Application of bile acids and animal bile products in the preparation of radiotherapy sensitizers

Abstract

The present invention relates to the pharmaceutical field, specifically to the use of bile acids and animal bile products in the preparation of tumor radiosensitizers. By examining the docking characteristics of bile acids with the EphA2 receptor binding site, their inhibitory effects on EphA2 enzyme activity, their inhibitory effects on EphA2 phosphorylation, and their radiosensitization effects on radiotherapy-resistant tumor cells, it was found that bile acids, including taurocholic acid, ursodeoxycholic acid, and tauroursodeoxycholic acid, can inhibit EphA2 enzyme activity and EphA2 phosphorylation within the nanomolar to micromolar concentration range, and can inhibit radioresistance induced by elevated EphA2 expression and / or EphA2 phosphorylation within the micromolar concentration range. Therefore, bile acids, taurocholic acid, ursodeoxycholic acid, and tauroursodeoxycholic acid have promising application as tumor radiosensitizers.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceuticals, and particularly relates to the application of bile acid substances and animal bile products in the preparation of radiotherapy sensitizers. Background Art

[0002] Bile acids are a family of steroidal acids found in animals, each containing a bile acid nucleus. They can be divided into free and conjugated bile acids based on whether they are bound to glycine, taurine, or other amino acids. Zhou Bo et al. (Zhou Bo. Identification of Medicinal Substances in Blue Fox (Alopex lagopus) Bile and Study of Its Medicinal Effects [D]. Northeast Forestry University, 2015) and Liu Hongyuan et al. (CN114306349A) reported that bile products, as the main organic component of animal bile, exhibit numerous pharmacological activities, including antibacterial, antioxidant, anti-inflammatory, tumor cell proliferation inhibition, cough suppression, and improved glucose metabolism. Bile acids are relatively safe. When administered systemically to rodents, the median lethal dose (LD50) exceeds 5 g / kg body weight by oral gavage, and the median lethal dose (LD50) for rodents is approximately 300 to 500 mg / kg body weight by parenteral administration.

[0003] Tsutomu Matsubara et al. (Matsubara T, Li F, Gonzalez F J. FXR signaling in the enterohepatic system[J]. Molecular and cellular endocrinology, 2013, 368(1-2): 17-29.) and Massimiliano Tognolini et al. (Tognolini M, Incerti M, Pala D, et al. Target Hopping as a Useful Tool for the Identification of Novel EphA2Protein–Protein Antagonists[J]. ChemMedChem, 2014, 9(1): 67-72.) reported that known affinity receptors for bile acids include farnesoid X receptor, bile acid receptor TGR5, pregnane X receptor, vitamin D receptor, and Ephrin receptor family A2 (EphA2).Regarding this type of receptor, Salvatore Modica et al. (Modica S, Murzilli S, Salvatore L, Schmidt DR, Moschetta A. Nuclear bile acid receptor FXR protects against intestinal tumorigenesis. Cancer Res 2008; 68: 9589-94.) reported that farnesoid X receptor is involved in regulating intestinal tumor growth; Chen et al. (Chen Y, Huang W, Chen F, et al. Pregnane X receptorsregulate CYP2C8 and P-glycoprotein to impact on the resistance of NSCLC cells to Taxol[J]. Cancer Medicine, 2016, 5(12): 3564-3571.) reported that pregnane X receptor regulates the sensitivity of tumors to some chemotherapeutic drugs. This receptor is highly expressed in intestinal epithelial cells and hepatocytes; Zheng et al. (Zheng W, Duan B, Zhang Q, et al. Vitamin D-induced vitamin D receptor expressioninduces tamoxifen sensitivity in MCF-7 stem cells via suppression of Wnt / β-catenin signaling[J]. Bioscience Reports, 2018) Vitamin D receptors are also involved in regulating the sensitivity of tumors to some chemotherapy drugs; Emmanouil Fokas et al. reported (Fokas E, Kamlah F, Hänze J, et al. EphA2 blockade enhances the anti-endothelial effect of radiation and inhibitsirradiated tumor cell-induced migration of endothelial cells[J]. Thoraciccancer, 2010, 1(4): 153-162.) EphA2 receptors are associated with radiotherapy resistance. The above studies suggest that bile acid-affinity receptors may be involved in the occurrence and development of tumors and their resistance to radiotherapy and chemotherapy.

[0004] While research on the effects of bile acids on the activity of the farnesoid X receptor, bile acid receptor TGR5, pregnane X receptor, and vitamin D receptor is relatively extensive, research on the interaction between bile acids and the EphA2 receptor and its association with radioresistance has been relatively recent. Currently, there are relatively few clinically approved radiosensitizers, with only a few, such as sodium glycidazole. The existence of tumor radioresistance has led to an urgent need for radiosensitizers in clinical practice. Summary of the Invention

[0005] To alleviate radiotherapy resistance, a new radiotherapy sensitizer is provided. The present invention investigates the affinity targets and radiotherapy sensitization activity of bile acids and provides the use of bile acids and animal bile products in the preparation of radiotherapy sensitizers.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] The present invention provides the use of bile acid substances and animal bile products in the preparation of tumor radiotherapy sensitizers.

[0008] The bile acid may contain only one active ingredient, preferably any one of cholic acid, taurocholic acid, ursodeoxycholic acid, and tauroursodeoxycholic acid, or a pharmaceutically acceptable salt thereof. Upon exposure of tumor cells to these bile acids, any one of these bile acids, at a micromolar concentration, can enhance the killing effect of radiotherapy on tumor cells to a certain extent.

[0009] The bile acid substance can be a mixture, that is, a mixture consisting of at least two of bile acid, taurocholic acid, ursodeoxycholic acid, tauroursodeoxycholic acid, or their pharmaceutically acceptable salts.

[0010] The animal bile product may be an animal bile product in which any one of cholic acid, taurocholic acid, ursodeoxycholic acid, or tauroursodeoxycholic acid is the primary component (highest content) of the bile acids contained in the animal bile product. For example, among the bile acids contained in the animal bile product, any one of cholic acid, taurocholic acid, ursodeoxycholic acid, or tauroursodeoxycholic acid accounts for more than 50% by weight of the total bile acids contained.

[0011] The animal bile product is preferably any one of a bear bile product and a blue fox bile product.

[0012] In a preferred embodiment, the animal bile product is a liquid or solid product of bear-drained bile.

[0013] In another preferred embodiment, the animal bile product is a liquid or solid product of blue fox bile.

[0014] Furthermore, the animal bile product is a dried solid product of blue fox bile. Preferably, the dried solid product of blue fox bile is a solid product obtained by drying blue fox bile at 37°C for 48 hours.

[0015] The above-mentioned bile acids, taurocholic acid, ursodeoxycholic acid, and tauroursodeoxycholic acid can bind to the adenosine triphosphate (ATP) binding site of the EphA2 receptor, inhibit the enzyme activity and phosphorylation of the EphA2 receptor, enhance the killing effect of radiotherapy on tumor cells, and reduce the radiotherapy resistance of tumor cells.

[0016] The aforementioned radiosensitizer is a radiosensitizer for tumors expressing EphA2 receptors, such as any one of lung cancer, nasopharyngeal cancer, ovarian cancer, breast cancer, and melanoma.

[0017] In a preferred embodiment, the tumor is lung cancer. Further preferably, the lung cancer is non-small cell lung cancer.

[0018] The preparation of the radiosensitizer can be any one of an inhalation preparation, an injection preparation, an external preparation, and an oral preparation. Furthermore, the preparation of the radiosensitizer can be an intratumoral injection preparation.

[0019] In addition, the bile acid substance or animal bile product can be combined with at least one of celecoxib and sodium glycidazole to form a compound as a radiotherapy sensitizer.

[0020] Beneficial effects:

[0021] First, the bile acid, taurocholic acid, ursodeoxycholic acid, and tauroursodeoxycholic acid of the present invention can enhance the killing effect of radiotherapy on tumor cells. Studies have shown that they also have a radiosensitizing effect on radiotherapy-resistant tumor cells.

[0022] Secondly, the blue fox bile of the present invention is abundant in source and is cheap and easy to obtain as a radiotherapy sensitizer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the molecular docking diagram of bile acid and the ATP binding site of EphA2 receptor;

[0024] Figure 2 This is the molecular docking diagram of chenodeoxycholic acid and the ATP binding site of EphA2 receptor;

[0025] Figure 3 This is the molecular docking diagram of deoxycholic acid and the ATP binding site of EphA2 receptor;

[0026] Figure 4This is the molecular docking diagram of taurocholic acid and the ATP binding site of EphA2 receptor;

[0027] Figure 5 This is the molecular docking diagram of taurochenodeoxycholic acid and the ATP binding site of EphA2 receptor;

[0028] Figure 6 This is the molecular docking diagram of taurodeoxycholic acid and the ATP binding site of EphA2 receptor;

[0029] Figure 7 This is the molecular docking diagram of tauroursodeoxycholic acid and the ATP binding site of EphA2 receptor;

[0030] Figure 8 This is the molecular docking diagram of ursodeoxycholic acid and the ATP binding site of EphA2 receptor;

[0031] Figure 9 The log concentration-EphA2 enzyme activity response curve of 8 bile acids;

[0032] Figure 10 This is a bar graph showing the effects of eight bile acids at different concentrations on the phosphorylated EphA2 content in prostate cancer cells;

[0033] Figure 11 The effect of different bile acids on the survival rate of radioresistant lung cancer A549 cells after 2Gy irradiation;

[0034] DMSO is dimethyl sulfoxide, DASA is dasatinib, CELE is celecoxib, TUDCA is tauroursodeoxycholic acid, TCA is taurocholic acid, TCDCA is taurochenodeoxycholic acid, TDCA is taurodeoxycholic acid, CDCA is chenodesoxycholic acid, UDCA is ursodeoxycholic acid, DCA is deoxycholic acid, CA is cholic acid, # indicates p < 0.05 compared with DMSO, * indicates p < 0.05 compared with TUDCA, A indicates p < 0.05 compared with A549 cells, and B indicates p < 0.05 compared with DMSO-treated radioresistant A549 cells. DETAILED DESCRIPTION

[0035] The following examples are provided to facilitate understanding of the present invention. The instruments, reagents, and software used in the examples are all commercially available. Although the research contents of the following examples are independent of each other, the references and numbers are continuous and related. Example 1

[0036] 1 Research background and purpose

[0037] Ephrin A1 is one of the natural ligands of the EphA2 receptor. After binding to the EphA2 receptor, Ephrin A1 can induce EphA2 phosphorylation and participate in the regulation of tumor cell radiosensitivity. Two bile acids, lithocholic acid and cholanic acid, have been found to be able to hinder the protein-protein interaction (PPI) between Ephrin A1 and the EphA2 receptor. Among them, the 103rd Arg amino acid residue of the EphA2 receptor is the key residue for the binding of Ephrin A1 to the EphA2 receptor. Lithocholic acid and cholanic acid can prevent Ephrin A1 from approaching the 103rd Arg amino acid residue of the EphA2 receptor, thereby exerting a PPI inhibitory effect. [1,2] .

[0038] However, in the investigation of PPI inhibitors of compounds with similar structures to lithocholic acid, the inhibitory effect of the compounds on the PPI of the ephrin A1-EphA2 receptor was not related to the structural similarity of lithocholic acid. Among the eight substances with similar structures to lithocholic acid, two had significant inhibitory effects on the PPI of the ephrin A1-EphA2 receptor. The remaining six substances, such as ursodeoxycholic acid (UDCA, CAS 128-13-2), although similar in structure to lithocholic acid and forming a salt bridge with or close to the 103rd amino acid residue of the EphA2 receptor, did not significantly inhibit the PPI of the ephrin A1 and EphA2 receptors. [1] .

[0039] Whether other bile acids can prevent Ephrin A1 from accessing the Arg 103 residue of the EphA2 receptor, thereby exerting its PPI inhibitory effect, requires further investigation. The purpose of this study was to investigate whether other bile acids have the ability to inhibit Ephrin A1-EphA2 receptor PPI using molecular docking and other methods.

[0040] 2 Methods

[0041] Refer to the methods reported in the literature [1] The inhibitory effects of tauroursodeoxycholic acid (TUDCA, CAS: 14605-22-2), taurocholic acid (TCA, CAS: 81-24-3), taurochenodeoxycholic acid (TCDCA, CAS: 516-35-8), taurodeoxycholic acid (TDCA, CAS: 516-50-7), chenodesoxycholic acid (CDCA, CAS: 474-25-9), deoxycholic acid (DCA, CAS: 83-44-3), and cholic acid (CA, CAS: 81-25-4) on the PPI of ephrin A1-EphA2 receptors were investigated. Lithocholic acid was used as the positive control compound in the in vitro assay.

[0042] 2.1 Molecular docking test

[0043] The protein structure database (PDB) of the 3D structure of the EphA2 receptor-Ephrin A1 protein complex is coded as 3HEI. [1] , Maestro software package was used to optimize and process proteins and ligands, and docking box parameters were set according to the coordinates.

[0044] 2.2 Inhibitory effect of bile acids on Ephrin A1-EphA2 PPI

[0045] A water blank control and a negative control (solvent without test compound, i.e., 1% dimethyl sulfoxide in water) were routinely set up. For compound testing, a 96-well plate was incubated overnight at 4°C with EphA2-Fc in phosphate buffer (1 μg / mL, 100 μL / well). After washing with phosphate buffer (phosphate buffer containing 0.05% Tween 20, pH 7.5), the plate was incubated with blocking buffer (phosphate buffer containing 0.5% bovine serum albumin, pH 7.4) at 37°C for 1 hour. Test compounds and lithocholic acid were prepared in 1% dimethyl sulfoxide and added to the 96-well plate in a concentration gradient from 0 mmol / L to 10 mmol / L (final lithocholic acid concentration of 50 μmol / L). The plate was incubated at 37°C for 1 hour. Subsequently, 200 ng / mL of biotinylated Ephrin-A1-Fc was added and the plate was incubated at 37°C for 4 hours. After washing the plate again, 100 μL / well of horseradish peroxidase-labeled streptavidin (dissolved in phosphate buffer containing 0.5% bovine serum albumin, pH 7.4, 0.05 μg / mL) was added and incubated at 37°C for 20 min. After washing, TMB (3,3',5,5'-tetramethylbenzidine) was used for color development. After adding HCl to terminate the reaction, the absorbance was measured at 450 nm (OD value, blank wells were set to zero).

[0046] The OD value corresponding to the negative control was considered 100% for ephrin A1-EphA2 receptor binding. The OD value after addition of each test compound and lithocholic acid was calculated as the percentage of the OD value corresponding to the negative control. The minimum ephrin A1-EphA2 receptor binding rate (Bmin) for each compound within the test concentration range was determined. One-way ANOVA was used to compare the Bmin values ​​for each compound, and IC50 values ​​were calculated using GraphPad 8.0 software.

[0047] 3 Results

[0048] 3.1 Docking test of different bile acids with the Ephrin A1 binding cassette of the EphA2 receptor

[0049] In addition to lithocholic acid, seven bile acids, including TUDCA, TCA, CDCA, TCDCA, TDCA, CA, and DCA, were tested for docking with the Ephrin A1 binding box of the EphA2 receptor. As shown in Table 1, all seven bile acids can enter the binding box and approach the Arg 103 residue of EphA2. TUDCA and CDCA can form a salt bridge with the Arg 103 residue.

[0050] 3.2 Inhibitory effects of different bile acids on PPI of Ephrin A1-EphA2 receptors

[0051] Since TUDCA, TCA, CDCA, TCDCA, TDCA, CA, and DCA can all enter the binding box and are close to the 103rd Arg amino acid residue of EphA2, the ability of seven bile acids to inhibit the binding of Ephrin A1 to the EphA2 receptor was investigated. The inhibitory effect of different bile acids on the PPI of the ephrin A1-EphA2 receptor was determined by ELISA. As shown in Table 1, the addition of the positive control carbolic acid significantly inhibited the binding of ephrin A1 to the EphA2 receptor under the test conditions. Except for TUDCA, the minimum binding rate Bmin of the ephrin A1-EphA2 receptor after the addition of the other test compounds was higher than 92%, and the inhibition rate was lower than 20%, indicating that there was no inhibitory effect on the PPI of the ephrin A1-EphA2 receptor. [1] .

[0052] IC50 values ​​were calculated using Graphpad 8.0 software. The IC50 of TUDCA in inhibiting the binding of ephrin A1 to EphA2 receptors was 2.395 mmol / L. Its IC50 was > 2 mmol / L, and the inhibition rate was less than 20% at 100 μmol / L, so it was ineffective. [1] .

[0053] Table 1 Inhibitory effects of different bile acids on Ephrin A1-EphA2 PPI

[0054]

[0055] Notes: Y: Yes; N: No; #: p < 0.05 compared with negative; "-": Not applicable or not tested; " / ": Inhibition rate is less than 20% at the tested concentration, invalid [1] ;A: IC50>2mM, the inhibition rate is less than 20% at 100μmol / L, which is ineffective.

[0056] 4 Conclusion

[0057] Molecular docking and in vitro PPI assays for the Ephrin A1-EphA2 receptor revealed that among the seven bile acids (TUDCA, TCA, CDCA, TCDCA, TDCA, CA, and DCA), TUDCA exhibited some inhibitory activity against the Ephrin A1-EphA2 receptor PPI. However, its IC50 value was too high, indicating a lack of in vivo application value. However, structural modification of TUDCA as a lead compound may yield promising compounds. Example 2

[0058] 1 Research background and purpose

[0059] In addition to the Ephrin A1 ligand binding site, the EphA2 receptor also has multiple binding pockets such as the ATP binding site, nucleotide binding site, and ribose binding site. These sites are located differently from the Ephrin A1 ligand binding site, and the activities caused by activating or inhibiting these sites are also different. For example, the Ephrin A1 ligand binding site of the EphA2 receptor is located in the extracellular domain, while multiple binding pockets such as the ATP binding site are located in the intracellular domain near the β-folded structure. When the ligand binding site of the EphA2 receptor is activated, its activity is directed at the ligand-dependent phosphorylation of the EphA2 receptor, while the enzymatic activity of the EphA2 receptor does not depend on the binding of the ligand binding site. For example, cholic acid and lithocholic acid inhibit the ligand-dependent phosphorylation of EphA2, but do not inhibit the enzymatic activity of EphA2. [2] .

[0060] The binding site of dasatinib to the EphA2 receptor is a non-ligand binding site such as the ATP binding site. Currently, extensive structural optimization has been carried out using kinase inhibitors such as dasatinib and nilotinib as lead compounds, resulting in a variety of compounds with good affinity and selectivity for the ATP binding site of the EphA2 receptor. [2] .

[0061] After docking bile acids with the Ephrin A1 ligand binding site of the EphA2 receptor and investigating their in vitro PPI inhibitory activity, this study aimed to further investigate the docking characteristics of bile acids with the ATP binding site of the EphA2 receptor and test their effects on EphA2 enzyme activity and phosphorylation.

[0062] 2 Methods

[0063] 2.1 Docking test of bile acids and the ATP binding site of the EphA2 receptor

[0064] The 3D structure of the EphA2 receptor-small molecule complex is encoded in the Protein Data Bank (PDB) as 5NKB. Computer-assisted batch docking screening was performed using iGEMDOCK software according to the user manual. Test compounds included TUDCA, TCA, CDCA, TCDCA, TDCA, UDCA, CA, and DCA. Binding parameters for eight bile acids at the EphA2 receptor ATP binding site were obtained.

[0065] 2.2 Determination of the inhibitory effect of bile acids on EphA2 enzyme activity

[0066] Reference Methods [3]Recombinant human EphA2 enzyme activity was measured using the EphaA2 Assay Kit (BPS Bioscience) using Poly (Glu:Tyr, 1:4) as a substrate according to the kit's instructions. Staurosporine (1 μmol / L) and cholanic acid (1 μmol / L) were used as positive controls, and 1% dimethyl sulfoxide (DMSO) in water were used as the drug solvent. Kinase buffer served as a blank control. The inhibitory effects of test compounds at various concentrations on EphA2 enzyme activity were determined, and IC50 values ​​were calculated using GraphPad Prism.

[0067] 2.3 Inhibitory effect of bile acids on EphA2 phosphorylation

[0068] References [1, 4] Method: A water blank control and a negative control (solvent without test compound, i.e., 1% dimethyl sulfoxide aqueous solution) were routinely set up. PC3 prostate cancer cells were seeded in 12-well plates at a concentration of 10 5 Cells were cultured to approximately 70% confluence and serum-starved overnight. Test compounds and the positive control, dasatinib, were prepared in 1% dimethyl sulfoxide (DMSO) in water. After serum starvation overnight, cells were pretreated with various concentrations of test compounds or dasatinib (final concentration 1 μmol / L) or solvent (1% DMSO in water) for 20 minutes. Cells were stimulated with 0.25 μg / mL Ephrin-A1-Fc for 20 minutes, dissolved in phosphate-buffered saline, added to RIPA lysis buffer, resuspended, and lysed at 4°C for 30 minutes. The supernatant was centrifuged and the protein concentration was determined using a BCA kit. The phosphorylated EphA2 content was determined using a phospho-EphA2 IC ELISA kit. The absorbance was measured at 450 nm (OD value, with blank wells set to zero).

[0069] The OD value of the negative control well was considered as 100% for phosphorylated EphA2 content, and the percentage of the OD value of the test compound and dasatinib wells to the OD value of the negative control well was considered as the relative phosphorylated EphA2 content. The relative phosphorylated EphA2 content of each compound well was compared by one-way analysis of variance.

[0070] 3 Results

[0071] 3.1 Molecular docking characteristics of bile acids and the ATP binding site of the EphA2 receptor

[0072] iGEMDOCK software was used for batch molecular docking to investigate the docking characteristics of 8 bile acids with the ATP binding site of the EphA2 receptor. Docking fitness was obtained and interaction analysis was performed to collect amino acid residues that interacted with the EphA2 receptor through hydrogen bonds, charge energy, and van der Waals forces. The docking diagrams of 8 bile acids with the ATP binding site of the EphA2 receptor can be found in Figures 1 to 8 The binding sites of the eight bile acids are all located in the ATP binding pocket near the β-sheet structure of the EphA2 receptor 3D structure.

[0073] The docking parameters of the eight bile acids at the ATP-binding site of the EphA2 receptor are shown in Table 2. The docking fitness values ​​ranged from -101.913 to -89.1952, and they interacted with amino acid residues such as Ile at position 619 of the EphA2 receptor. Analysis of the docking scores and interacting amino acid residues suggests that the eight bile acids may inhibit EphA2 enzymatic activity and phosphorylation.

[0074] 3.2 Inhibitory effect of bile acids on EphA2 enzyme activity

[0075] Based on batch molecular docking tests, the inhibitory effects of eight bile acids on EphA2 enzyme activity were determined over a gradient concentration range. Under the experimental conditions, the positive control, staurosporine, inhibited EphA2 enzyme activity by 82%. The negative control, cholanoic acid, had no significant inhibitory effect on EphA2 enzyme activity. The log concentration-EphA2 enzyme activity response curves for the eight bile acids are shown in Figure 2. Figure 9 The calculated IC50 values ​​are shown in Table 2. The IC50 values ​​of the eight bile acids for inhibiting EphA2 enzyme activity vary greatly, but the IC50 values ​​are still within the nanomolar to micromolar concentration range.

[0076] Table 2 Docking characteristics of different bile acids with the ATP binding site of the EphA2 receptor and their inhibitory effects on EphA2 enzyme activity

[0077]

[0078] Remarks “-”: not applicable or not tested; a : The table shows the amino acid number, i.e. the position of the amino acids in the protein sequence; b : IC50 values ​​are in μmol / L, with 95% confidence intervals in square brackets.

[0079] 3.3 Inhibitory effect of bile acids on EphA2 phosphorylation

[0080] After determining the inhibitory effect of bile acids on EphA2 enzyme activity, the inhibitory effect of different bile acids on EphA2 phosphorylation was determined in vitro. Figure 10 As shown in Figure 3 . Dasatinib significantly inhibited EphA2 phosphorylation, with an inhibition rate approaching 90%. Among the various bile acids, TUDCA, TCA, TCDCA, TDCA, UDCA, and CA showed significant inhibitory effects on EphA2 phosphorylation. TUDCA exhibited a slightly greater inhibitory effect on EphA2 phosphorylation than TCA and UDCA, but the difference was not statistically significant.

[0081] 4 Conclusion

[0082] Molecular docking, enzyme activity assays, and receptor phosphorylation assays revealed that the bile acids tested docked effectively with the non-ligand binding site (ATP binding site) of the EphA2 receptor, inhibiting EphA2 enzyme activity at nanomolar to micromolar concentrations. TUDCA, TCA, TCDCA, TDCA, UDCA, and CA showed significant inhibitory effects on EphA2 phosphorylation, with IC50 values ​​below cytotoxic concentrations. These substances, their acceptable salts, and compounds derived from their structural engineering as lead compounds may have potential to enhance tumor chemoradiotherapy and chemotherapy sensitization. Example 3

[0083] 1 Research background and purpose

[0084] When tumor cells are irradiated, their EphA2 expression and phosphorylation levels increase. EphA2 activation and phosphorylation have been shown to be associated with decreased radiosensitivity. Inhibitors targeting EphA2 have demonstrated unique advantages in radiosensitization. [5-7] .

[0085] Given the inhibitory effects of TUDCA, TCA, UDCA, TCDCA, TDCA, and CA on EphA2 enzyme activity and phosphorylation. This section uses non-small cell lung cancer A549 cells as a model to investigate the radiosensitization effects of TUDCA, TCA, UDCA, TCDCA, TDCA, and CA on non-small cell lung cancer. A549 cells express EphA2, and when irradiated, the levels of EphA2 and EphA2 phosphorylation are higher than those in normal cells and A549 cells that have not been irradiated. [5-7] .

[0086] 2 Methods

[0087] Non-small cell lung cancer A549 cells ATCC CCL-185 were inoculated in RPMI1640 medium containing 10% fetal bovine serum and cultured using the method described in the literature. [8] Radiotherapy-resistant non-small cell lung cancer A549 cells (A549IR cells) were induced and cultured at 37°C in an incubator containing 5% CO2.

[0088] A549IR cells were cultured to the logarithmic growth phase, digested with 0.25% trypsin, and pipetted into a cell suspension. 10 μl of the cell suspension was added with an equal amount of 0.4% trypan blue staining solution and counted under a microscope. The cell concentration was adjusted to 3 × 10 3 The drug was prepared in 1% DMSO (dimethyl sulfoxide) aqueous solution at 100 µl / well in a 6-well plate. A549IR cells were treated with 1% DMSO aqueous solution or drug for 6 hours, then irradiated with 2 Gy of radiation. The cells were cultured for 14 days and the radiosensitivity parameter, SF2, was determined using a colony formation assay. Non-radioresistant A549 cells were treated with 1% DMSO aqueous solution instead of drug and SF2 was determined using the same method. Final drug concentrations (μmol / L) in the wells were: TUDCA 12.5, TCA 2.5, UDCA 400, TCDCA 80, TDCA 210, CA 155, and celecoxib (CELE, positive control) 10.

[0089] One-way ANOVA was used to compare SF2 in cells treated with different drugs.

[0090] 3 Results

[0091] This study investigated the effects of TUDCA, TCA, UDCA, TCDCA, TDCA, and CA on the radiosensitivity parameter SF2 of radiotherapy-resistant A549 non-small cell lung cancer cells. Among various radiosensitivity parameters, SF2 was less affected by inoculation time and inoculation density and was relatively stable. Figure 11 In radioresistant A549 cells, pretreatment with TUDCA, TCA, UDCA, and CA significantly reduced SF2. Among them, SF2 in radioresistant A549 cells pretreated with CA remained significantly higher than in A549 cells without radioresistance. Pretreatment with TCDCA and TDCA also reduced SF2 in radioresistant A549 cells, but the differences were not significant.

[0092] Comparison of the concentration-response relationship between drug treatment and EphA2 phosphorylation and the inhibition of radioresistance in A549 cells revealed inconsistent trends. For example, while TCDCA and TDCA significantly inhibited EphA2 phosphorylation, they did not significantly reduce SF2 in radioresistant A549 cells, suggesting that bile acids have complex effects on tumor cells. However, the radiosensitizing effects of TUDCA, TCA, UDCA, and CA can be at least partially attributed to their inhibition of EphA2 enzyme activity and phosphorylation.

[0093] 4 Conclusion

[0094] TUDCA, TCA, UDCA and CA have inhibitory effects on the radioresistance of A549 cells at the micromolar level. Example 4

[0095] 1 Research background and purpose

[0096] Blue Fox ( Alopex lagopus ) is one of the economic animals that has been artificially bred on a large scale and can provide humans with precious fur. After the blue fox is bred for its skin, its organs, including the fox gallbladder, are discarded as waste. Studies have found that blue fox bile contains bile acids such as TUDCA and UDCA, which are similar to the composition of bear bile. Blue fox bile and its dried products are expected to become a substitute for bear bile, which can not only improve the comprehensive utilization value of blue foxes, but also alleviate the shortage of natural bear bile. [9] .

[0097] Given that TUDCA, TCA, UDCA, and CA have inhibitory effects on the radioresistance of A549 cells at the micromolar level, this section uses non-small cell lung cancer A549 cells as a model to investigate the inhibitory effect of blue fox bile products on the radioresistance of A549 cells.

[0098] 2 Methods

[0099] 2.1 Preparation of Blue Fox Bile Products

[0100] Blue fox gallbladder was obtained from a fox farm in Suihua, Heilongjiang Province. Bile was extracted from the gallbladder of a freshly killed blue fox and dried at 37°C for 48 hours to obtain bile powder. The TUDCA sodium content per 250mg of bile powder was determined to be approximately 33.41mg. [9] TUDCA is the most abundant bile acid in bile powder. Dissolve the bile powder in 1% dimethyl sulfoxide (DMSO) solution, filter through a 0.22 μm microporous filter via sonication, and use the filtrate as the blue fox bile stock solution. Based on the bile powder, the stock solution concentration is 1 mg / mL.

[0101] 2.2 Study on the radiotherapy resistance of blue fox bile products

[0102] Radioresistant non-small cell lung cancer A549 cells (A549IR) were established using the method of Example 3. Cells were treated with either a 1% dimethyl sulfoxide (DMSO) aqueous solution or a blue fox bile stock solution. Colony formation assays were performed to measure the radiosensitivity parameter SF2 in A549IR and uninduced radioresistant A549 cells to investigate the effect of the blue fox bile stock solution on SF2 in radioresistant A549 cells. The bile concentration per well was 60 mg / L, calculated as dry bile powder. A 1% DMSO aqueous solution control was established.

[0103] One-way ANOVA was used to compare SF2 in cells treated with different drugs.

[0104] 3 Results

[0105] The SF2 values ​​for A549 cells without radioresistance (treated with 1% DMSO aqueous solution) and A549IR cells treated with 1% DMSO aqueous solution and blue fox bile were 0.43±0.08, 0.64±0.06, and 0.42±0.11, respectively. The SF2 value for A549IR cells treated with blue fox bile dry powder was significantly lower than that for A549IR cells treated with 1% DMSO aqueous solution. Example 5

[0106] 1 Research background and purpose

[0107] Bile acids are already in clinical use. For example, TUDCA and UDCA are used orally for the treatment of cholesterol gallstones. CA has also been used orally for the treatment of congenital defects in bile acid synthesis. When used as a radiosensitizer, CA should generally be administered within 12 hours before radiotherapy. This study investigated the radiosensitizing effect of CA administered before radiotherapy on tumor-bearing mice.

[0108] 2 Methods

[0109] 2.1 Establishment of nude mouse transplant tumor model

[0110] Non-small cell lung cancer A549 cells were cultured in vitro to the logarithmic growth phase, digested with 0.25% trypsin, pipetted, and centrifuged. A small amount of cell suspension was taken and stained with an equal amount of 0.4% trypan blue dye. The cells were counted under a microscope and the cell suspension was adjusted to 1×10 with complete culture medium. 7 The mice were inoculated with 0.2 ml of the solution under the right armpit of female nude mice.

[0111] 2.2 Experimental Animal Grouping and Treatment

[0112] CA is prepared with 0.9% sodium chloride injection and is prepared immediately before use.

[0113] The diameter of the transplanted tumor in nude mice was measured with a vernier caliper. 3 ~300mm 3 Afterwards, mice meeting the criteria were randomly divided into groups based on tumor volume (n=6). The administration volume was 0.2 ml per mouse. The model group and the radiotherapy-only group received an intravenous injection of 0.9% sodium chloride solution before radiotherapy; the CA group and the CA+radiotherapy group received an intravenous injection of CA 15 mg / kg before radiotherapy. Four hours after drug injection, the radiotherapy-only and CA+radiotherapy groups received a single 8 Gy radiotherapy fraction.

[0114] The tumor diameter was measured and weighed before administration. The tumor diameter was measured once every 7 days after administration, and the tumor volume (TV) was calculated. After measuring the tumor diameter twice, the mice were killed by dislocation, and the tumor tissue was removed and weighed.

[0115] The formula for calculating tumor volume is: V = 1 / 2×a×b2 , where a and b represent the tumor length and width respectively. The relative tumor volume (RTV) is calculated based on the measurement results using the following formula: RTV = Vt / V0. V0 is the tumor volume measured before administration, and Vt is the tumor volume measured at different time points (days, expressed as days) after administration.

[0116] 3 Results

[0117] Within 14 days after administration, no deaths or obvious abnormalities were observed in the nude mice in each group. As shown in Table 3, the RTV in the CA+radiotherapy group was lower than that in the model and CA groups on day 8 after administration; and lower than that in the model, CA, and radiotherapy-alone groups on day 15 after administration. Tumor weight in the CA+radiotherapy group was lower than that in the model, CA, and radiotherapy-alone groups on day 15 after administration.

[0118] Table 3 Effects of CA on radiosensitivity of A549 transplanted tumors in nude mice

[0119]

[0120] Note: The day of drug administration was defined as day 1. A: compared with the model group, p < 0.05; B: compared with the CA group, p < 0.05; C: compared with the radiotherapy group, p < 0.05.

[0121] 4 Conclusion

[0122] A single intravenous injection of CA before radiotherapy is beneficial to improve the radiosensitivity of tumor-bearing mice.

[0123] Discussion and Conclusion

[0124] EphA2 receptors are involved in the development and progression of some tumors and are also related to tumor radiotherapy sensitivity. Previously, some authors investigated the affinity of lithocholic acid, cholanic acid and their structural analogs to the ligand binding site of the EphA2 receptor and their inhibition of the PPI of the ephrin A1-EphA2. Lithocholic acid, cholanic acid and several structural analogs can inhibit the PPI of Ephrin A1-EphA2, but UDCA did not significantly inhibit the PPI of Ephrin A1-EphA2. [1] Because the bile acids studied are incomplete, this study investigated the inhibitory effects of seven bile acids, including TUDCA, TCA, CDCA, TCDCA, TDCA, CA, and DCA, on Ephrin A1-EphA2 PPI. With the exception of TUDCA, which had a slight inhibitory effect, TCA, CDCA, TCDCA, TDCA, CA, and DCA did not significantly inhibit Ephrin A1-EphA2 PPI.

[0125] However, in the investigation of affinity for the EphA2 receptor ATP binding site, EphA2 receptor enzyme activity, and EphA2 receptor phosphorylation, TUDCA, TCA, TCDCA, TDCA, UDCA, and CA were found to have significant inhibitory effects on EphA2 phosphorylation, suggesting that TUDCA, TCA, TCDCA, TDCA, UDCA, and CA may inhibit EphA2 phosphorylation by binding to the EphA2 receptor ATP binding site, thereby inhibiting downstream events of EphA2 phosphorylation, including tumor radioresistance.

[0126] Therefore, we further investigated the effects of TUDCA, TCA, TCDCA, TDCA, UDCA, and CA on tumor radiosensitivity in vitro. TUDCA, TCA, UDCA, and CA demonstrated radiosensitization effects on non-small cell lung cancer A549 cells. Although TCDCA and TDCA significantly inhibited EphA2 phosphorylation, they did not significantly reduce SF2 in radioresistant A549 cells. Furthermore, we investigated the in vitro radiosensitization effects of blue fox bile products and the radiosensitization effects of CA on nude mouse xenograft tumor models. Blue fox bile products exhibited similar in vitro radiosensitization effects to TUDCA, and CA also demonstrated a certain radiosensitization effect on nude mouse xenograft tumor models.

[0127] The study found that the effects of some bile acids on radiosensitivity differ from those based on EphA2 phosphate activity. Further research on other literature revealed that farnesoid X receptor is also related to tumor radiosensitivity.

[10] Some bile acids are strong agonists of farnesoid X receptors, which can activate farnesoid X receptors and reduce tumor radiosensitivity. Among them, TCDCA and TDCA are weak agonists of farnesoid X receptors.

[11] CA has a very weak activating effect on farnesoid X receptors, and concentrations as high as 100 μmol / L do not activate farnesoid X receptors. [12,13] TCA has a weaker activating effect on farnesoid X receptor, with an EC50 of 0.59 mmol / L to 1 mmol / L.

[14] UDCA does not activate or antagonize farnesoid X receptors

[15] ; TUDCA is a farnesoid X receptor antagonist

[16] Non-small cell lung cancer A549 expresses both farnesoid X receptor and EphA2 receptor

[17] Therefore, combined with research results from both domestic and international studies, we speculate that the potent radiosensitizing effect of TUDCA may not only rely on its inhibition of EphA2 phosphorylation but also be related to its antagonistic effect on the farnesoid X receptor. IC50 analysis suggests that the radiosensitizing effects of TCA, UDCA, and CA may rely on their inhibition of EphA2 phosphorylation. However, TCDCA and TDCA failed to significantly modulate tumor cell radiosensitivity, possibly due to their complex pathways of action.

[0128] In summary, combined with existing research, the above studies show that TUDCA, TCA, UDCA, and CA have potential radiosensitization effects in tumor cells that express both farnesoid X receptor and EphA2 receptor.

[0129] Although the examples used bile acids or blue fox bile products to treat cells and investigate their biological activity, or administered them via intravenous injection, the effects of drug metabolism through the intestine have not been determined, nor have the effects of other animal bile products, such as bear bile products, been determined. However, based on the content of Examples 1-5, one skilled in the art can reasonably speculate that the aforementioned bile acids and other animal bile products would have similar effects when administered via inhalation, transdermal administration, oral administration, intratumoral injection, and other routes.

[0130] The above embodiments are not intended to limit the scope of protection of the invention. For technicians, various obvious changes or modifications can be made based on the above embodiments, which should still be regarded as the scope of protection of the present invention.

[0131] Reference sources noted in the examples:

[0132] [1]Tognolini M, Incerti M, Pala D. Target Hopping as a Useful Tool for the Identification of Novel EphA2 Protein-Protein Antagonists[J]. ChemMedChem, 2014, 9:67-62.

[0133] [2]Xiao T, Xiao Y, Wang W, et al. Targeting EphA2 in cancer[J]. Journal of Hematology & Oncology, 2020, 13(1): 1-17.

[0134] [3] Shi Y, Challa S, Sang P, et al. One-bead–two-compound thioetherbridged macrocyclic γ-AApeptide screening library against EphA2[J]. Journalof medicinal chemistry, 2017, 60(22): 9290-9298.

[0135] [4] Hassan-Mohamed I, Giorgio C, Incerti M, et al. UniPR 129 is acompetitive small molecule Eph-ephrin antagonist blocking in vitroangiogenesis at low micromolar concentrations[J]. British journal ofpharmacology, 2014, 171(23): 5195-5208.

[0136] [5] Fokas E, Kamlah F, Hänze J, et al . EphA2 blockade enhances theanti-endothelial effect of radiation and inhibits irradiated tumor cell‐induced migration of endothelial cells[J]. Thoracic cancer, 2010, 1(4): 153-162.

[0137] [6] Din SU, Ashamalla H, Gilbert K ST, et al . Ionizing RadiationInduces EphA2 Serine 897 Phosphorylation in a MAPK / RSK Dependent Manner andInhibition of Eph Radiosensitizes A549 Cells[J]. International Journal ofRadiation Oncology, Biology, Physics, 2016, 96(2): E588.

[0138] [7] Gong S, Li Y, Lv L, et al . Restored microRNA-519a enhances theradiosensitivity of non-small cell lung cancer via suppressing EphA2[J]. GeneTherapy, 2021: 1-13.

[0139] [8] Lee YS, Oh JH, Yoon S, et al. Differential gene expression profiles of radioresistant non–small-cell lung cancer cell lines established by fractionated irradiation: Tumor protein p53-inducible protein 3 conferssensitivity to ionizing radiation[J]. International Journal of RadiationOncology Biolog Physics, 2010, 77(3): 858-866.

[0140] [9] Zhou Bo. Identification of medicinal substances in blue fox (Alopex lagopus) bile and research on its medicinal effects[D]. Northeast Forestry University, 2015.

[0141]

[10] Zhao Congzhao. Study on the effect and mechanism of chenodeoxycholic acid on the radiosensitivity of cholangiocarcinoma cells[D]. Suzhou University.

[0142]

[11] Waise TMZ, Lim YM, Danaei Z, et al. Small intestinaltaurochenodeoxycholic acid-FXR axis alters local nutrient-sensingglucoregulatory pathways in rats[J]. Molecular metabolism, 2021, 44: 101132.

[0143]

[12] Fujino T, Une M, Imanaka T, et al. Structure-activityrelationship of bile acids and bile acid analogs in regard to FXR activation[J]. Journal of lipid research, 2004, 45(1): 132-138.

[0144]

[13] Wang H, Chen J, Hollister K, et al. Endogenous bile acids areligands for the nuclear receptor FXR / BAR[J]. Molecular cell, 1999, 3(5): 543-553.

[0145]

[14] Pathak P, Xie C, Nichols R G, et al . Intestine farnesoid Xreceptor agonist and the gut microbiota activate G-protein bile acidreceptor‐1 signaling to improve metabolism[J]. Hepatology, 2018, 68(4): 1574-1588.

[0146]

[15] Zhang Y, LaCerte C, Kansra S, et al. Comparative potency ofobeticholic acid and natural bile acids on FXR in hepatic and intestinal invitro cell models[J]. Pharmacology research & perspectives, 2017, 5(6):e00368.

[0147]

[16] Sun L, Xie C, Wang G, et al. Gut microbiota and intestinal FXRmediate the clinical benefits of metformin[J]. Nature medicine, 2018, 24(12):1919-1929.

[0148]

[17] Inoue K, Kawahito Y, Tsubouchi Y, et al. Increased farnesoid Xreceptor expression in non-small cell lung cancer[J]. Haigan, 2001, 41(4):293-297.

Claims

1. The use of bile acid substances in the preparation of tumor radiotherapy sensitizers, characterized in that: The bile acid substance is selected from at least one of bile acid, taurocholic acid, ursodeoxycholic acid, tauroursodeoxycholic acid, or a pharmaceutically acceptable salt thereof; the tumor is a tumor expressing the EphA2 receptor; the bile acid substance can bind to the adenosine triphosphate binding site of the EphA2 receptor, inhibit the enzymatic activity of the EphA2 receptor and the phosphorylation of the EphA2 receptor, thereby exerting a radiosensitization effect; the preparation of the radiosensitizer is any one of an inhalation preparation, an injection preparation, a topical preparation, and an oral preparation.

2. The use according to claim 1, characterized in that The bile acid substance is selected from at least two of cholic acid, taurocholic acid, ursodeoxycholic acid, tauroursodeoxycholic acid, or pharmaceutically acceptable salts thereof.

3. The use according to claim 1, characterized in that The radiotherapy sensitizer further contains at least one of celecoxib and sodium glycidazole.

4. The use of blue fox bile products in the preparation of tumor radiotherapy sensitizers, characterized in that: The blue fox bile product is a bile dry powder obtained by drying blue fox bile at 37°C for 48 hours. The content of sodium tauroursodeoxycholate in the bile dry powder is: 33.41 mg of sodium tauroursodeoxycholate per 250 mg of bile dry powder; the bile acid substances in the bile dry powder can bind to the adenosine triphosphate binding site of the EphA2 receptor, inhibit the enzymatic activity of the EphA2 receptor and the phosphorylation of the EphA2 receptor, and exert a radiotherapy sensitization effect; the tumor is a tumor expressing the EphA2 receptor.

Images