-target combination of PF-06471553 (Pf) can significantly inhibit lipid increase in drug-resistant cells and reduce lipid droplets in drug-resistant cells.

[0069] 2) Cellular CCK8 assay

[0070] Dual-targeted drug-resistant cell lines were seeded into 96-well plates and divided into a control group (Vehicle), a PF-06471553 (Pf) group (20 μM), an Encorafenib + Cetuximab group (2 μM, with a molar ratio of Encorafenib to Cetuximab of 1:1), and a drug treatment group as described in Example 2. Each group was treated with the drugs for 96 hours. Cell proliferation was assessed using CCK8 assay. Figure 11 As shown, the combination of dual targets with PF-06471553 (Pf) can significantly inhibit the proliferation of drug-resistant cells and restore their sensitivity.

[0071] 3) Flow cytometry evaluation after cell treatment

[0072] Dual-targeted drug-resistant cell lines were seeded into 96-well plates and randomly divided into a control group (Vehicle), a PF-06471553(Pf) group (20 μM), an Encorafenib + Cetuximab group (2 μM, with a molar ratio of Encorafenib to Cetuximab of 1:1), and a drug treatment group as described in Example 2. Each group was treated with the drugs for 48 hours. After 48 hours of treatment, the level of apoptosis was assessed by flow cytometry. Figure 12 As shown, the combination of dual targets with PF-06471553 (Pf) can significantly promote apoptosis in drug-resistant cells. Figure 13 ).

[0073] In summary, considering the limited efficacy of PF-06471553 or Encorafenib + Cetuximab alone in BRAF-V600E resistant cell lines, the combined treatment with PF-06471553 significantly enhanced the efficacy of Encorafenib + Cetuximab. These data suggest that the combined use of PF-06471553 and Encorafenib + Cetuximab synergistically enhances the cytotoxic effect of Encorafenib + Cetuximab and reverses the resistance of resistant cells to the dual-target drug.

[0074] Example 4

[0075] In order to further explore whether the drugs in Example 2 can increase the sensitivity of drug-resistant tumors to dual-target drugs in the in vivo environment, a mouse xenograft tumor model PDXs of patient-derived dual-target drug-resistant tumors was constructed to evaluate the combined effect of PF-06471553 in combination with Encorafenib + Cetuximab.

[0076] 1. Construction of mouse PDX model

[0077] Surgical tumor specimens from patients with BRAF V600E-mutant colorectal cancer were obtained, and tumor fragments were subcutaneously inoculated into the groin area of ​​4-week-old female nude mice. When the tumor size reached 200 mm... 3 At this time, as a control point for the efficacy of subsequent drug administration, mice were randomly assigned to a cohort and administered the drug or carrier daily. The four groups of mice received the treatments specified in the following four categories:

[0078] The control group received normal saline, and the experimental groups (Pf group, Enc+Cet group and Enc+Cet+Pf group) were administered the same dose by gavage daily.

[0079] Pf group, PF-06471553 alone, 20 mg / kg (calculated based on mouse body weight, the same below), once daily by gavage;

[0080] The Enc+Cet group and the Encorafenib+Cetuximab group were administered at a molar concentration ratio of 1:1 (Encorafenib:Cetuximab), 20 mg / kg, once daily by gavage.

[0081] The Enc+Cet+Pf group received the drug from Example 2, namely the dual-target drug combined with PF-06471553, at a dose of 20 mg / kg, administered by gavage once daily.

[0082] Mice were sacrificed 3 weeks after treatment, and tumor size was counted and analyzed.

[0083] 2. Test Results

[0084] 1) Size and weight of mouse tumors

[0085] like Figure 14 and Figure 15 As shown, co-treatment with dual-targeted drugs in combination with PF-06471553 (Pf) significantly inhibited tumor growth, resulting in a reduction in tumor volume and weight.

[0086] 2) Lipid levels in mouse blood

[0087] After the final tumor measurement, peripheral blood was drawn from the orbital fossa to test triglyceride levels, and the results were as follows: Figure 16As shown, peripheral blood triglyceride testing revealed that TG levels were significantly reduced after co-treatment with dual-targeted drugs combined with PF-06471553 (Pf).

[0088] 3) Tumor growth data transplanted in mice

[0089] like Figure 17 As shown, compared to the control group, the tumors in the PF-06471553(Pf) treatment group were almost not inhibited; while the dual-targeted drug treatment group did not show a trend of inhibiting tumor growth; the combined use of dual-targeted drugs and PF-06471553(Pf) significantly inhibited tumor growth, showing the strongest tumor growth inhibition among the four groups. Figure 18 As shown, we found that mice tolerated the combination therapy well compared to other treatments, and the weight loss in the combination group was negligible compared to other groups.

[0090] 4) Lipid content in tumors

[0091] We also used tissue section staining to visualize intratumoral lipid content, and IHC and IF analyses to examine protein expression in each tumor group. Figure 19 and Figure 20 As shown, Ki67 and TUNEL staining revealed that tumor tissue treated with dual-targeted drugs combined with PF-06471553 showed reduced proliferation and increased apoptosis, effectively alleviating the drug resistance problem of dual-targeted drugs. Compared with the control group or the dual-targeted drug treatment group alone, the PF-06471553 (Pf) combined with dual-targeted drug treatment group significantly reduced the protein expression level of MOGAT3, reduced the expression of Ki67 proliferation index, and increased apoptosis TUNEL.

[0092] In summary, the in vivo test results confirm that the combined use of PF-06471553 (Pf) and dual-targeted drugs can also reverse drug resistance in vivo, a result consistent with the in vitro test results.

[0093] In summary, this application's embodiments delve into the specific mechanisms by which lipid metabolism promotes resistance to dual-target drugs. In a PDX animal model, the combination of dual-target drugs and a small molecule inhibitor of MOGAT3 exhibited a synergistic effect, reversing drug resistance. Therefore, providing a combination drug of dual-target drugs and a small molecule inhibitor of MOGAT3 could offer a novel treatment option for BRAF V600E-mutant advanced colorectal cancer.

[0094] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

Claims

1. A combination therapy for treating BRAF V600E-mutant advanced colorectal cancer, characterized in that, Its active ingredients consist of the following substances: Encorafenib, Cetuximab and a small molecule inhibitor of MOGAT3; the small molecule inhibitor of MOGAT3 is PF-06471553.

2. The combination drug for BRAF V600E-mutant advanced colorectal cancer according to claim 1, characterized in that, The molar ratio of Encorafenib, Cetuximab, and PF-06471553 in the combination drug is 1:1:10.

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