Use of AZD1152 and gemcitabine in the preparation of a medicament for treating cholangiocarcinoma

Abstract

The present invention belongs to the technical field of cancer pharmaceuticals, and specifically relates to the use of AZD1152 and gemcitabine in the preparation of a drug for treating cholangiocarcinoma. AZD1152 and gemcitabine exhibit a synergistic effect in the treatment of cholangiocarcinoma. The combination of AZD1152 with gemcitabine can significantly inhibit cholangiocarcinoma cell proliferation and tumor growth, with efficacy superior to that of AZD1152 or gemcitabine alone.

Description

l Use of AZDl 152 and gemcitabine in the preparation of a medicament for treating Cholangiocarcinoma TECHNICAL FIELD The present invention belongs to the technical eld of cancer pharmaceuticals, specically relating to the use of AZD1152 and gemcitabine in the preparation of a medicament for treating cholangiocarcinoma. BACKGROUND Cholangiocarcinoma is a highly aggressive malignant tumor originating from biliary epithelial cells, with an incidence rate that has been increasing year by year. Due to its insidious early symptoms and difculty in diagnosis, approximately 70% of patients are already in advanced stages or have developed metastases at the time of diagnosis. Currently, the rst-line standard treatment for advanced Cholangiocarcinoma is mainly based on gemcitabine combined with platinum-based drugs. However, the objective response rate (OR) is only about 15 - 30%, and the median overall survival (OS) is generally less than 12 months. Existing chemotherapy regimens are associated with high drug resistance and signicant toxic side effects, highlighting the urgent need for the development of more effective treatment strategies. In recent years, studies have found that abnormal expression of Aurora kinase B is associated with chemoresistance in Cholangiocarcinoma, suggesting it may be a potential target to overcome the therapeutic limitations 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 reported in the prior art. SUMMARY Based on this, the present invention has found that AZDl 152 and gemcitabine have a synergistic effect in the treatment of cholangiocarcinoma. They can be formulated into a medicament for treating Cholangiocarcinoma, signicantly inhibiting Cholangiocarcinoma tumor cells and tumor growth, with efcacy signicantly superior to that of monotherapy. To achieve the above purpose, the present invention may adopt the following technical solutions: In one aspect, the present invention provides a pharmaceutical composition comprising AZD1152 and gemcitabine. In another aspect, the present invention provides a medicament comprising the above pharmaceutical composition. Preferably, the dosage form of the above medicament includes oral solution, injection, granule, tablet, powder, capsule, emulsion, spray, or patch. In another aspect, the present invention provides the use of the above pharmaceutical composition in the preparation of a medicament for treating cholangiocarcinoma. Preferably, in the above use, the pharmaceutical composition has the following functions: (a) it has the effect of inhibiting the growth of Cholangiocarcinoma cells; (b) it has the effect of inhibiting Cholangiocarcinoma tumor growth. Preferably, in the above use, the Cholangiocarcinoma cells include one or more selected from QBC939, HuCCTl, or RBE. Preferably, in the above use, the dosage form of the medicament includes oral solution, injection, granule, tablet, powder, capsule, emulsion, spray, or patch. The benecial effects of the present invention include: AZD1152 and gemcitabine exhibit a synergistic effect in the treatment of cholangiocarcinoma. AZD1152 in combination with gemcitabine can signicantly inhibit Cholangiocarcinoma cells and tumor growth, and the effect is superior to that of AZDl 152 or gemcitabine alone. BRIEF DESCRIPTION OF THE FIGURES Figure 1A shows the changes in cell activity after treating human cholangiocarcinoma cell lines with different concentrations of AZD1 152; Figure 1B shows the changes in cell volume and clone formation ability after treating human cholangiocarcinoma cell lines with different concentrations of AZD1 152; Figure 2A shows the heat map of the combined effects of different concentrations of AZD1 152 and gemcitabine on human cholangiocarcinoma cell lines; Figure 2B shows the scatter plot of the combined effects of different concentrations of AZD1 152 and gemcitabine on human cholangiocarcinoma cell lines; Figure 2C shows the inhibition of colony formation by the AZD1152-gemcitabine combination; Figure 2D shows the effect of the AZDllSZ-gemcitabine combination on cell apoptosis; Figure 3 shows the inhibition of CCA organoids by the AZD1 152-gemcitabine combination; Figure 4A shows the schematic diagram of the in vivo efcacy verication process of the PDX model; Figure 4B shows the changes in tumor volume of the PDX model after different medications; Figure 4C shows the tumor conditions of the PDX model after different medications. DETAILED DESCRIPTION OF THE INVENTION The examples given are intended to better illustrate the present invention, but the content of the present invention is not limited to the examples given. Therefore, those familiar with the art may make nonessential improvements and adjustments to the implementation scheme according to the above invention content, which still belongs to the protection scope of the present invention. The terms used herein are only used to describe specic embodiments and are not intended to limit the present disclosure. Unless the context has a signicantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "including", "having", "including" 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 specication 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. In a rst aspect, the embodiment of the present invention provides a pharmaceutical composition, comprising AZD1152 and gemcitabine. It should be noted that AZD1152 and gemcitabine have a synergistic therapeutic effect on cholangiocarcinoma. AZD1152 in combination with gemcitabine can signicantly inhibit cholangiocarcinoma cells and tumor growth, and the effect is superior to that of AZD1 152 or gemcitabine alone. It should be noted that, in the pharmaceutical composition of the present invention, AZD1152 and gemcitabine may be packaged separately or may be formulated into the same substance. In addition, the pharmaceutical composition of the present invention may further include other active substances capable of treating cholangiocarcinoma. In a second aspect, the embodiment of the present invention provides a medicament, which comprises the above pharmaceutical composition. It should be noted that the pharmaceutical composition of the present invention may be formulated into medicaments of different dosage forms by adding different excipients to adapt to different use scenarios. The dosage form of the medicament may include oral solution, injection, granule, tablet, powder, capsule, emulsion, spray, or patch, etc. In addition, the excipients used in the above different dosage forms are all wellknown to those skilled in the art. For example, tablet preparation mainly uses diluents (such as starch, dextrin, sucrose, or mannitol, etc.), absorbents (such as calcium sulfate, dibasic calcium phosphate, or light magnesium oxide, etc.), binders (such as povidone, syrup, or hydroxypropyl methylcellulose, etc.), wetting agents (such as water, etc.), or disintegrants (such as dry starch, sodium starch glycolate, or crospovidone, etc.); for example, liquid oral preparations mainly use solubilizers, suspending agents, emulsiers, or colorants, etc. In a third aspect, the embodiment of the present invention provides the use of the above pharmaceutical composition in the preparation of a medicament for treating cholangiocarcinoma. In some specic examples, in the above use, the pharmaceutical composition has the following functions: (a) it has the effect of inhibiting the growth of cholangiocarcinoma cells; (b) it has the effect of inhibiting cholangiocarcinoma tumor growth. In some specic examples, in the above use, the cholangiocarcinoma cells include one or more selected from QBC939, HuCCTl, or RBE. In some specic examples, in the above use, the dosage form of the medicament includes oral solution, injection, granule, tablet, powder, capsule, emulsion, spray, or patch. It should be noted that, as mentioned above, in the above use, the pharmaceutical composition may also be formulated into medicaments of different dosage forms by adding different excipients to adapt to different use scenarios. The dosage form of the medicament may include oral solution, injection, granule, tablet, powder, capsule, emulsion, spray, or patch, etc. In addition, the excipients used in the above different dosage forms are all wellknown to those skilled in the art. For example, tablet preparation mainly uses diluents (such as starch, dextrin, sucrose, or mannitol, etc.), absorbents (such as calcium sulfate, dibasic calcium phosphate, or light magnesium oxide, etc.), binders (such as povidone, syrup, or hydroxypropyl methylcellulose, etc.), wetting agents (such as water, etc.), or disintegrants (such as dry starch, sodium starch glycolate, or crospovidone, etc.); for example, liquid oral preparations mainly use solubilizers, suspending agents, emulsiers, or colorants, etc. To better understand the present invention, the following specic examples further illustrate the content of the invention, but the content of the invention is not limited to the following examples. In the following examples, statistical analysis was performed using R software (version 4.3.1). Normally distributed data were analyzed by two-tailed Student s t-test or one-way ANOVA. For non-normally distributed data, the Mann - Whitney U test (for comparison between two groups) or the Kruskal - Wallis H test (for comparison among multiple groups) was used. The correlation between prognosis and AURKB expression was assessed using the log-rank test and Cox regression analysis. For publicly available prognostic data, the Survminer R package was used to analyze the correlation between AURKB expression values and the prognosis of CCA patients. A p-value < 0.05 was considered statistically signicant; ns: not signicant, *: P < 0.05, **: P < 0.01, ***: P < 0.001. In the following examples, the human embryonic kidney cell line HEK-293T and human cholangiocarcinoma cell lines QBC939, HuCCTl, and RBE were purchased from the China Center for Type Culture Collection. HEK-293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), while QBC939, HuCCTl, RBE, HCCC-9810, Huh28, TFKl, and SK-CHA-l cells were cultured in RPMI 1640 medium containing 10% FBS. In the following examples, for primary mouse cholangiocarcinoma cell lines, tumor tissues were isolated and minced from HTViinduced KRASGlzD / sgpl9 mouse cholangiocarcinoma models. The tissue was digested at 37 ° C for 45 minutes in RPMI 1640 medium containing 2.5 mg / mL collagenase IV (Biosharp, BS165) 0.1 mg / mL DNase I (Biosharp, BSl37), and 40 ug / mL Dispase II. After red blood cell removal, the cells were expanded in RPMI 1640 medium supplemented with 1% penicillin streptomycin and 10% FBS. All cells were cultured in a humidied incubator at 37 ° C with 5% C02. Mycoplasma contamination testing conrmed that all cell lines were negative. All cells were cryopreserved using serumfree rapid cell freezing solution (DesignGene Biotechnology, DGlOOS, Wuhan). Cell proliferation experiments were performed as follows: CCK-8 assay: Cells were seeded in 96-well plates at a density of 1,000 cells / 100 uL per well. After attachment, 10 uL of CGK-8 reagent (Vazyme, A311) was added and incubated at 37° C for 1 hour. Absorbance at 450 nm was measured using a microplate reader. Colony formation assay: Cells were seeded at a density of 1,000 cells / 2 mL per well in 6-well plates. Medium was refreshed once after ~5 days, and cells were cultured for another 10 - 14 days. Colonies were xed with 4% paraformaldehyde and stained with O. 1% crystal violet. Drug cytotoxicity assay: Depending on cell growth rates, 1 - 2 >< 104 cells were seeded in 6-well plates. After 48 hours, drugs were added, and cells were cultured for 7 _ 10 days. Colonies were xed with 4% paraformaldehyde and stained with 0.1% crystal violet. Drug combination index (CI) was calculated as follows: AZD1152 and gemcitabine were each applied in concentration gradients to QBC939 and HuCCTl cell lines for CCK-8 assays. CI values were calculated using CompuSyn software based on the Chou-Talalay method, analyzing dose-effect curves. A CI value <1 indicates synergism, =1 indicates additive effect, and >1 indicates antagonism. CCA organoid culture was performed as follows: Freshly resected CCA tissue samples were collected in the operating room and transported on ice to the laboratory in sterile DMEM or RPMI1640 medium containing antibiotics. Samples were washed ve times in a biosafety cabinet, minced with sterile scissors, and digested for 30 minutes at 37° C in a solution of 2.5 mg / mL collagenase IV, 0.1 mg / mL DNase I, and 40 ug / mL Dispase II to form small cell clusters (avoiding full single-cell dissociation). Digested tissue was embedded in Matrigel BME2 (basement membrane extract, type 2), and upon solidication, expansion medium was added. The expansion medium was composed of Advanced DMEM / F-12 (Gibco, 12634010) supplemented with 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 uM A83-01, 10 uM Y-27632, 500 ng / mL Rspondin-l, 10 uM forskolin, and 1% penicillin-streptomycin. Medium was refreshed twice per week. Example 1: Drug treatment Cholangiocarcinoma cell lines QBC939, HuCCTl, and RBE were treated with various concentrations of AZD1 152. After 96 hours, CCK-8 assays were conducted to calculate cell viability. As shown in Figures 1A and 1B, the results demonstrated that AZD1152 inhibited cholangiocarcinoma cell proliferation in a dose-dependent manner. Morphological changes of cholangiocarcinoma cells were observed under an optical microscope after treatment. High concentrations of AZD1152 led to increased cell volume and decreased mitotic activity (Figure 1B). Example 2: Validation of drug synergy (In Vitro) (1) Cell Proliferation assay CCK8 Assay: Cells were seeded at 1,000 cells / well in 96-well plates and treated with gradient concentrations of AZD1152 and gemcitabine. After 96 hours of incubation, the medium was discarded, and CCK-8 reagent was added. The plates were incubated at 37° C for 1 hour, and absorbance was measured at 450 nm using a microplate reader to calculate cell viability. As shown in Figures 2A and 2B, the results indicated a CI < l (e.g., CI = 0.6 in HuCCTl cells), demonstrating a signicant synergistic effect. In addition, based on the cell growth rate, 1 - 2 >< 104 cells were seeded in 6 well plates. After 48 hours, drugs were added, and the cells were cultured in medium for an additional 7 - 10 days. Cells were then xed with 4% paraformaldehyde and stained with 0.1% crystal violet for imaging. As shown in Figure 2C, the AZD1152 _ gemcitabine combination signicantly inhibited cell viability and colony formation. (2) Cell apoptosis assay Flow cytometry was used to evaluate apoptosis in CCA cells treated with AZD1152, gemcitabine, or their combination. Both supernatant and adherent cells (1 _ 5 >< 105, without EDTA trypsin digestion) were collected, centrifuged at 300g at 4° C for 5 minutes, and washed twice with pre-cooled PBS. Cells were resuspended in 100 uL Binding Buffer. Then, 5 uL Annexin V-FITC and 5 uL PI staining solution were added, and samples were incubated at room temperature for 10 minutes. Subsequently, 400 uL of 1 >< Binding Buffer was added and mixed gently. The samples were analyzed by ow cytometry within 1 hour. The results showed that low-dose combination therapy induced stronger apoptosis (Figure 2D). Example 3: Verication in CCA organoid model Freshly resected CCA tissue samples were collected in the operating room and placed in sterile DMEM or RIPA-1640 medium supplemented with double antibiotics. Samples were transported on ice to the laboratory and washed ve times under a biosafety cabinet. Tissue was minced with sterile scissors and digested at 37° C for 30 minutes using a solution containing 2.5 mg / mL collagenase IV, 0.1 mg / mL DNase I, and 40 ug / mL Dispase 11, to form small cell clusters while avoiding complete single-cell dissociation. The digested tissue was seeded into Matrigel BME2 (basement membrane extract, type 2). After Matrigel solidied, expansion medium was added. The expansion medium contained multiple growth factors and supplements, including Advanced DMEM / F-12 (Gibco, 12634010) supplemented with 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 uM A8301, 10 uM Y 27632, 500 ng / mL Rspondinl, 10 uM forskolin, and 1% penicillinstreptomycin. Medium was changed twice weekly. Once organoids had formed, they were redigested and treated with drugs: AZD1152 (0.1 _ 1 uM) and gemcitabine (1 - 10 uM). Treated organoids were seeded into 48-well plates. When organoids grew to an appropriate size, images were acquired and diameters measured. As shown in Figure 3, AZD1152 monotherapy suppressed organoid growth, and combination with gemcitabine demonstrated a synergistic effect. Example 4: In Vivo efcacy verication (PDX Model) Fresh post-operative cholangiocarcinoma tumor tissues from patients were cut into small pieces (~3 mm in diameter) and implanted subcutaneously into the axilla of NCG mice, forming the F0 generation. Once tumors grew to 100 _ 150 mm3 in volume, they were passaged into NCG mice to obtain the F1 generation. The F2 generation was used for drug administration experiments. Ten days after F2 implantation, treatment was initiated. AZD1152 was prepared in a solvent mixture of 5% DMSO + 40% PEG300 + 5% Tween + 50% ddHzO and administered intraperitoneally at 25 mg / kg every three days. Gemcitabine was dissolved in saline and administered intraperitoneally at 20 mg / kg on the same schedule. (See Figure 4A for protocol illustration.) Tumor growth was monitored regularly, with measurements of tumor length (L) and width (W). Tumor volume was calculated using the formula: V = 0.5 >< L >< W2 . As shown in Figures 4B and 4C, the combination therapy group exhibited signicantly greater tumor growth inhibition compared to either monotherapy group. The above embodiments are provided for illustrative purposes only and do not limit the scope of the present invention. While the invention has been described in detail with reference to preferred embodiments, those skilled in the art will appreciate that modications or equivalent substitutions can be made without departing from the spirit and scope of the invention, which should be encompassed by the appended claims.

Claims

1. Pharmaceutical composition, characterized by comprising AZD1152 and gemcitabine.

2. Pharmaceutical agent, characterized by comprising the pharmaceutical composition according to claim 1.

3. Pharmaceutical agent according to claim 2, characterized in that the dosage form of the pharmaceutical agent an oral solution, injection, granules, tablet, powder, capsules, emulsion, spray or patch included.

4. Use of the pharmaceutical composition according to claim 1 in the preparation of a drug for the treatment of cholangiocarcinoma.

5. Use according to claim 4, characterised in that the pharmaceutical composition comprises the following has functions: (a) inhibiting the growth of cholangiocarcinoma cells; (b) inhibiting the growth of cholangiocarcinoma tumors.

6. Use according to claim 5, characterised in that the cholangiocarcinoma cells contain one or more of QBC939, HuCCTl or RBE included.

7. Use according to claim 5 or 6, characterised in that the dosage form of the medicinal product an oral solution, injection, granules, tablets, powder, capsules, emulsion, spray or patch. 1 / 7 Figure1A Figure1B