Application of AZD1152 and gemcitabine in preparation of medicine for treating bile duct cancer

Abstract

The invention belongs to the technical field of cancer drugs, and particularly relates to application of AZD1152 and gemcitabine to preparation of drugs for treating bile duct cancer. The AZD1152 and the gemcitabine have the effect of synergistically treating the bile duct cancer, the AZD1152 and the gemcitabine can remarkably inhibit the growth of bile duct cancer cells and tumors in cooperation with the AZD1152, and the effect is better than that of single AZD1152 or gemcitabine.

Description

Technical Field

[0001] The present invention belongs to the technical field of cancer drugs, and particularly relates to the use of AZD1152 and gemcitabine in preparing drugs for treating bile duct cancer. Background Art

[0002] Cholangiocarcinoma is a highly invasive malignant tumor originating from bile duct epithelial cells, with an increasing incidence. Due to its insidious early symptoms and difficulty in diagnosis, approximately 70% of patients have already progressed to the advanced stage or metastasis by the time of diagnosis.

[0003] The current standard first-line treatment for advanced cholangiocarcinoma is primarily gemcitabine combined with a platinum-based drug, but the objective response rate (ORR) is only approximately 15-30%, and the median overall survival (OS) is generally less than 12 months. Existing chemotherapy regimens are associated with high drug resistance and significant side effects, necessitating the development of more effective treatment strategies.

[0004] In recent years, studies have found that abnormal expression of Aurora kinase B is associated with chemotherapy resistance in cholangiocarcinoma, suggesting that it may be a potential target for overcoming the efficacy bottleneck of gemcitabine. Although combination therapy strategies have been widely explored, the synergistic effect of AZD1152 and gemcitabine in the treatment of cholangiocarcinoma has not yet been studied. Summary of the Invention

[0005] Based on this, the present invention found that AZD1152 and gemcitabine have a synergistic effect in treating cholangiocarcinoma, and can be prepared into a drug for treating cholangiocarcinoma, which can significantly inhibit cholangiocarcinoma tumor cells and tumor growth, and the effect is significantly better than single drug use.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides a pharmaceutical composition comprising AZD1152 and gemcitabine.

[0008] Another aspect of the present invention provides a medicine comprising the above pharmaceutical composition.

[0009] Preferably, the dosage forms of the above-mentioned medicine include oral solution, injection, granule, tablet, powder, capsule, emulsion, spray or patch.

[0010] In another aspect, the present invention provides a use of the above-mentioned pharmaceutical composition in preparing a drug for treating bile duct cancer.

[0011] Preferably, in the above-mentioned use, the pharmaceutical composition has the following functions:

[0012] (a) It has the effect of inhibiting the growth of bile duct cancer cells;

[0013] (b) It has the effect of inhibiting the growth of bile duct cancer tumors.

[0014] Preferably, in the above use, the cholangiocarcinoma cells include one or more of QBC939, HuCCT1 or RBE.

[0015] Preferably, in the above use, the dosage form of the drug includes oral solution, injection, granules, tablets, powders, capsules, emulsions, sprays or patches.

[0016] The beneficial effects of the present invention include: AZD1152 and gemcitabine have a synergistic effect in treating bile duct cancer, and AZD1152 synergistically with gemcitabine can significantly inhibit bile duct cancer cells and tumor growth, and the effect is better than that of single AZD1152 or gemcitabine. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1A The changes in cell activity of human cholangiocarcinoma cell lines after treatment with different concentrations of AZD1152;

[0018] Figure 1B Changes in cell volume and clone formation ability of human cholangiocarcinoma cell lines after treatment with different concentrations of AZD1152;

[0019] Figure 2A This is a heat map of the combined effects of different concentrations of AZD1152 and gemcitabine on human cholangiocarcinoma cell lines;

[0020] Figure 2B The figure is a scatter plot of the combined effects of different concentrations of AZD1152 and gemcitabine on human cholangiocarcinoma cell lines;

[0021] Figure 2C The inhibition of colony formation by the AZD1152-gemcitabine combination;

[0022] Figure 2D The effect of AZD1152-gemcitabine combination on cell apoptosis;

[0023] Figure 3 The AZD1152-gemcitabine combination inhibits CCA organoids;

[0024] Figure 4A Schematic diagram of the in vivo efficacy validation process for PDX models;

[0025] Figure 4B The changes in tumor volume of PDX models after different drug administration;

[0026] Figure 4C The tumor conditions of the PDX model after different drug treatments. DETAILED DESCRIPTION

[0027] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0028] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "comprise" and the like are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0029] In a first aspect, an embodiment of the present invention provides a pharmaceutical composition comprising AZD1152 and gemcitabine.

[0030] It should be noted that AZD1152 and gemcitabine have a synergistic effect in treating cholangiocarcinoma. AZD1152 and gemcitabine can significantly inhibit cholangiocarcinoma cells and inhibit tumor growth, and the effect is better than that of single AZD1152 or gemcitabine.

[0031] It should be noted that in the pharmaceutical composition of the present invention, AZD1152 and gemcitabine can be packaged separately or prepared as the same substance. In addition, the pharmaceutical composition of the present invention can also contain other active substances that can treat bile duct cancer.

[0032] In a second aspect, an embodiment of the present invention provides a medicine comprising the above-mentioned pharmaceutical composition.

[0033] It should be noted that the pharmaceutical composition of the present invention can also be added with different excipients to prepare different dosage forms of drugs suitable for different usage scenarios. The dosage forms of the drugs may include oral liquids, injections, granules, tablets, powders, capsules, emulsions, sprays or patches, etc.; in addition, the excipients of the above-mentioned different dosage forms are all well known to those skilled in the art. For example, the preparation of tablets mainly uses diluents (such as starch, dextrin, sucrose or glycosides, etc.), absorbents (calcium sulfate, calcium hydrogen phosphate or light magnesium oxide, etc.), binders (povidone, syrup or hydroxypropyl methylcellulose, etc.), wetting agents (water, etc.) or disintegrants (dry starch, sodium hydroxymethyl starch or cross-linked polyvinylpyrrolidone, etc.); for example, the preparation of liquid oral preparations mainly uses solubilizers, suspending agents, emulsifiers or colorants, etc.

[0034] In a third aspect, an embodiment of the present invention provides a use of the above-mentioned pharmaceutical composition in the preparation of a drug for treating bile duct cancer.

[0035] In some specific examples, in the above uses, the pharmaceutical composition has the following functions:

[0036] (a) It has the effect of inhibiting the growth of bile duct cancer cells;

[0037] (b) It has the effect of inhibiting the growth of bile duct cancer tumors.

[0038] In some specific examples, in the above use, the cholangiocarcinoma cells include one or more of QBC939, HuCCT1, or RBE.

[0039] In some specific examples, in the above uses, the dosage form of the drug includes oral solution, injection, granules, tablets, powders, capsules, emulsions, sprays or patches.

[0040] It should be noted that, as mentioned above, the pharmaceutical composition for the above-mentioned use can also be added with different excipients to prepare different dosage forms of drugs to be suitable for different usage scenarios. The dosage forms of the drugs may include oral liquids, injections, granules, tablets, powders, capsules, emulsions, sprays or patches, etc.; in addition, the excipients of the above-mentioned different dosage forms are all well known to those skilled in the art. For example, the preparation of tablets mainly uses diluents (such as starch, dextrin, sucrose or glycerol, etc.), absorbents (calcium sulfate, calcium hydrogen phosphate or light magnesium oxide, etc.), binders (povidone, syrup or hydroxypropyl methylcellulose, etc.), wetting agents (water, etc.) or disintegrants (dry starch, sodium hydroxymethyl starch or cross-linked polyvinylpyrrolidone, etc.); for example, the preparation of liquid oral preparations mainly uses solubilizers, suspending agents, emulsifiers or colorants, etc.

[0041] In order to better understand the present invention, the content of the present invention is further explained below with reference to specific examples, but the content of the present invention is not limited to the following examples.

[0042] In the following examples, statistical analysis was performed using R software (version 4.3.1). Normally distributed data were analyzed using a two-tailed Student's t-test or one-way ANOVA. For non-normally distributed data, the Mann-Whitney U test (for comparing two groups) or the Kruskal-Wallis H test (for comparing multiple groups) was used. The correlation between prognosis and AURKB expression was evaluated by log-rank test and Cox regression analysis. For publicly available prognostic data, the SurvminerR package was used to analyze the correlation between AURKB expression values and the prognosis of CCA patients. P values < 0.05 were considered statistically significant; ns: not significant, *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0043] In the examples below, the human embryonic kidney cell line HEK-293T and the human cholangiocarcinoma cell lines QBC939, HuCCT1, and RBE were purchased from the China Center for Type Culture Collection. HEK-293T cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), while QBC939, HuCCT1, RBE, HCCC-9810, Huh28, TFK1, and SK-CHA-1 cells were cultured in RPMI1640 supplemented with 10% FBS.

[0044] In the following example, for a primary mouse cholangiocarcinoma cell line, KRAS was induced by HTVi. G12D In the sgp19 mouse cholangiocarcinoma model, tumor tissue was isolated and minced, and then digested in RPMI 1640 medium containing 2.5 mg / mL collagenase IV (Biosharp, BS165), 0.1 mg / mL DNase I (Biosharp, BS137), and 40 μg / mL Dispase II at 37°C for 45 minutes. After red blood cell removal, cells were expanded in RPMI 1640 medium supplemented with 1% penicillin-streptomycin and 10% FBS.

[0045] In the following examples, all cells were cultured in a humidified incubator at 37°C and 5% CO2. Mycoplasma contamination testing confirmed that all cell lines were negative. All cells were cryopreserved using serum-free rapid cell freezing medium (DesignGene Biotechnology, DG100S, Wuhan).

[0046] In the following example, the steps for a cell proliferation experiment are as follows: For the Cell Counting Kit-8 (CCK-8) assay, cells were seeded at a density of 1000 cells / 100 μL in a 96-well plate. After the cells adhered, 10 μL of CCK-8 reagent (Vazyme, A311) was added, and the cells were incubated at 37°C for 1 hour. The absorbance at 450 nm was measured using a microplate reader. For the 5-ethynyl-2'-deoxyuridine (EdU) assay, cells were seeded at a density of 5000 cells / 200 μL in a 96-well plate. After 48 hours, 50 μM EdU (RiboBio, C10310-1) was added, and the cells were incubated at 37°C for an additional 2 hours. The cells were then fixed, permeabilized, and stained, and finally, DNA staining was performed. In the colony formation assay, cells were seeded in 6-well plates at a density of 1000 cells / 2 mL. The medium was changed after about 5 days and cultured for 10-14 days. After fixation with 4% formaldehyde, the cells were stained with 0.1% crystal violet. In the drug cytotoxicity assay, 1-2×10 4Cells were seeded in 6-well plates, drugs were added 48 hours later, cultured in culture medium for 7-10 days, fixed with 4% formaldehyde, and stained with 0.1% crystal violet.

[0047] In the following example, the Combination Index (CI) is calculated as follows: A CCK8 assay was performed in QBC939 and HuCCT1 cell lines using a concentration gradient of AZD1152 and gemcitabine. CompuSyn software was used to analyze the dose-effect curves based on the Chou-Talalay method, and the CI value for the combined use of the two drugs was calculated. A CI value less than 1 indicates a synergistic effect, a value equal to 1 indicates an additive effect, and a value greater than 1 indicates an antagonistic effect.

[0048] In the following example, the CCA organoid culture method is as follows: obtain a freshly resected CCA tissue sample in the operating room, place it in sterile DMEM or RIPA-1640 medium containing double antibodies, transport it to the laboratory on ice, and wash it five times in a biosafety cabinet. The tissue is then minced with sterile scissors and digested with 2.5 mg / mL collagenase IV, 0.1 mg / mL DNase I, and 40 μg / mL Dispase II at 37°C for 30 minutes to form small cell clusters (avoid single cell dissociation). The digested tissue is inoculated in Matrigel BME2 (basement membrane extract, type 2), and expansion medium is added after the Matrigel solidifies. The expansion medium consisted of various cytokines and other components, including 1% GlutaMAX, 1% HEPES, 2% B27, 1% N2, 1.25 mM NAC, 10 mM nicotinamide, 100 ng / mL WNT3a, 50 ng / mL EGF, 100 ng / mL FGF10, 25 ng / mL HGF, 10 nM gastrin, 25 ng / mL Noggin, 5 μM A83-01, 10 μM Y-27632, 500 ng / mL R-spondin-1, 10 μM Forskolin, and 1% penicillin-streptomycin in Advanced DMEM / F-12 (Gibco, 12634010), and the medium was changed twice a week.

[0049] Example 1: Drug treatment

[0050] Human cholangiocarcinoma cell lines QBC939, HuCCT1, and RBE were treated with different concentrations of AZD1152. CCK8 assay was performed 96 hours later to calculate cell viability. The results are shown in Table 1. Figure 1A and Figure 1BThe results showed that AZD1152 inhibited the growth of bile duct cancer cells in a dose-dependent manner. The morphological changes of bile duct cancer cells after drug treatment were observed under an optical microscope. High concentrations of AZD1152 led to an increase in cell volume and a decrease in cell division ( Figure 1B ).

[0051] Example 2: Verification of drug synergistic effect (in vitro)

[0052] (1) Cell proliferation assay

[0053] CCK-8 assay: 1000 cells were seeded into each well of a 96-well plate, and gradient concentrations of AZD1152 and gemcitabine were added. After incubation for 96 hours, the medium was discarded, and CCK-8 reagent was added. The cells were incubated at 37°C for 1 hour, and the absorbance at 450 nm was measured on a microplate reader to calculate the cell viability. Figure 2A and Figure 2B As shown, the results showed that CI < 1 (such as CI = 0.6 in HuCCT1), indicating significant synergistic effect.

[0054] In addition, depending on the growth rate of cells, 1-2×10 4 Cells were seeded into 6-well plates; after 48 hours, drugs were added and the cells continued to grow in culture medium for 7 to 10 days. Subsequently, the cells were fixed with 4% formaldehyde and stained with 0.1% crystal violet and photographed. Figure 2C As shown, the results showed that the AZD1152-gemcitabine combination significantly inhibited cell viability and colony formation.

[0055] (2) Cell apoptosis experiment

[0056] Flow cytometry was used to detect the apoptotic effects of AZD1152 and gemcitabine alone or in combination on CCA cells. 5 (EDTA-free trypsin digestion), centrifuge at 300g, 4°C for 5 minutes, discard the supernatant, wash the cells twice with pre-chilled PBS, resuspend the cells in 100μl Binding Buffer, add 5μl Annexin V-FITC and 5μl PI staining solution to each sample, incubate at room temperature for 10 minutes, add 400μl 1× Binding Buffer, and mix gently. After staining, the samples were analyzed by flow cytometry within 1 hour. The results showed that the low-dose combination of the two drugs induced stronger apoptosis ( Figure 2D ).

[0057] Example 3: CCA organoid model validation

[0058] Freshly resected CCA tissue samples were obtained in the operating room and placed in sterile double-antibody medium DMEM or RIPA-1640 medium. These samples were transported to the laboratory on ice and washed five times in a biosafety cabinet. Subsequently, the tissue was cut into small pieces using sterile scissors and digested with 2.5 mg / mL collagenase IV, 0.1 mg / mL DNase I, and 40 μg / mL Dispase II at 37°C for 30 minutes to form small cell clusters while avoiding single cell dissociation. The digested tissue was inoculated into Matrigel BME2 (basement membrane extract, type 2). After the Matrigel solidified, expansion medium was added. The culture medium contains a variety of cytokines and other components, including 1% GlutaMAX, 1% HEPES, 2% B27, 1% N2, 1.25mM NAC, 10mM nicotinamide, 100ng / mL WNT3a, 50ng / mL EGF, 100ng / mL FGF10, 25ng / mL HGF, 10nM gastrin, 25ng / mL Noggin, 5μM A83-01, 10μM Y-27632, 500ng / mL R-spondin-1, 10μM Forskolin, and 1% penicillin-streptomycin in Advanced DMEM / F-12 (Gibco, 12634010). The culture medium is changed twice a week. Once organoids have formed, they are re-digested and treated with drugs (organoids are treated with AZD1152 (0.1–1μM) and gemcitabine (1–10μM). The treated organoids are then seeded into 48-well plates. Once the organoids have grown to the appropriate size, images are captured and their diameters are measured.

[0059] The results are as follows Figure 3 As shown, the results showed that AZD1152 alone inhibited organoid growth and showed a synergistic effect when combined with gemcitabine.

[0060] Example 4: In vivo efficacy verification (PDX model)

[0061] Fresh tumor tissues from patients with cholangiocarcinoma after surgery were collected, cut into small pieces with a diameter of about 3 mm, and inoculated into the armpits of NCG mice, which is the F0 generation. After the tissue grows to a volume of 100-150 cubic millimeters, the tissue is further cut into small pieces and passaged to NCG mice to obtain the F1 generation. By the F2 generation, the formal drug administration experiment can be started. After the F2 generation is inoculated, drug administration begins on the 10th day. AZD1152 solution (solvent is 5% DMSO + 40% PEG300 + 5% Tween + 50% ddH2O) (25 mg / kg) and gemcitabine solution (solvent is normal saline) (20 mg / kg) are intraperitoneally injected once every three days (without time interval) (refer to Figure 4A). Tumor growth was then monitored, and the length (L) and width (W) of the tumor were measured. The tumor volume was calculated using the formula V = 0.5 × L × W2. Figure 4B and Figure 4C As shown, the results showed that the tumor growth inhibition of the combination group was significantly better than that of the single drug.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. A pharmaceutical composition, characterized in that Including AZD1152 and gemcitabine.

2. A drug, characterized in that Comprising the pharmaceutical composition according to claim 1.

3. The drug according to claim 2, characterized in that The dosage forms of the drug include oral solution, injection, granules, tablets, powder, capsules, emulsion, spray or patch.

4. Use of the pharmaceutical composition according to claim 1 in preparing a drug for treating bile duct cancer.

5. The use according to claim 4, characterized in that The pharmaceutical composition has the following functions: (a) It has the effect of inhibiting the growth of bile duct cancer cells; (b) It has the effect of inhibiting the growth of bile duct cancer tumors.

6. The use according to claim 5, characterized in that The cholangiocarcinoma cells include one or more of QBC939, HuCCT1, or RBE.

7. The use according to claim 5 or 6, characterized in that The dosage forms of the drug include oral solution, injection, granules, tablets, powder, capsules, emulsion, spray or patch.

Images