Use of chrysosplenetin in combination with 5-fluorouracil in the preparation of drugs against colorectal cancer

Abstract

The application relates to application of a combination of populosidone and 5-fluorouracil in preparation of colorectal cancer drugs. The populosidone and 5-fluorouracil (5-FU) are combined to prepare a medicinal composition, the populosidone can greatly improve the inhibition capacity of 5-FU on colorectal cancer tumor cells, the combination has a synergistic effect and improves the sensitivity, the inhibition effect on tumors can be achieved under lower-dose 5-FU, and the combination does not have a synergistic inhibiting proliferation reaction on human normal colon epithelial cells.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of salinomycin combined with 5-fluorouracil in the preparation of anti-colorectal cancer drugs. Background Technology

[0002] Aspergillus tinctoriae, a compound derived from the traditional Chinese medicines Artemisia scoparia and Alpinia oxyphylla. Alpinia oxyphylla Natural coumarin compounds extracted from plants such as *Miq.* have attracted attention due to their wide range of biological activities. Existing non-clinical studies (in vitro and animal experiments) have shown that salicornin possesses various pharmacological effects, including anti-inflammatory, antioxidant, hepatoprotective, and immunomodulatory properties.

[0003] Colorectal cancer is one of the leading causes of cancer-related deaths worldwide. Pyrimidine antimetabolites, particularly 5-fluorouracil (5-FU), have been cornerstone chemotherapy regimens for colorectal cancer since their introduction in the 1950s, used for adjuvant therapy, neoadjuvant therapy, and palliative care in advanced stages. They exert their anti-tumor effects by inhibiting thymidine synthase (TS) and misincorporation into nucleic acids, thus interfering with DNA synthesis and repair in tumor cells.

[0004] However, the clinical application of 5-FU has long faced two major bottlenecks, severely limiting its efficacy and impacting patients' quality of life: First, inherent or acquired resistance. A significant proportion of patients are initially insensitive to 5-FU (inherent resistance) or gradually develop resistance during treatment (acquired resistance), leading to treatment failure. The mechanism is complex, involving multiple factors such as TS overexpression, DNA mismatch repair dysfunction, and blocked apoptosis pathways. Second, significant dose-limiting toxicity. 5-FU also has a killing effect on rapidly proliferating normal tissues, often leading to myelosuppression (neutropenia, thrombocytopenia), severe gastrointestinal mucositis (stomatitis, diarrhea), hand-foot syndrome, and other toxic side effects. These toxicities force treatment interruption or dose reduction, affecting efficacy and even endangering patients' lives.

[0005] To overcome the aforementioned bottlenecks, a "sensitization and toxicity reduction" strategy is often adopted in clinical practice. Regarding "sensitization," a common practice is to combine 5-FU with other chemotherapy drugs with different mechanisms of action (such as oxaliplatin and irinotecan) (e.g., the FOLFOX and FOLFIRI regimens), or with targeted drugs (such as bevacizumab and cetuximab). While these combination regimens improve efficacy to some extent, they often involve an additive or altered toxicity profile, and the overall toxic side effects are not reduced, sometimes even becoming more complex and severe. Regarding "toxicity reduction," apart from using conventional supportive care drugs (such as antiemetics and white blood cell boosters), there is a lack of ideal drugs that can specifically counteract the toxicity of 5-FU at the pathophysiological level.

[0006] The prior art (patent application number: CN202510597333.5, a drug for treating solid tumors and its application) mentions that F1 / F3 combined with 5-FU can synergistically inhibit the proliferation of solid tumor cells, providing a novel drug for the treatment of solid tumors, but does not involve the sensitizing and detoxifying effects of 5-FU.

[0007] Prior art (patent application number: CN202110525771.2, Application of uricase in the preparation of antitumor drug sensitizers) mentions that the combined use of uricase and 5-FU has a significantly better inhibitory effect on cancer cells than 5-FU treatment alone, indicating that uricase can be used to prepare antitumor drug sensitizers. Prior art (patent application number: Application of KPT-330 in combination with 5-FU or oxaliplatin in anti-gastric cancer drugs) mentions that the combined use of KPT-330 with oxaliplatin and 5-FU in gastric cancer, compared with the single-drug treatment group, can promote tumor cell apoptosis to improve chemotherapy sensitivity and improve resistance to oxaliplatin and 5-FU. None of the above patents involve the attenuation of the toxicity of 5-FU.

[0008] The prior art (patent application number: CN202210170225.6, Use of PLX3397 in the Treatment of Colorectal Cancer) mentions that the present invention has found through research that oral administration of PLX3397 can significantly inhibit the malignant proliferation of subcutaneous xenografts of colorectal cancer and significantly reduce liver metastasis of colorectal cancer. Furthermore, the combined use of PLX3397 and 5-FU can have a synergistic effect. This suggests that PLX3397 can be formulated into a drug for treating colorectal cancer, or for inhibiting the proliferation of colorectal cancer cells, or for reducing liver metastasis of colorectal cancer cells. Simultaneously, PLX3397 has low toxicity and side effects in the treatment of colorectal cancer, thus providing a new approach and direction for the treatment of colorectal cancer, and has significant application value. Although the low toxicity and side effects of PLX3397 are mentioned, and the synergistic effect of its combination with 5-FU is mentioned, it does not address whether the combination of the two can reduce the toxic effects of 5-FU. Summary of the Invention

[0009] To address the long-standing problems in the clinical application of the pyrimidine antimetabolite 5-fluorouracil (5-FU) in the prior art, this invention provides the application of salinomycin combined with 5-fluorouracil in the preparation of an anti-colorectal cancer drug; the molar concentration ratio of salinomycin and 5-fluorouracil used in combination is 1:1; the colorectal cancer is derived from human colorectal cancer cells HCT116 and LoVo; salinomycin, alone or in combination with other drugs, with the addition of excipients, is formulated into an anti-tumor drug preparation.

[0010] The advantages of this invention are: 1. The combined use of drugs can enhance the inhibitory effect of 5-FU on tumor cells. The combination has a synergistic effect and improves sensitivity. The tumor-inhibiting effect can be achieved at a lower dose of 5-FU. Therefore, the salicylic acid of this invention can enhance the inhibitory effect of 5-FU on tumors.

[0011] 2. The combined use of these drugs does not synergistically inhibit the proliferation of normal human colonic epithelial cells NCM460.

[0012] 3. The combination of drugs significantly inhibits the growth of transplanted tumors. Not only does it have no significant effect on the weight of mice, but the salicornin can also partially alleviate the increase of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) caused by 5-FU, as well as the decrease of neutrophils. Therefore, the salicornin of the present invention can reduce the toxicity of 5-FU in vivo for the chemotherapy of colorectal cancer. Attached Figure Description

[0014] Figure 1 This is a diagram showing the toxicity experiment of salicylic acid and 5-FU on human colorectal cancer cells (SRB). Figure 2 A graph showing the drug combination index (CI) and synergistic effect score of salicornin and 5-FU on human colorectal cancer cells; Figure 3 This is a dose-response curve of salicylic acid and 5-FU on human colorectal cancer cells. Figure 4 This is a diagram showing the toxicity of salicylic acid and 5-FU to normal human colonic epithelial cells (SRB). Figure 5 The drug combination index (CI) of salicylic acid and 5-FU on normal human colonic epithelial cells is shown in the figure. Figure 6 Figure showing the body weight of mice in an in vivo tumor transplantation experiment using salicornin and 5-FU. Figure 7 This is an image of a mouse tumor in an in vivo experiment using salicornin and 5-FU to treat xenografted tumors in mice. Figure 8The graph shows the tumor growth curves of mice in vivo xenografts in an experiment involving salicornin and 5-FU. Figure 9 Image showing the tumor weight of mice in an in vivo xenograft experiment using salicornin and 5-FU. Figure 10 The graph shows the serum aspartate aminotransferase (AST) content in mice with xanthocyanin and 5-FU in vivo xanthocyanidins. Figure 11 The graph shows the serum alanine aminotransferase (ALT) levels in mice with xanthocyanin and 5-FU in vivo xanthocyanidins. Figure 12 This is a graph showing the effect of salicylic acid and 5-FU on the serum neutrophil content of mice with xenograft tumors in vivo. Detailed Implementation

[0015] To provide a more detailed description of the present invention, the following embodiments are provided for further explanation. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0016] Cell and drug sources: HCT116: CL-0096, Wuhan Punosei Life Science Technology Co., Ltd. LoVo: CL-0144, Wuhan Punosei Life Science Technology Co., Ltd. NCM460: BNCC339288, Beina Biotechnology Sausage flavonoids (T3S1775, Taoshu Biotechnology) 5-FU (T0984, Taoshu Biotechnology) Example 1

[0017] Experiments on the effects of flavonoids combined with 5-FU on human colorectal cancer cells and normal human colonic epithelial cells. SRB cytotoxicity assay Human colorectal cancer cells in logarithmic growth phase, HCT116 (3000 cells / 100 μL / well) and LoVo (3000 cells / 100 μL / well), were digested and seeded in 96-well plates. Different concentrations of ascorbic acid (0.625 μM, 1.25 μM, 2.5 μM, 5.0 μM, 10 μM, 20 μM), 5-FU (0.625 μM, 1.25 μM, 2.5 μM, 5.0 μM, 10 μM, 20 μM) and the Comb group (0.625 μM+0.625 μM, 1.25 μM+1.25 μM, 2.5 μM+2.5 μM, 5.0 μM+5.0 μM, 10 μM+10 μM, 20 μM+20 μM) were used. After 72 hours of administration of the drug alone or in combination, the plate was fixed with pre-cooled 50% TCA (final concentration 10%) for 1 hour. The fixative was discarded, the plate was washed and air-dried, stained with 0.4% SRB dye for 10 minutes, the dye was discarded, the plate was washed with 1% acetic acid solution, air-dried, dissolved in 10 mM unbuffered Tris alkaline solution, placed on a micro-shaker for 10 minutes, and the absorbance at OD515nm was detected by an ELISA reader. The effect of the drug on the survival rate of tumor cells was calculated.

[0018] Human colorectal cancer cells in logarithmic growth phase, HCT116 (3000 cells / 100 μL / well) and LoVo (3000 cells / 100 μL / well), were digested and seeded in 96-well plates. Cells were treated with a fixed concentration of ascorbic acid (1.5 μM) and different concentrations of 5-FU (0.008, 0.04, 0.2, 1, 5, 25 μM) alone or in combination for 72 hours. After treatment, pre-cooled 50% TCA (final concentration 10%) was added for fixation for 1 hour. The fixative was discarded, the plates were washed and air-dried, stained with 0.4% SRB dye for 10 minutes, the dye was discarded, the plates were washed with 1% acetic acid solution, air-dried, dissolved in 10 mM unbuffered Tris alkaline solution, and placed on a micro-shaker for 10 minutes. The absorbance at OD515 nm was measured using a microplate reader, and the effect of the drugs on tumor cell survival was calculated.

[0019] Human normal colonic epithelial cells (NCM460) in logarithmic growth phase (3000 cells / 100 μL / well) were digested and seeded in 96-well plates. Different concentrations of ascorbic acid (0.625 μM, 1.25 μM, 2.5 μM, 5.0 μM, 10 μM, 20 μM), 5-FU (0.625 μM, 1.25 μM, 2.5 μM, 5.0 μM, 10 μM, 20 μM), and the Comb group (0.625 μM+0.625 μM, 1.25 μM+1.25 μM, 2.5 μM+2.5 μM, 5.0 μM+5.0 μM, 10 μM+10 μM, 20 μM+20 μM) were administered alone or in combination. After 72 hours, pre-cooled 50%... Fix with TCA (final concentration 10%) for 1 hour, discard the fixative, wash the plate and air dry, stain with 0.4% SRB dye for 10 minutes, discard the dye, wash the plate with 1% acetic acid solution, air dry, dissolve in 10 mM unbuffered Tris alkaline solution, place on a micro-shaker for 10 minutes, and detect the OD515nm absorbance value with an ELISA reader to calculate the effect of the drug on the survival rate of normal human colonic epithelial cells.

[0020] Combination Index (CI) and Synergy Score of Drug Combinations Based on the results of the previous SRB cytotoxicity experiment, 1) the Chou-Talalay method (http: / / www.combosyn.com / ) was used to calculate the drug combination index (CI). The Chou-Talalay method can quantitatively analyze the interaction of two or more drugs using the CI index. The CI formula is: CI = d1 / D1 + d2 / D2 (D1 and D2 are the doses that produce an effect when the drugs act alone, and d1 and d2 are the equivalent doses when the drugs act together). When CI > 1, it indicates that the drug combination produces an antagonistic effect; CI = 1 indicates an additive effect; and CI < 1 indicates a synergistic effect. 2) The SynergyFinder program was used to perform drug combination synergistic analysis. The cell viability of cells treated with single and combined drugs was entered into the SynergyFinder online tool (https: / / synergyfinder.fimm.fi / v3 / ) in matrix format, and the HSA and BLISS models were used to analyze the synergistic score of the drug combination.

[0021] Experimental study on the effects of 5-FU enhanced by salicylic acid on colorectal cancer xenografts. Xenograft mouse experiment HCT116 cell lines in the logarithmic growth phase were used to prepare cells with a density of 102. 7Cells were suspended in PBS at a concentration of 100 µL / mL. In a clean bench for animal experiments, 16 BALB / c nude mice (4-6 weeks old) were subcutaneously injected into both hind limbs. The mice were divided into six groups of four: control (Ctrl), salicylic acid (10 mg / kg / d, ip) (TEC), 5-fluorouracil (20 mg / kg / tid, ip) (5-FU), and salicylic acid + 5-FU (Comb). The administration period was 20 days, during which tumor volume, mouse weight, and overall health were observed. On day 21, the animals were euthanized by CO2 inhalation. Tumors were dissected, photographed, and weighed. Peripheral blood was collected from the mice; 100 µL was used to detect neutrophil count, and the remainder was used to collect serum for AST and ALT detection.

[0022] AST and ALT detection Whole blood samples were collected from mice, allowed to coagulate at room temperature, and then centrifuged to separate serum. Serum samples were stored at −80℃ for later use. Alanine aminotransferase (ALT) (catalog number C009-2-1, Nanjing Jiancheng Biotechnology Institute) and aspartate aminotransferase (AST) assay kits (catalog number C010-2-1, Nanjing Jiancheng Biotechnology Institute) were used, strictly following the kit instructions. Absorbance values ​​were measured using a microplate reader, and ALT and AST activities were calculated. Results are expressed as U / L.

[0023] neutrophil count Peripheral blood (50–100 μL) was collected from nude mice and placed in EDTA anticoagulant tubes. Red blood cells were lysed using ACK erythrocyte lysis buffer (catalog number 00-4333-57, eBioscience). Subsequently, a mouse neutrophil flow cytometry kit (catalog number 19762, STEMCELL) was used, with fluorescently labeled anti-mouse CD11b (catalog number 60001, STEMCELL) and Ly6G (catalog number 60031, STEMCELL) antibodies added, and incubated at 4°C in the dark for 30 minutes. After washing, flow cytometry was performed. Neutrophils were defined as CD11b⁺Ly6G⁺ cells. The absolute neutrophil count (K / µL) was calculated by multiplying the total white blood cell count obtained from a complete blood count by the proportion of neutrophils obtained from flow cytometry.

[0024] Experimental results Experimental results of the combined use of flavonoids and 5-FU on human colorectal cancer cells and normal human colonic epithelial cells SRB cytotoxicity assay results The experimental data were processed according to the following steps: (1) The median absorbance value of the blank control group was used as the background value; columns 10 and 11 were used as negative control wells (Control). (2) The original absorbance values ​​of each experimental well and negative control well were subtracted from the background value to obtain the corrected absorbance values. (3) The average value of the corrected absorbance values ​​of the negative control group was used as the benchmark (defined as 100% cell viability), and the cell survival rate of each drug treatment group was calculated according to the following formula:

[0025] OD 实验孔 The optical density values ​​of the pores in the experimental group reflect the light absorption of the cells after the experimental treatment.

[0026] OD 空白孔 : Optical density values ​​of the blank control group wells, used to subtract background interference (such as the absorbance of the culture medium and reagents themselves). OD 阴性对照孔 The optical density value of the negative control well represents the light absorption level of cells without experimental treatment (or only under negative treatment), serving as a baseline reference for cell viability.

[0027] Human colorectal cancer cells HCT116 and LoVo were treated with different concentrations of ethylcarboxylic acid (TEC) and 5-FU. The cell viability was then assessed using an SRB toxicity assay after obtaining 96-well plate readings of HCT116 and LoVo cells. C1 was used as the background value to calculate the cell viability. The results are shown in Tables 1 to 7 below. Figure 1 As shown; Table 1. HCT116 cell readings in 96-well plates

[0028] Table 2 Corrected absorbance of HCT116 cells under different drug groups and concentrations

[0029] Table 3. Cell viability of HCT116 cells under different drug groups and concentrations.

[0030] Table 4. LoVo cell readings in 96-well plates

[0031] Table 5 Corrected absorbance of LoVo cells under different drug groups and concentrations

[0032] Table 6. Cell viability of LoVo cells under different drug groups and concentrations.

[0033] Table 7 IC values ​​for HCT116 and LoVo 50

[0034] The results showed that the combination of drugs could enhance the inhibitory effect of 5-FU on tumor cells.

[0035] In human colorectal cancer cells HCT116 and LoVo, cells were treated with a fixed concentration of ascorbic acid and different concentrations of 5-FU. The cell viability was then assessed using an SRB toxicity assay after obtaining 96-well plate readings of HCT116 and LoVo cells. Using E4 as the background value, cell viability was calculated. The results are shown in Tables 8 to 11 below. Figure 3 As shown; Table 8. Readings of LoVo cells and HTC116 cells in 96-well plates.

[0036] Table 9 Corrected absorbance of LoVo cells and HTC116 cells under different drug groups and concentrations.

[0037] Table 10 Cell viability of LoVo cells and HTC116 cells under different drug groups and concentrations

[0038] Table 11 IC values ​​for HCT116 and LoVo 50

[0039] The results showed that 5-FU had an effect on the IC50 of HCT116 and LoVo cells. 50 The values ​​decreased by 16.8 times and 25.6 times, respectively, indicating that the combination therapy has a significant sensitizing effect and can achieve tumor inhibition at a low dose of 5-FU.

[0040] Human normal colonic epithelial cells NCM460 were treated with different concentrations of ethylcarboxypic acid (TEC) and 5-FU. The cell viability was then determined using an SRB toxicity assay after obtaining 96-well plate readings of NCM460 cells. B1 was used as the background value to calculate the cell viability. The results are shown in Tables 12 to 15 below. Figure 4 As shown in Table 12; NCM460 cell 96-well plate reading table

[0041] Table 13 Corrected absorbance of NCM460 cells under different drug groups and concentrations

[0042] Table 14. Cell viability of NCM460 cells under different drug groups and concentrations.

[0043] Table 15 NCM460 ICs 50

[0044] The results showed that salicornin did not synergistically inhibit its proliferation with 5-FU.

[0045] Combination Index (CI) and Synergy Score Results of Drug Combinations The CI index and synergy scores (HSA and BLISS) for drug combination therapy both showed the sensitivity and synergistic effect of the combination. Figure 2 ); and showed antagonistic effects of the combination on normal human colonic epithelial cells NCM460 (); Figure 5 ).

[0046] Experimental results of using 5-FU enhanced by salicylic acid on colorectal cancer xenografts * Experimental results in the figure P <0.05,** P <0.01, *** P <0.001; ns=no significance.

[0047] Results of xenograft mouse experiments In vivo experiments on mice with xenografts showed that the combination of drugs had no significant effect on the body weight of the mice. Figure 6 The combination of the two drugs significantly inhibited the growth of transplanted tumors. Figure 7 , Figure 8 , Figure 9 ).

[0048] AST and ALT test results In vivo experiments using the combined drugs on transplanted tumor mice showed that salicylic acid could also partially alleviate the elevation of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) induced by 5-FU. Figure 10 , Figure 11 ).

[0049] Neutrophil count results In vivo experiments on xenograft mice using a combination of drugs showed that the combination of drugs resulted in a decrease in neutrophils. Figure 12 ) In summary, salinomycin can significantly enhance the inhibitory effect of 5-FU on colorectal cancer tumor cells; the combination therapy has a synergistic effect and improves sensitivity; it can achieve tumor inhibition at a relatively low dose of 5-FU; and the drug does not inhibit the proliferation of normal human colonic epithelial cells; salinomycin can sensitize and reduce the toxicity of 5-FU in vivo for chemotherapy of colorectal cancer.

Claims

1. Application of salicornin combined with 5-fluorouracil in the preparation of anti-colorectal cancer drugs.

2. The application of salicin combined with 5-fluorouracil as described in claim 1 in the preparation of an anti-colorectal cancer drug, characterized in that, The molar ratio of the combined use of salicylic acid and 5-fluorouracil is 1:

1.

3. The application of salicin combined with 5-fluorouracil as described in claim 1 in the preparation of anti-colorectal cancer drugs, characterized in that, The colorectal cancer mentioned originates from human colorectal cancer cells HCT116 and LoVo.

4. The application of salicin combined with 5-fluorouracil as described in claim 1 in the preparation of an anti-colorectal cancer drug, characterized in that, Saussureanin, alone or in combination with other drugs and with the addition of excipients, can be formulated into anti-tumor drug preparations.

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