A pharmaceutical composition for treating colorectal cancer

Abstract

The application belongs to the field of anti-tumor, and particularly relates to a pharmaceutical composition for treating colorectal cancer. The application finds that the combination of compounds HIT211363271 and TAS-116 can significantly inhibit the proliferation, migration and glycolysis related phenotype of colorectal cancer cells, promote the apoptosis of colorectal cancer cells, and inhibit the growth of colorectal cancer organoids, and no obvious systemic toxicity is found, so the combination can be used for preparing a drug for treating colorectal cancer, and provides a new candidate scheme for treating colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of anti-tumor therapy, specifically relating to a pharmaceutical composition for treating colorectal cancer. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive system, characterized by high incidence, high risk of recurrence and metastasis, and a heavy disease burden on patients. In recent years, with population aging, changes in dietary structure, and lifestyle, the incidence and mortality rates of colorectal cancer remain high globally. Clinically, the main treatments for colorectal cancer include surgery, chemotherapy, targeted therapy, and immunotherapy. However, for patients with locally advanced, metastatic, and recurrent colorectal cancer, there are still problems such as limited efficacy, easy development of drug resistance, and poor prognosis. Therefore, developing new therapeutic drugs, optimizing combination therapy strategies, and improving treatment efficacy remain important research directions in the field of colorectal cancer.

[0003] Tumor metabolic reprogramming is one of the important biological characteristics of malignant tumors. Numerous studies have shown that colorectal cancer cells often exhibit significant abnormalities in glucose metabolism during their development and progression. Enhanced glycolysis not only provides energy for rapid tumor cell proliferation but also promotes tumor progression, metastasis, and treatment resistance by providing biosynthetic substrates, regulating redox balance, and altering the tumor microenvironment. Therefore, interventions targeting abnormal tumor glucose metabolism and related processes, and the exploration of more effective combination therapy regimens, are considered important potential directions for the treatment of colorectal cancer. Summary of the Invention

[0004] To address the aforementioned technical issues, preliminary screening of this invention revealed that compound HIT211363271 exhibits anti-colorectal cancer activity in cell phenotypes, glycolysis-related phenotypes, and organoid models. Further research showed that combining compound HIT211363271 with compound TAS-116 significantly inhibited colorectal cancer cell proliferation, migration, and glycolysis-related phenotypes, promoted apoptosis, and inhibited organoid growth, providing experimental evidence for subsequent drug development and translational research. Specifically, this includes the following: In a first aspect, the present invention provides a pharmaceutical composition for antitumor purposes, wherein the active ingredients of the pharmaceutical composition include compound HIT211363271 or a pharmaceutically acceptable salt thereof and compound TAS-116 or a pharmaceutically acceptable salt thereof; the structural formula of compound HIT211363271 is shown in formula (I) below; the structural formula of compound TAS-116 is shown in formula (II) below.

[0005] Equation (I);

[0006] Formula (II).

[0007] Preferably, in one embodiment, the ratio of compound HIT211363271 or a pharmaceutically acceptable salt thereof to compound TAS-116 or a pharmaceutically acceptable salt thereof is 1:1 based on the in vivo weight.

[0008] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipients.

[0009] Preferably, the pharmaceutical composition can be formulated into any one of the following dosage forms: tablets, granules, capsules, oral liquids, injections, or suspensions.

[0010] In a second aspect, the present invention provides the use of the pharmaceutical composition described in the first aspect in the preparation of antitumor drugs.

[0011] Preferably, the tumor is colorectal cancer.

[0012] Preferably, the pharmaceutical composition inhibits the proliferation of colorectal cancer cells.

[0013] Preferably, the pharmaceutical composition inhibits the migration of colorectal cancer cells.

[0014] Preferably, the pharmaceutical composition promotes apoptosis in colorectal cancer cells.

[0015] Preferably, the pharmaceutical composition inhibits the glycolytic phenotype of colorectal cancer cells.

[0016] Preferably, the pharmaceutical composition inhibits the growth of colorectal cancer organoids.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: (1) This invention proposes and systematically verifies the application scheme of HIT211363271 and TAS-116 in combination for the treatment of colorectal cancer.

[0018] (2) The present invention uses four groups of drugs: Vehicle, HIT211363271, TAS-116 and Combo. The results show that the combination of drugs significantly inhibits the proliferation, colony formation, migration and glycolysis of colorectal cancer cells and promotes apoptosis compared with any single drug.

[0019] (3) The present invention further confirmed in organoid models and nude mouse xenograft models that the combination therapy has a better anti-tumor effect than the single drug.

[0020] (4) The present invention also conducted safety evaluations on body weight, organs, HE and blood biochemistry, etc. The results showed that the combined use scheme did not show obvious systemic toxicity under experimental conditions and has further development potential. Attached Figure Description

[0021] Figure 1 Figure 1 shows the results of the combined action analysis of HIT211363271 and TAS-116 and its effect on the proliferation-related phenotypes of colorectal cancer cells. Among them, A is the result of the combined action analysis of the two drugs based on the ZIP model using the SynergyFinder online platform; B is the result of the cell viability change detection by CCK-8 assay; C is the result of the colony formation assay; and D is the result of the change in DNA synthesis capacity detected by EdU assay.

[0022] Figure 2 The results of the combined use of HIT211363271 and TAS-116 on the migration and apoptosis of colorectal cancer cells are shown in the figure. Among them, A is the result of the scratch assay to detect the changes in cell migration ability in each group; B is the result of Annexin V / PI flow cytometry to detect the changes in cell apoptosis in each group.

[0023] Figure 3 The results of the combined use of HIT211363271 and TAS-116 on the glycolysis-related phenotypes of colorectal cancer cells are shown in the figure. Among them, A is the result of the detection of changes in glucose consumption in each group; B is the result of the detection of changes in lactate production in each group; and C is the result of the detection of changes in ATP content in each group.

[0024] Figure 4 The results of the combined use of HIT211363271 and TAS-116 on the growth of colorectal cancer organoids are shown in the figure; the growth morphology of colorectal cancer organoids in the Vehicle group, 5-Fu group, HIT211363271 group, TAS-116 group and Combo group are shown at different time points.

[0025] Figure 5 The images show the tumor-suppressing effect and histological results of the combination of HIT211363271 and TAS-116 in a subcutaneous xenograft model of colorectal cancer. Among them, A is a schematic diagram of the animal experiment process; B is a growth curve of xenografts in nude mice in each group; C is a statistical graph of tumor weight at the experimental endpoint; D is a solid image of the dissected tumor in each group; and E is an image of the immunohistochemical detection results of the xenograft tissue.

[0026] Figure 6The following figures show the safety evaluation results of HIT211363271 combined with TAS-116 in a subcutaneous xenograft model of colorectal cancer. Among them, A is the result of weight change of nude mice in each group; B is the statistical graph of weight of heart, liver, spleen, lung and kidney; C is the result of H&E staining of major organs; and D is the result of serum ALT, AST, Cr and BUN detection. Detailed Implementation

[0027] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods used are conventional methods in the art, and the reagents, materials and instruments used are commercially available.

[0028] In this embodiment of the invention, human colorectal cancer cell lines HCT116 and Caco2, as well as normal intestinal epithelial cells HIEC, were selected. The HCT116 and Caco2 cells were mainly used for in vitro morphological experiments, functional experiments, and drug combination evaluation; the HIEC cells could be used as a reference control for normal intestinal epithelial cells.

[0029] The colorectal cancer organoids used were derived from patient tissue isolates and cultured, and stably passaged. The related research has received ethical approval (approval number 2026-054). During culture, the organoid suspension was mixed with pre-cooled matrix gel at a volume ratio of 3:7 and inoculated into preheated 24-well culture plates. The plates were incubated at 37 °C for 30 min to allow the matrix gel to solidify. Then, complete colorectal cancer organoid culture medium was added, and the plates were cultured at 37 °C with 5% CO2. The culture medium was changed every 2 days. After the organoids reached stable growth, subsequent drug treatments were performed according to the experimental design.

[0030] The HIT211363271, TAS-116, and 5-Fu were all dissolved in DMSO to prepare stock solutions, with the HIT211363271 stock solution concentration being 50 mM, the TAS-116 stock solution concentration being 10 mM, and the 5-Fu stock solution concentration being 50 mM. They can be further diluted to prepare corresponding working solutions as needed for subsequent in vitro experiments, organoid experiments, and animal experiments.

[0031] The preferred experimental animals were 4–5 week old female BALB / c-Nude nude mice, housed in an SPF-grade animal facility, used to establish a subcutaneous xenograft model and evaluate the efficacy and safety of the drug. Animal experiments were conducted after ethical approval, with ethical approval number MEC120250027.

[0032] Unless otherwise specified, the reagents and consumables used in the embodiments of this invention can all be commercially available products conventional in the art, mainly including cell culture-related reagents, cell function detection-related reagents, metabolic detection-related reagents, organoid culture-related reagents, animal drug administration solution-related reagents, and histological detection-related reagents. Specifically, cell culture-related reagents may include penicillin-streptomycin antibiotics, RPMI 1640 medium, fetal bovine serum, PBS phosphate buffer, and trypsin digestion solution; cell function detection-related reagents may include CCK-8 assay kits, crystal violet staining solution, EdU cell proliferation assay kits, and Annexin V / PI apoptosis assay kits; metabolic detection-related reagents may include glucose assay kits, lactate assay kits, and ATP assay kits; organoid culture-related reagents may include matrix gel, colorectal cancer organoid culture medium, and related auxiliary reagents; animal drug administration solution-related reagents may include DMSO, physiological saline, and PEG300; and histological detection-related reagents may include tissue fixation solution, paraffin embedding reagents, HE staining reagents, antigen retrieval solution, DAB chromogenic solution, and hematoxylin counterstain solution.

[0033] Unless otherwise specified, the experimental instruments and equipment used in the embodiments of this invention can be conventional commercial equipment in the field, including but not limited to: clean bench, cell culture incubator, inverted microscope, fluorescence microscope, flow cytometer, low temperature high speed centrifuge, microplate reader, multifunctional enzyme reader, tissue slicer and fully automated biochemical analyzer, etc.

[0034] The preferred in vivo phenotypic and functional experiments for the combination regimen of this invention are set up as follows: a solvent control group (Vehicle group), a HIT211363271 group, a TAS-116 group, and a combination group (Combo group), to evaluate the effects of single drugs and combination therapy on the proliferation, migration, apoptosis, and glycolysis-related phenotypes of colorectal cancer cells. Specifically, the CCK-8 assay is used to detect changes in cell viability; the colony formation assay and EdU assay are used to evaluate cell proliferation capacity; the scratch assay is used to evaluate cell migration capacity; Annexin V / PI double staining flow cytometry is used to evaluate the level of apoptosis; and the detection of glucose consumption, lactate production, and ATP content is used to evaluate changes in glycolysis-related phenotypes. All of the above assays can be performed according to the corresponding kit instructions or conventional methods.

[0035] To evaluate the antitumor effect of the combination therapy of this invention in vivo, a subcutaneous xenograft tumor model in nude mice was established. After tumor formation and reaching a predetermined volume, animals were randomly assigned to groups and drug administration began. The groups included a Vehicle group, a HIT211363271 group, a TAS-116 group, a Combo group, and a 5-Fu positive control group. During drug administration, the long and short diameters of the tumor were measured periodically, and the tumor volume was calculated as V = 1 / 2 × long diameter × short diameter². At the experimental endpoint, the tumor was dissected and weighed. To evaluate in vivo safety, mouse weight and general condition were recorded periodically during drug administration. At the experimental endpoint, major organs were collected for HE staining observation, and serum ALT, AST, Cr, and BUN were collected to evaluate liver and kidney function. If necessary, immunohistochemical analysis of the xenograft tissue could be performed to further evaluate the antitumor effect of the combination therapy at the tissue level.

[0036] Each in vitro experiment of this invention is preferably performed at least three times independently; animal experiments are conducted according to the predetermined sample size. Experimental data are expressed as mean ± standard deviation. Comparisons between two groups are preferably performed using a two-tailed Student's t-test, and comparisons among multiple groups are preferably performed using one-way ANOVA and appropriate post-hoc testing methods. A p-value < 0.05 is used as the criterion for statistical significance.

[0037] HIT211363271 was purchased from Shenzhen Taoshu Biotechnology Co., Ltd., manufacturer number C464-1425. According to the basic information provided by the supplier, the molecular formula of the compound is C. 24 H 29 FN4O4S has a relative molecular mass of 488.58. TAS-116 was purchased from MedChemExpress, catalog number HY-15785. According to the basic information provided by the supplier, the molecular formula of the compound is C2. 25 H 26 N8O has a relative molecular mass of 454.53.

[0038] Example 1: Analysis of the combination of HIT211363271 and TAS-116 and its effect on proliferation-related phenotypes of colorectal cancer cells. To evaluate the combined effect of HIT211363271 and TAS-116 and its influence on proliferation-related phenotypes of colorectal cancer cells, solvent control group (Vehicle group), HIT211363271 group, TAS-116 group and combination group (Combo group) were set up in HCT116 and Caco2 cells.

[0039] First, a synergistic effect analysis of the combined drug therapy was performed: cells were seeded in 96-well plates, with 1 × 10⁶ cells per well. 4Cells were collected and, after cell adhesion, were treated with TAS-116 alone, HIT211363271 alone, or a combination of both drugs. The concentration gradients of TAS-116 were set at 0, 0.05, 0.1, 0.2, 0.3, and 0.5 μM, and the concentration gradients of HIT211363271 were set at 0, 5, 10, 20, and 40 μM. The two drugs were combined in a matrix to form 30 combined treatment conditions. To eliminate solvent interference, the final concentration of DMSO was kept consistent under each treatment condition by supplementing DMSO. After 48 h of drug administration, cell viability was assessed using the CCK-8 assay, and the inhibition rate under each treatment condition was calculated. The combined drug effects were analyzed using the SynergyFinder online platform, with inhibition as the readout and baseline correction performed. The zero-interaction power (ZIP) model was used to calculate the synergistic effect score of the two drugs in combination, and a combined response heatmap was plotted.

[0040] Based on the synergistic effect analysis results of the combined drug therapy, representative effective concentration combinations of HIT211363271 and TAS-116 were further selected for subsequent phenotypic validation. Specifically, a Vehicle group, a HIT211363271 group, a TAS-116 group, and a Combo group were established. In the Vehicle group, DMSO was added as a solvent control; in the HIT211363271 group, HIT211363271 was added to a final concentration of 40 μM; in the TAS-116 group, TAS-116 was added to a final concentration of 300 nM; and in the Combo group, both HIT211363271 and TAS-116 were added to final concentrations of 20 μM and 200 nM, respectively. Proliferation-related phenotypic experiments were conducted under these conditions.

[0041] CCK-8 time curve experiment: HCT116 and Caco2 cells were seeded at 1000 cells / well in 96-well plates, with 6 replicates per group and blank wells. After cell attachment, DMSO was added to the Vehicle group as a solvent control; HIT211363271 was added to the HIT211363271 group to a final concentration of 40 μM; TAS-116 was added to the TAS-116 group to a final concentration of 300 nM; and the Combo group was simultaneously added to the HIT211363271 and TAS-116 to final concentrations of 20 μM and 200 nM, respectively. The final DMSO concentration was kept consistent across groups by supplementing with DMSO, and the final culture volume for each well was 100 μL. Cell viability was assessed on days 0, 1, 2, 3, and 4 after cell administration. Each time point was detected using an independent 96-well plate. 10 μL of CCK-8 working solution was added to each well, and the cells were incubated at 37 °C for 2 h before measuring the absorbance at 450 nm and plotting cell growth curves.

[0042] Clonogenesis assay: HCT116 and Caco2 cells were digested and prepared into single-cell suspensions, and seeded at 1000 cells / well in 6-well plates. After cell attachment, DMSO was added as a solvent control in the Vehicle group; HIT211363271 was added to a final concentration of 40 μM; TAS-116 was added to a final concentration of 300 nM; and the Combo group was simultaneously treated with HIT211363271 and TAS-116 to final concentrations of 20 μM and 200 nM, respectively. The final DMSO concentration was maintained across all groups by supplementing with additional DMSO, and the final volume of each well was 2 mL. Cells were cultured for 10–14 days, with the culture medium changed every 3 days and the corresponding drugs added concurrently. After the formation of visible clones, the culture medium was discarded, the cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet for 20 min, washed, dried and photographed, and the number of clones was counted using ImageJ software.

[0043] EdU experiment: HCT116 and Caco2 cells were mixed at 3×10⁻⁶ 4Cells were seeded per well in 24-well plates. After cell attachment, DMSO was added to the Vehicle group as a solvent control; HIT211363271 was added to the HIT211363271 group to a final concentration of 40 μM; TAS-116 was added to the TAS-116 group to a final concentration of 300 nM; and the Combo group was treated with both HIT211363271 and TAS-116 to final concentrations of 20 μM and 200 nM, respectively. The final DMSO concentration was maintained consistent across all groups by adding more DMSO, and the final culture volume for each well was 1 mL. After culturing for 48 h, EdU incorporation was detected according to the kit instructions. Specifically, after incubating with EdU working solution for 2 h, the culture medium was discarded, the solution was fixed with 4% paraformaldehyde for 15 min, permeabilized with permeabilizing solution for 15 min, and then incubated with click reaction solution at room temperature in the dark for 40 min. Hoechst 33342 was then added and incubated at room temperature in the dark for 10 min. Finally, the results were observed under an inverted fluorescence microscope and the EdU positivity rate was calculated using ImageJ software.

[0044] The results are as follows Figure 1 As shown, HIT211363271 and TAS-116 exhibited a synergistic effect in HCT116 and Caco2 cells. Further phenotypic experiments revealed that both the HIT211363271 and TAS-116 groups inhibited colorectal cancer cell proliferation to varying degrees compared to the Vehicle group, with the Combo group showing a more significant inhibitory effect; clonogenic and EdU assays also showed consistent results. These results indicate that the combination of HIT211363271 and TAS-116 significantly inhibits colorectal cancer cell proliferation compared to either drug alone.

[0045] Example 2: Effects of the combination of HIT211363271 and TAS-116 on the migration and apoptosis of colorectal cancer cells. To evaluate the effects of the combination of HIT211363271 and TAS-116 on the migration and apoptosis of colorectal cancer cells, the grouping and drug treatment conditions were the same as in Example 1, and the results were detected by scratch assay and Annexin V / PI double staining flow cytometry.

[0046] Scratch assay: HCT116 and Caco2 cells were subjected to a 1×10⁻⁶ scratch test. 6Cells were seeded per well in 6-well plates and cultured at 37 °C in a 5% CO2 incubator until confluence reached 90% or higher. After vertical scratching with a sterile pipette tip, cells were gently washed with PBS to remove detached cells. Culture medium containing 1% serum was then added for further incubation. For the Vehicle group, DMSO was added as a solvent control; for the HIT211363271 group, HIT211363271 was added to a final concentration of 40 μM; for the TAS-116 group, TAS-116 was added to a final concentration of 300 nM; and for the Combo group, both HIT211363271 and TAS-116 were added to final concentrations of 20 μM and 200 nM, respectively. The final DMSO concentration was maintained across all groups by supplementing with DMSO, and the final culture volume for each well was 2 mL. Images were acquired at 0 h, 24 h, and 48 h. The scratch area was measured using ImageJ software, and the scratch healing rate was calculated using the following formula: Scratch healing rate (%) = [(0 h scratch area - scratch area at each time point) / 0 h scratch area] × 100%.

[0047] Apoptosis experiment: HCT116 and Caco2 cells were cultured at 6 × 10⁻⁶ cells per cell line. 5 Cells were seeded per well in 6-well plates and cultured at 37 °C in a 5% CO2 incubator for 24 h before drug treatment. The Vehicle group received DMSO as a solvent control; the HIT211363271 group received HIT211363271 to a final concentration of 40 μM; the TAS-116 group received TAS-116 to a final concentration of 300 nM; and the Combo group received both HIT211363271 and TAS-116 to final concentrations of 20 μM and 200 nM, respectively. The final DMSO concentration was maintained across all groups by supplementing with DMSO, and the final culture volume for each well was 2 mL. After 48 h of treatment, cells were collected from all groups, washed with pre-cooled PBS, resuspended in 1× Binding Buffer according to the kit instructions, and incubated with Annexin V Alexa Fluor APC and PI staining solution for 15 min in the dark. Immediately afterwards, flow cytometry was used for detection, collecting at least 10,000 valid cell events from each group, and the data were analyzed using appropriate analysis software. The sum of the early apoptosis rate and the late apoptosis rate was used as the total apoptosis rate.

[0048] The results are as follows Figure 2As shown, compared with the Vehicle group, both the HIT211363271 group and the TAS-116 group reduced the migration rate of colorectal cancer cells and increased the total apoptosis rate, while the Combo group showed a stronger inhibitory effect on migration and a more significant pro-apoptotic effect. These results indicate that the combination of HIT211363271 and TAS-116 can more significantly inhibit the migration of colorectal cancer cells and promote their apoptosis than either drug alone.

[0049] Example 3: Effect of combined use of HIT211363271 and TAS-116 on glycolysis-related phenotypes in colorectal cancer cells To evaluate the effect of the combination of HIT211363271 and TAS-116 on glycolysis-related phenotypes of colorectal cancer cells, the grouping and drug treatment conditions were the same as in Example 1, and the glucose consumption, lactate production and ATP content of cells in each group were detected.

[0050] HCT116 and Caco2 cells were mixed at a rate of 6 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates and cultured at 37 ℃ in a 5% CO2 incubator. After cell adhesion, the cells were treated with the following drugs: DMSO was added to the Vehicle group as a solvent control; HIT211363271 was added to the HIT211363271 group to a final concentration of 40 μM; TAS-116 was added to the TAS-116 group to a final concentration of 300 nM; and the Combo group was treated with both HIT211363271 and TAS-116 to final concentrations of 20 μM and 200 nM, respectively. The final DMSO concentration was maintained consistent across all groups by adding DMSO, and the final culture volume for each well was 2 mL. After 48 h of treatment, the cell culture supernatant was collected for glucose and lactate detection, and cells were also collected and lysed for ATP detection. The culture supernatant was diluted as needed to the detection range of the kits, and the glucose and lactate content in the culture supernatant was detected using glucose and lactate detection kits, respectively. Glucose consumption was calculated based on the difference between the initial glucose concentration in the blank culture medium and the residual glucose concentration in the cell culture supernatant. Lactate production was calculated based on the difference between the lactate concentration in the cell culture supernatant and the initial lactate concentration in the blank culture medium. Intracellular ATP levels were detected using an ATP assay kit and normalized to the total protein content.

[0051] The results are as follows Figure 3 As shown, compared with the Vehicle group, both the HIT211363271 group and the TAS-116 group reduced glucose consumption, lactate production, and ATP levels, with the Combo group showing the greatest decrease. These results indicate that the combination of HIT211363271 and TAS-116 can more significantly inhibit the glycolytic phenotype of colorectal cancer cells than either drug alone.

[0052] Example 4: Effect of the combination of HIT211363271 and TAS-116 on the growth of colorectal cancer organoids After colorectal cancer organoids were cultured to full expansion and stable growth as described above, they were treated with drug-containing medium prepared using complete colorectal cancer organoid culture medium according to the experimental design. The experiment included four groups: Vehicle, 5-Fu, HIT211363271, TAS-116, and Combo. The Vehicle group received DMSO as a solvent control; the HIT211363271 group received HIT211363271 at a final concentration of 40 μM; the TAS-116 group received TAS-116 at a final concentration of 300 nM; the Combo group received both HIT211363271 and TAS-116 at final concentrations of 20 μM and 200 nM, respectively; and the 5-Fu positive control group received 5-Fu at a final concentration of 20 μM. To eliminate the influence of solvents, the final solvent concentrations were kept consistent across all groups. At 0, 24, 48, 72 and 96 h after treatment, the morphological changes, growth status and survival of organoids were observed and recorded using an inverted microscope to evaluate the effects of different drug treatments on the growth of colorectal cancer organoids.

[0053] The results are as follows Figure 4 As shown, compared with the Vehicle group, both the HIT211363271 group and the TAS-116 group inhibited organoid formation and growth to varying degrees, while the Combo group showed a more significant inhibitory effect on organoid morphology and overall growth status; the 5-Fu positive control group also showed an inhibitory effect. These results indicate that the combination of HIT211363271 and TAS-116 also has superior anti-colorectal cancer effects at the organoid level compared to single-agent therapy.

[0054] Example 5: Antitumor effect and histological examination of HIT211363271 combined with TAS-116 in a subcutaneous xenograft model of colorectal cancer. To evaluate the antitumor effect of the combination therapy of this invention in vivo, a subcutaneous xenograft tumor model was established in nude mice. HCT116 cells were prepared into a single-cell suspension, at a concentration of 3.0 × 10⁶ cells per mouse. 6One cell line, 150 μL in volume, was subcutaneously inoculated into the right back of BALB / c nude mice. After tumor formation, drug administration began on day 10 post-inoculation. Four groups were established: Vehicle, 5-FU, HIT211363271, TAS-116, and Combo, with five mice in each group. All groups received intraperitoneal injection, once every 3 days for a total of 6 administrations. The drug solution for each group was prepared using 5% DMSO, 40% PEG300, and 55% physiological saline, with a total injection volume of 100 μL per mouse per administration. The HIT211363271 group was administered HIT211363271 at 20 mg / kg; the TAS-116 group was administered TAS-116 at 20 mg / kg; the 5-FU group was administered 5-FU at 20 mg / kg; the Combo group was administered both HIT211363271 at 10 mg / kg and TAS-116 at 10 mg / kg, and the two drugs were mixed and administered intraperitoneally in a single injection; the Vehicle group received the same volume of blank drug solution as the other drug groups, which consisted of 5% DMSO, 40% PEG300, and 55% physiological saline, without the active ingredient. Tumor volume was measured periodically during the administration period, and the general condition of the mice was recorded. Tumor volume was calculated using the following formula: TV = 0.5 × a × b², where a is the longest diameter of the tumor and b is the shortest diameter of the tumor. Mice were sacrificed 48 h after the last administration, and the tumors were dissected, weighed, and photographed.

[0055] Further immunohistochemical analysis was performed on the transplanted tumor tissue. Tumor tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and then sectioned to a thickness of 4 μm. After dewaxing, hydration, antigen retrieval, and blocking, the sections were incubated overnight at 4°C with the corresponding primary antibody. The next day, after washing with PBS, the sections were incubated at room temperature for 30 min with the corresponding secondary antibody, developed using DAB staining, and counterstained with hematoxylin. The sections were dehydrated, cleared, mounted, and observed and images were acquired under a microscope. The detected indicators included Ki67, Cleaved-caspase-3, LDHA, E-cadherin, and β-catenin.

[0056] The results are as follows Figure 5As shown in the middle AE, compared with the Vehicle group, both the HIT211363271 group and the TAS-116 group inhibited tumor growth, while the Combo group showed more significant inhibition of tumor volume growth and a more significant decrease in tumor weight at the endpoint. Further immunohistochemical analysis of the transplanted tumor tissue revealed that the combination group significantly reduced the expression of Ki67, LDHA, and β-catenin, and increased the expression of Cleaved-caspase-3 and E-cadherin compared to the single-drug groups. These results indicate that the combination of HIT211363271 and TAS-116 also has a superior tumor-suppressive effect than single-drug therapy in vivo, and exhibits better inhibition of proliferation, pro-apoptosis, and regulation of metabolism / invasion-related molecules at the tissue level.

[0057] Example 6: Safety evaluation of HIT211363271 used in conjunction with TAS-116 Based on Example 5, the safety of each group of mice was evaluated. The safety evaluation included weight monitoring, weighing of major organs, H&E staining, and detection of serum ALT, AST, Cr, and BUN. Specifically, during the animal administration period, mouse weight was recorded every 3 days, and general conditions such as mental state, feeding, activity, and fur condition were observed. At the experimental endpoint, major organs such as the heart, liver, spleen, lungs, and kidneys were collected, surface connective tissue was removed, and the organs were weighed and fixed in 4% paraformaldehyde fixative for subsequent H&E staining observation. Simultaneously, at the experimental endpoint, blood samples were collected using the orbital blood sampling method, and serum was collected after centrifugation for the detection of ALT, AST, Cr, and BUN.

[0058] The results are as follows Figure 6 As shown in the results of AD, the overall body weight of mice in each group remained stable, and no significant abnormal changes were observed in the weight of major organs. H&E staining results showed no significant pathological damage to major organs, and no significant abnormal changes were observed in blood biochemical indicators related to liver and kidney function. This suggests that the combination therapy described in this invention did not show significant systemic toxicity under experimental conditions.

[0059] This invention verified the feasibility of combining HIT211363271 and TAS-116 for the treatment of colorectal cancer through in vitro experiments, organoid experiments, and in vivo animal experiments. The results showed that the combination regimen significantly inhibited the proliferation, migration, and glycolysis-related phenotypes of colorectal cancer cells compared to single-agent therapy, promoted apoptosis, and exhibited superior tumor-suppressive effects in organoid and nude mouse xenograft models; simultaneously, no significant systemic toxicity was observed under the experimental conditions. Therefore, the combination technology provided by this invention can be used for the development of combination therapies for colorectal cancer, pharmaceutical compositions, and related translational products, and has promising prospects for industrial application.

Claims

1. A pharmaceutical composition for antitumor purposes, characterized in that, The active ingredients of the pharmaceutical composition include compound HIT211363271 or a pharmaceutically acceptable salt thereof and compound TAS-116 or a pharmaceutically acceptable salt thereof; the structural formula of compound HIT211363271 is shown in formula (I) below; the structural formula of compound TAS-116 is shown in formula (II) below; Equation (I); Formula (II).

2. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or excipients.

3. The pharmaceutical composition according to claim 2, characterized in that, The pharmaceutical composition can be formulated into any of the following dosage forms: tablets, granules, capsules, oral liquids, injections, or suspensions.

4. The use of the pharmaceutical composition according to any one of claims 1-3 in the preparation of an antitumor drug.

5. The application as described in claim 4, characterized in that, The tumor is colorectal cancer.

6. The application as described in claim 5, characterized in that, The pharmaceutical composition inhibits the proliferation of colorectal cancer cells.

7. The application as described in claim 5, characterized in that, The pharmaceutical composition inhibits the migration of colorectal cancer cells.

8. The application as described in claim 5, characterized in that, The pharmaceutical composition promotes apoptosis in colorectal cancer cells.

9. The application as described in claim 5, characterized in that, The pharmaceutical composition inhibits the glycolytic phenotype of colorectal cancer cells.

10. The application as described in claim 5, characterized in that, The pharmaceutical composition inhibits the growth of colorectal cancer organoids.

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