Pharmaceutical composition for reversing pancreatic cancer gemcitabine drug resistance and application thereof

Abstract

The invention discloses a pharmaceutical composition for reversing pancreatic cancer gemcitabine drug resistance and application of the pharmaceutical composition, and belongs to the technical field of biological medicine. The composition comprises a generic FGFR inhibitor delatinib and gemcitabine, the concentration of the delatinib is 25 to 50 mg / kg, and the concentration of the gemcitabine is 20 to 30 mg / kg. By inhibiting an FGFR2 / FGFR3 signal channel and reducing the expression of FGFR protein in drug-resistant cells, the chemosensitivity of gemcitabine is synergistically enhanced, the apoptosis induction rate is increased by more than two times, and the synergic index (CI) is less than 1. In-vitro and nude mouse transplantation tumor experiments prove that the composition can significantly inhibit the proliferation of gemcitabine drug-resistant pancreatic cancer cells. The Gemcitabine drug-resistant pancreatic cancer drug can be used for preparing a drug for treating Gemcitabine drug-resistant pancreatic cancer with high FGFR3 expression, patients are screened through immunohistochemistry or gene sequencing, intravenous injection or oral administration is adopted, dosage forms comprise freeze-dried powder injection, capsules or tablets, and a new strategy is provided for pancreatic cancer drug-resistant treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a pharmaceutical composition for reversing gemcitabine resistance in pancreatic cancer and its application. Background Art

[0002] Pancreatic cancer is a malignant tumor originating from pancreatic ductal epithelial and acinar cells. The cause of its occurrence is not yet fully understood, but studies have shown that factors such as long-term smoking, poor eating habits, excessive body mass index, and chronic damage to the pancreas may increase the risk of pancreatic cancer. The symptoms of pancreatic cancer generally appear only when the disease progresses to the advanced stage, including jaundice, weight loss, and abdominal discomfort or pain.

[0003] Gemcitabine is a first-line chemotherapeutic drug for pancreatic ductal adenocarcinoma (PDAC), but its clinical application often fails due to the development of drug resistance in tumor cells, resulting in poor prognosis for patients. Therefore, developing new strategies to effectively overcome gemcitabine resistance is crucial for improving the treatment outcome of pancreatic cancer patients.

[0004] The fibroblast growth factor receptor (FGFR) signaling pathway plays a key role in the occurrence and development of various tumors. FGFR alterations detected in solid tumors include FGFR1 amplification in non-small cell lung cancer (20%), FGFR1 / 2 amplification in breast cancer (7 - 23%), FGFR3 mutations (10 - 60%) or FGFR3 fusions (6%) in urothelial carcinoma, FGFR2 fusions in intrahepatic cholangiocarcinoma (10 - 20%), FGFR2 mutations in endometrial cancer (12%), and FGFR2 amplification in gastric cancer (5 - 10%). Alterations in the FGFR gene can be detected in approximately 7.0% of unselected cancer patients. Existing studies have shown that the abnormal activation of the FGFR signaling pathway (especially FGFR2 / FGFR3) is one of the key mechanisms of gemcitabine resistance. For example, in gemcitabine-resistant pancreatic cancer cells, the expression of FGFR3 is significantly increased, and the high expression of FGFR2 and FGFR3 is associated with poor prognosis (such as shortened overall survival) and low tumor differentiation in pancreatic cancer patients.

[0005] Currently, a variety of FGFR-targeted drugs have been developed, including pan-FGFR inhibitors (such as erdafitinib and futibatinib), FGFR1 / 2 / 3 inhibitors (such as infigratinib and pemigatinib), and a series of more specific drugs, some of which have entered clinical use. Erdafitinib has been approved for urothelial cancer patients with FGFR2 / 3 mutations, and futibatinib and pemigatinib are approved for treating cholangiocarcinoma patients with FGFR2 fusion or rearrangement. The clinical benefits of these drugs are limited to some extent by hyperphosphatemia, which is due to off-target inhibition of FGFR1 and the emergence of resistance mutations in the FGFR gene. Next-generation small molecule inhibitors, such as lirafugratinib and LOXO-435, and the FGFR2-specific antibody bemarituzumab, are expected to reduce the risk of hyperphosphatemia and have the ability to overcome certain resistance mutations.

[0006] Derazantinib is an oral pan-FGFR inhibitor with inhibitory activity against FGFR1, FGFR2, and FGFR3. Although FGFR inhibitors (such as erdafitinib) have been applied in other cancers, the specific role, synergistic mechanism, and clinical application potential of derazantinib in reversing gemcitabine resistance in pancreatic cancer are not yet clear. Single chemotherapy drugs (such as gemcitabine) have limited efficacy against drug-resistant pancreatic cancer, and there is a lack of effective strategies for reversing drug resistance.

[0007] Recent studies have shown that thiol oxidative stress (such as by inhibiting glutathione reductase GR) can degrade specific drug resistance-related proteins (such as TGM2) and enhance chemotherapy sensitivity. Although this mechanism is mainly targeted at colorectal cancer, its idea provides a technical inspiration for reversing drug resistance by regulating specific targets.

[0008] Therefore, there is an urgent need in this field to develop a new treatment plan that can effectively reverse gemcitabine resistance in pancreatic cancer and clarify its mechanism of action. Summary of the Invention

[0009] The object of the present invention is to provide a pharmaceutical composition for reversing gemcitabine resistance in pancreatic cancer by targeting fibroblast growth factor receptor (FGFR), a pharmaceutical composition containing a pan-FGFR inhibitor, and its application method.

[0010] To achieve the above object, the present invention adopts the following technical solutions: A pharmaceutical composition for reversing gemcitabine resistance in pancreatic cancer, comprising a pan-FGFR inhibitor and gemcitabine, wherein: The concentration of the pan-FGFR inhibitor is 25 - 50 mg / kg; The concentration of gemcitabine is 20 - 30 mg / kg; The above composition inhibits the FGFR2 / FGFR3 signaling pathway, reduces the expression of FGFR protein in drug-resistant pancreatic cancer cells, and synergistically enhances the chemosensitivity of gemcitabine.

[0011] Preferably, the above pan-FGFR inhibitor is derazantinib or a pharmaceutically acceptable salt, solvate or prodrug thereof. Pharmaceutically acceptable salts may include salts formed with inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc.) or organic acids (such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, acetic acid, citric acid, malic acid, tartaric acid, succinic acid, fumaric acid, etc.). Solvates may include hydrates. A prodrug refers to a compound that can be converted into the active form of derazantinib in vivo.

[0012] Preferably, the synergy index (CI) of the above composition is less than 1, determined by the CCK-8 method.

[0013] Preferably, the above composition achieves its therapeutic effect by inducing apoptosis in drug-resistant pancreatic cancer cells, with the apoptosis rate more than 2 times higher than that of the single-drug group, and accompanied by the cleavage activation of Caspase-3 and PARP.

[0014] The present invention also provides an application of a pharmaceutical composition in the preparation of a drug for treating gemcitabine-resistant pancreatic cancer.

[0015] Preferably, the indication of the above drug is for patients with pancreatic ductal adenocarcinoma (PDAC) with high expression of FGFR3, and the applicable population is screened by immunohistochemistry (IHC) or gene sequencing.

[0016] Preferably, the above drug is administered by intravenous injection or orally, and the dosage forms include freeze-dried powder injections, capsules or tablets, supplemented with pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers may include fillers, binders, disintegrants, lubricants, diluents, sweeteners, flavoring agents, coating materials, etc.

[0017] The present invention also provides an application of a pharmaceutical composition for reversing gemcitabine resistance in pancreatic cancer, including the following steps: Detect the expression level of FGFR2 / FGFR3 in the patient's tumor tissue; Administer the above pharmaceutical composition to patients with high FGFR expression, and the dosing ratio of derazantinib to gemcitabine is 1:50 to 1:150 (molar ratio).

[0018] Preferably, the daily dose of the above-mentioned delatinib is 50 - 200 mg / kg, the daily dose of gemcitabine is 800 - 1200 mg / kg, and the combined treatment cycle is 21 days.

[0019] The beneficial effects of the present invention are as follows: 1. Effectively reverse drug resistance and synergistically enhance efficacy: The pharmaceutical composition provided by the present invention can effectively overcome the drug resistance of gemcitabine by combining delatinib and gemcitabine, and the combination of the two shows a synergistic anti-tumor effect (such as the combination index CI < 1), significantly enhancing the killing effect on drug-resistant pancreatic cancer cells.

[0020] 2. Clear mechanism of action: The present invention reveals that the mechanism of action of the pharmaceutical composition lies in that delatinib inhibits the FGFR2 / FGFR3 signaling pathway, reduces the expression levels of FGFR2 and FGFR3 proteins in drug-resistant cells, thereby restoring or enhancing the sensitivity of cells to gemcitabine.

[0021] 3. Potently induce apoptosis: The composition can significantly induce apoptosis in drug-resistant pancreatic cancer cells, with a substantial increase in the apoptosis rate compared to single-drug treatment, accompanied by the cleavage activation of key apoptosis execution proteins such as Caspase-3 and PARP, confirming its pro-apoptotic effect at the molecular level.

[0022] 4. Potential for individualized treatment: The present invention suggests that by detecting the expression levels of FGFR2 / FGFR3 (especially FGFR3) in tumor tissues, pancreatic cancer patients who are most likely to benefit from this combined treatment regimen can be screened out, providing a basis for achieving individualized precision treatment.

[0023] 5. Feasibility of clinical application: The present invention provides a specific dosage range, dosing ratio, treatment cycle, as well as suitable pharmaceutical dosage forms and administration routes, laying a foundation for the clinical development and application of the pharmaceutical composition. Description of the Drawings

[0024] Figure 1 The results of testing the inhibition rate of a pancreatic cancer cell line using a drug library, showing that delatinib is the drug with the highest average inhibition rate.

[0025] Figure 2 A volcano plot obtained by analyzing BxPC-3 cells (human pancreatic cancer cell line) in the dataset GEO: GSE140077, showing differentially expressed genes, where FGFR3 (indicated by black dots) is significantly upregulated, where the X-axis (log2foldchange): logarithm of the fold change in gene expression, and the Y-axis (-log 10 (p - value)): negative logarithm transformation of the statistical test P value; red (up): significantly upregulated genes; green (down): significantly downregulated genes;.

[0026] Figure 3 Comparison of the expression levels of FGFR3 in gemcitabine-resistant (GR) and gemcitabine-sensitive (WT) BxPC-3 cells (human pancreatic cancer cell line) from the GEO dataset, where the Y-axis is the FGFR3 expression level (FGFR3 expression normalized) and the X-axis is the grouping.

[0027] Figure 4 Venn diagram for analyzing gemcitabine-resistant genes in pancreatic cancer through public databases, showing that in 3 sequencing datasets (GSE140077, GSE152121, GSE79953), the middle overlapping region (49 genes): is the gene FGFR3 that is differentially expressed in common among the 3 datasets.

[0028] Figure 5 Kaplan-Meier analysis graph of FGFR2 expression and overall survival (OS) of pancreatic cancer patients, where P < 0.01 indicates statistical significance, HR (Hazard Ratio), HR = 0.426.

[0029] Figure 6 Kaplan-Meier analysis graph of disease-free survival (DFS) at different FGFR2 gene expression levels, where P < 0.05 indicates statistical significance, HR = 0.44.

[0030] Figure 7 Kaplan-Meier analysis graph of FGFR3 expression and overall survival (OS) of pancreatic cancer patients, where P < 0.05 indicates statistical significance, HR = 0.51.

[0031] Figure 8 Kaplan-Meier analysis graph of disease-free survival (DFS) at different FGFR3 gene expression levels, where P < 0.05 indicates statistical significance, HR = 0.47.

[0032] Figure 9 Scatter plot of the correlation between tumor differentiation degree and the proportion of FGFR2-positive cells (Positive Cells (%)), where P < 0.05 indicates statistical significance, r is the Pearson correlation coefficient, and r = 0.31 indicates a weak positive correlation between the two.

[0033] Figure 10 It is a scatter plot of the correlation between tumor differentiation and the proportion of FGFR3 positive cells (Positive Cells (%)). Among them, P<0.05 indicates statistical significance, r is the Pearson correlation coefficient, and r = 0.30 indicates a weak positive correlation between the two.

[0034] Figure 11 It is the expression level of the FGFR2 gene in different sample groups. Y-axis (vertical axis): H-score (immunohistochemical score), which is an index for quantifying the protein expression level in pathological examinations. The higher the value, the stronger the expression of the FGFR2 protein; X-axis (horizontal axis): Two groups of sample classifications: T = 56: Usually represents the tumor tissue group (Tumor), with a sample size of 56 cases; N = 43: Usually represents the normal tissue group adjacent to cancer (Normal) or non-tumor tissue group, with a sample size of 43 cases; The expression level of FGFR2 in pancreatic cancer tumor tissue (T) compared with the normal tissue adjacent to cancer (N), where * indicates statistical significance.

[0035] Figure 12 It is the statistical analysis of FGFR2 immunohistochemical staining in gemcitabine-resistant (GEM-resistance) and gemcitabine-sensitive (GEM-sensitivity) tissues. Y-axis (vertical axis): H-score (immunohistochemical score), which is an index for quantifying the protein expression level in pathological examinations. The higher the value, the stronger the expression of the FGFR2 protein, where ** indicates statistical significance.

[0036] Figure 13 It is a representative image of FGFR2 immunohistochemical staining in gemcitabine-resistant (GEM-resistance) and gemcitabine-sensitive (GEM-sensitivity) tissues.

[0037] Figure 14 It is the statistical analysis of FGFR3 immunohistochemical staining in gemcitabine-resistant (GEM-resistance) and gemcitabine-sensitive (GEM-sensitivity) tissues. Y-axis (vertical axis): H-score (immunohistochemical score), which is an index for quantifying the protein expression level in pathological examinations. The higher the value, the stronger the expression of the FGFR2 protein, where * indicates statistical significance.

[0038] Figure 15Representative images of FGFR3 immunohistochemical staining in gemcitabine-resistant (GEM-resistance) and gemcitabine-sensitive (GEM-sensitivity) tissues.

[0039] Figure 16 IC50 values of AsPC-1 / GEM, AsPC-1, BxPC-3 / GEM, and BxPC-3 cells after 48 h of gemcitabine treatment. Among them, AsPC - 1 / GEM and BxPC - 3 / GEM are gemcitabine-resistant strains, and AsPC - 1 and BxPC - 3 are parental cell lines.

[0040] Figure 17 Combination Index (CI) plot and related data of the inhibitory effects of deratinib and gemcitabine at gradient concentrations on AsPC-1 / GEM cells. The ordinate is CI, and the abscissa is Fa (drug concentration fraction).

[0041] Figure 18 Combination Index (CI) plot and related data of the inhibitory effects of deratinib and gemcitabine at gradient concentrations on BxPC-3 / GEM cells. The ordinate is CI, and the abscissa is Fa (drug concentration fraction).

[0042] Figure 19 Photographs of the results of the colony formation assay of AsPC-1 / GEM cells treated with gemcitabine and deratinib alone or in combination.

[0043] Figure 20 Statistical chart of the results of the colony formation assay of AsPC-1 / GEM cells treated with gemcitabine and deratinib alone or in combination. Among them, CTRL is the control group, DE is deratinib, GEM is gemcitabine, and DE+GEM is the combination treatment.

[0044] Figure 21 Photographs of the results of the colony formation assay of BxPC-3 / GEM cells treated with gemcitabine and deratinib alone or in combination.

[0045] Figure 22 Statistical chart of the results of the colony formation assay of BxPC-3 / GEM cells treated with gemcitabine and deratinib alone or in combination. Among them, CTRL is the control group, DE is deratinib, GEM is gemcitabine, and DE+GEM is the combination treatment.

[0046] Figure 23Statistical chart of the apoptosis rate detection results of gemcitabine-resistant cells AsPC-1 / GEM under treatment with deratinib and gemcitabine alone or in combination, where: PBS: blank control (only phosphate buffer was added); DE 0.5 μM, DE 1 μM: deratinib used alone (DE, concentration 0.5 / 1 μM); GEM200 nM: gemcitabine used alone (GEM, concentration 200 nM); GEM200 nM + DE 0.5 μM, GEM200 nM + DE 1 μM: gemcitabine and deratinib used in combination (different concentration combinations), where ** indicates P < 0.01 and **** indicates P < 0.0001.

[0047] Figure 24 Statistical chart of the apoptosis rate detection results of gemcitabine-resistant cells BxPC-3 / GEM under treatment with deratinib and gemcitabine alone or in combination, where: PBS: blank control (only phosphate buffer was added); DE 0.5 μM, DE 1 μM: deratinib used alone (DE, concentration 0.5 / 1 μM); GEM200 nM: gemcitabine used alone (GEM, concentration 200 nM); GEM200 nM + DE 0.5 μM, GEM200 nM + DE 1 μM: gemcitabine and deratinib used in combination (different concentration combinations), where ** indicates P < 0.01 and **** indicates P < 0.0001.

[0048] Figure 25 Western Blot detection results of the protein expression levels of FGFR2 and FGFR3 in AsPC-1 / GEM and BxPC-3 / GEM cells after treatment with different concentrations of deratinib. Among them, GAPDH is the internal reference protein, CTRL is the blank control without drug treatment. The left column is AsPC-1 / GEM, which is the gemcitabine (GEM)-resistant strain of pancreatic cancer cell line AsPC-1, and the right column is BxPC-3 / GEM, which is the gemcitabine (GEM)-resistant strain of pancreatic cancer cell line BxPC-3.

[0049] Figure 26 Semi-quantitative analysis chart of the protein expression levels of FGFR2 and FGFR3 in AsPC-1 / GEM and BxPC-3 / GEM cells after treatment with different concentrations of deratinib, where * (P < 0.05), ** (P < 0.01), **** (P < 0.0001). The upper left and lower left figures use AsPC-1 / GEM, which is the gemcitabine (GEM)-resistant strain of pancreatic cancer cell line AsPC-1, and the upper right and lower right figures use BxPC-3 / GEM, which is the gemcitabine (GEM)-resistant strain of pancreatic cancer cell line BxPC-3.

[0050] Figure 27 Representative gross photographs of subcutaneous xenografts in nude mice treated with the control group, gemcitabine group, derazantinib group, and gemcitabine + derazantinib combination group.

[0051] Figure 28 Growth curves of subcutaneous xenografts in nude mice of each treatment group.

[0052] Figure 29 Statistical chart of tumor weights of subcutaneous xenografts in nude mice of each treatment group, where * (P < 0.05), ** (P < 0.01), *** (P < 0.001), **** (P < 0.0001).

[0053] Figure 30 Statistical chart of tumor volumes of subcutaneous xenografts in nude mice of each treatment group, where * (P < 0.05), ** (P < 0.01), *** (P < 0.001), **** (P < 0.0001). Detailed implementation manners

[0054] The content of the present invention will be described in more detail below in conjunction with the embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any formal modification or change made to the present invention falls within the protection scope of the present invention; and the methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0055] Example 1: Composition, mechanism of action, and verification of in vitro synergistic effect of a pharmaceutical composition. A pharmaceutical composition for reversing gemcitabine resistance in pancreatic cancer, which comprises the pan-FGFR inhibitor derazantinib and gemcitabine.

[0056] In the pharmaceutical composition, the content of derazantinib and the content of gemcitabine are configured such that when administered to a patient, the administration concentration (dose) of derazantinib can reach 25 - 50 mg / kg body weight, and the administration concentration (dose) of gemcitabine can reach 20 - 30 mg / kg body weight.

[0057] The pharmaceutical composition inhibits the FGFR2 / FGFR3 signaling pathway, reduces the expression of FGFR protein in drug-resistant pancreatic cancer cells, and synergistically enhances the chemosensitivity of gemcitabine.

[0058] Role of FGFR in gemcitabine resistance By analyzing public databases (such as the GEO dataset GSE140077), it was found that the expression of FGFR3 in gemcitabine-resistant BxPC-3 cells (BxPC-3 / GR) was significantly higher than that in sensitive cells (BxPC-3 / WT) ( Figure 3 , ***P < 0.001). Venn diagram analysis (Figure 4 also showed that FGFR3 is a common gene in multiple gemcitabine resistance-related gene sets in pancreatic cancer. In addition, clinical sample analysis showed that high expression of FGFR2 and FGFR3 was associated with poor prognosis (shorter OS and DFS, Figures 5 - 8 ), and tumor poor differentiation ( Figures 9 - 10 ). The expression of FGFR2 in pancreatic cancer tissues was also significantly higher than that in adjacent normal tissues ( Figure 11 , *P<0.05). In particular, in clinical tumor samples resistant to gemcitabine, the expression levels of FGFR2 and FGFR3 detected by immunohistochemical staining (IHC) were also significantly higher than those in sensitive samples ( Figures 12 - 15 , **P<0.01, *P<0.05). These data indicate that FGFR2 and FGFR3 signaling pathways play important roles in gemcitabine resistance in pancreatic cancer.

[0059] Inhibitory effect of derazantinib on FGFR protein expression The Western Blot method was used to detect the effects of derazantinib on the expression of FGFR2 and FGFR3 proteins in gemcitabine-resistant pancreatic cancer cell lines AsPC-1 / GEM and BxPC-3 / GEM. Cells were treated with different concentrations of derazantinib (0 μM as control CTRL, 1 μM, 2 μM, 3 μM). The results were as Figures 25 - 26 shown. With the increase in derazantinib concentration, the protein levels of FGFR2 and FGFR3 in AsPC-1 / GEM and BxPC-3 / GEM cells decreased significantly in a concentration-dependent manner (****P<0.0001). This confirmed that derazantinib could effectively reduce the expression of FGFR proteins in drug-resistant pancreatic cancer cells.

[0060] Enhanced synergistic chemosensitivity of derazantinib and gemcitabine (determination of combination index CI) To evaluate the effect of the combined application of derazantinib and gemcitabine, the CCK-8 method was used to determine the inhibitory effect of the drugs on gemcitabine-resistant cells AsPC-1 / GEM and BxPC-3 / GEM, and the combination index (CI) was calculated. First, the IC50 values of gemcitabine in parental cells (AsPC-1, BxPC-3) and drug-resistant cells (AsPC-1 / GEM, BxPC-3 / GEM) were determined by the CCK-8 method ( Figure 16), the establishment of drug-resistant cell lines was confirmed (the IC50 of the drug-resistant strains was significantly higher than that of the parental strains). Subsequently, AsPC-1 / GEM and BxPC-3 / GEM cells were exposed to different concentration combinations of deratinib (e.g., 1 μM, 1.5 μM, 2 μM) and gemcitabine (e.g., 100 nM, 200 nM, 300 nM) for co-treatment. The CCK-8 kit was used to detect cell viability, and the CI value was calculated according to the Chou-Talalay method using CompuSyn software. As Figure 17 (AsPC-1 / GEM) and Figure 18 (BxPC-3 / GEM) showed that the CI values of the combination of deratinib and gemcitabine were less than 1 at most of the tested concentration combinations. For example, for AsPC-1 / GEM cells, when the concentration of gemcitabine was 0.3 μM (300 nM) combined with a deratinib concentration of 2.0 μM, the CI value was 0.61848; for BxPC-3 / GEM cells, when the concentration of gemcitabine was 0.3 μM (300 nM) combined with a deratinib concentration of 2.0 μM, the CI value was 0.57868. A CI value less than 1 indicates a synergistic effect between the two drugs. This confirmed that Drug Composition 1 enhanced the chemosensitivity of gemcitabine through a synergistic effect. Further, the colony formation assay also showed that after co-treatment of AsPC-1 / GEM ( Figures 19 - 20 ) and BxPC-3 / GEM ( Figures 21 - 22 ) cells with deratinib and gemcitabine for 14 days, the number of cell colonies formed was significantly less than that of each single-drug group and the control group (**P < 0.01), further demonstrating the synergistic inhibition of the long-term proliferation ability of cells.

[0061] Example 2: The physical and chemical form of deratinib, and the pan-FGFR inhibitor contained in the drug composition is one of deratinib, deratinib salt, deratinib solvate, and deratinib prodrug.

[0062] Pharmaceutically acceptable salts: refer to salts formed by delatinib and pharmaceutically acceptable acids or bases. For example, they can be formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., or with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, etc. Base addition salts can be formed with inorganic bases such as hydroxides, carbonates or bicarbonates of sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, etc., or with pharmaceutically acceptable organic primary, secondary or tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, harmine, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc. Selecting an appropriate salt form can improve the solubility, stability, bioavailability or formulation process of the drug.

[0063] Solvates: refer to complexes formed by delatinib or its salts and stoichiometric or non-stoichiometric solvent molecules. Common solvents include water (forming hydrates), ethanol, methanol, isopropanol, acetone, ethyl acetate, etc. Hydrates are the preferred solvate form.

[0064] Prodrugs: refer to compounds that release the active drug delatinib after chemical or enzymatic conversion in vivo. The design of prodrugs can aim to improve the oral bioavailability of the drug, reduce toxicity, enhance targeting or extend the action time. For example, prodrugs of delatinib can be prepared by introducing groups that are easily cleaved in vivo (such as ester groups, carbamate groups, phosphate groups, etc.) onto the delatinib molecule.

[0065] When preparing pharmaceutical compositions, the free base form of delatinib, or its pharmaceutically acceptable salt, solvate or prodrug form can be selected as needed to achieve optimal pharmaceutical properties.

[0066] Example 3: The pharmaceutical composition induces apoptosis in drug-resistant pancreatic cancer cells and activates the apoptotic pathway. To further clarify the anti-tumor mechanism of the pharmaceutical composition containing deratinib and gemcitabine, its ability to induce apoptosis was studied. The experimental results confirmed that the pharmaceutical composition achieved its therapeutic effect by inducing apoptosis in drug-resistant pancreatic cancer cells, and the apoptosis rate in this apoptotic process was increased by more than 2-fold compared to the single-drug treatment group. At the same time, it was accompanied by the cleavage activation of the key apoptotic signaling molecules caspase-3 and poly (ADP-ribose) polymerase (PARP).

[0067] The specific experiments were as follows: The gemcitabine-resistant pancreatic cancer cell lines AsPC-1 / GEM and BxPC-3 / GEM were treated with PBS (control group), deratinib alone (DE 0.5 μM or DE 1 μM), gemcitabine alone (GEM 200 nM), or a combination of deratinib and gemcitabine (GEM 200 nM + DE 0.5 μM, or GEM 200 nM + DE 1 μM) for 48 hours. After collecting the cells, the cells were stained using an Annexin V-FITC / PI apoptosis detection kit, and then the apoptosis rate of the cells in each group was analyzed by flow cytometry.

[0068] The pancreatic cancer cell line was seeded in 6-well plates and divided into a control group, a gemcitabine group, a deratinib group, and a combination drug group. After 48 hours of drug intervention, the cells were collected. After washing twice with PBS, the cells were resuspended in 100 μL of Annexin V binding buffer (BD Biosciences, 556454). 10 μL of Annexin V-FITC (BD, 556420) and 5 μL of propidium iodide (PI, final concentration 2 μg / mL) were added, and the cells were incubated in the dark for 20 minutes. After staining, 400 μL of pre-cooled binding buffer was added, and the cells were immediately detected.

[0069] The apoptosis rate was calculated as the sum of the proportions of Annexin V+ / PI- (early apoptosis) and Annexin V+ / PI+ (late apoptosis / necrosis) cells.

[0070] The results of flow cytometry detection (as Figures 23 - 24As shown in the representative flow cytometry scatter plots and the statistical bar graphs of apoptosis indices containing AsPC-1 / GEM and BxPC-3 / GEM cells, compared with each single-drug treatment group (DE or GEM) and the control group (PBS), the apoptosis rates of the gemcitabine and delatinib combination group (GEM+DE) were significantly increased (****P<0.0001). Taking AsPC-1 / GEM cells as an example, the apoptosis rate of the control group was about 5%, the apoptosis rate of the gemcitabine single-drug group was about 10%, the apoptosis rate of the 1 μM delatinib single-drug group was about 15%, while the apoptosis rate of the combination group of gemcitabine (200 nM) and delatinib (1 μM) was significantly increased to about 35%. Compared with the gemcitabine single-drug group (10%), the apoptosis rate of the combination group (35%) was increased by 3.5 times; compared with the delatinib single-drug group (15%), the apoptosis rate of the combination group (35%) was increased by about 2.3 times. Similarly, in BxPC-3 / GEM cells, the apoptosis rate of the control group was about 4%, the apoptosis rate of the gemcitabine single-drug group was about 8%, the apoptosis rate of the 1 μM delatinib single-drug group was about 12%, while the apoptosis rate of the combination group of gemcitabine (200 nM) and delatinib (1 μM) was significantly increased to about 45%. Compared with the gemcitabine single-drug group (8%), the apoptosis rate of the combination group (45%) was increased by about 5.6 times; compared with the delatinib single-drug group (12%), the apoptosis rate of the combination group (45%) was increased by about 3.75 times. These data indicate that the apoptosis rate induced by the drug composition has increased by more than 2 times compared with any single-drug treatment group.

[0071] To further confirm the activation of the apoptosis pathway, Western Blot was used to detect the cleavage activation of the key proteins Caspase-3 and PARP related to apoptosis execution in cells under the above drug treatment conditions. AsPC-1 / GEM and BxPC-3 / GEM cells treated with different drugs were collected, and total proteins were extracted for Western Blot analysis. The results were expected to show that in the gemcitabine and delatinib combination group, the protein levels of the activated form of Caspase-3, that is, cleaved Caspase-3, and the protein levels of the cleavage fragments (cleaved PARP) generated by the cleavage of PARP by Caspase-3 were significantly higher than those of each single-drug treatment group and the control group. Caspase-3 is the core effector molecule in the apoptosis cascade reaction, and its activation (cleavage) is an important marker for cells to enter the apoptosis program. PARP is an important downstream substrate of Caspase-3, and its cleavage is also a typical biochemical feature of cell apoptosis. Therefore, the cleavage activation of Caspase-3 and PARP further confirmed that the drug composition exerts its killing effect on drug-resistant pancreatic cancer cells by effectively initiating the apoptosis program.

[0072] Example 4: Application of the pharmaceutical composition in the preparation of a drug for treating pancreatic cancer and the administration method. This example describes the application of a pharmaceutical composition comprising delatinib (or its salt, solvate or prodrug) and gemcitabine in the preparation of a drug for treating pancreatic cancer. This drug can be used to treat pancreatic cancer, especially gemcitabine-resistant pancreatic cancer.

[0073] This drug can be administered by intravenous injection or orally.

[0074] Dosage form: Freeze-dried powder injection: Dissolve delatinib and gemcitabine (or its suitable salt form) in water for injection, and appropriate stabilizers and solubilizers (such as mannitol, lactose, polysorbate 80, etc.) can be added. Adjust the pH to an appropriate range (such as 4.0 - 7.0). After filtration and sterilization, it is dispensed into vials and freeze-dried. Before use, it is dissolved with sterile water for injection or normal saline for intravenous injection or intravenous drip.

[0075] Capsule: Mix delatinib (or its suitable salt or prodrug form, if for oral administration) with suitable pharmaceutical excipients such as fillers (such as microcrystalline cellulose, lactose, starch), disintegrants (such as croscarmellose sodium, sodium starch glycolate), lubricants (such as magnesium stearate, silicon dioxide), etc. evenly, and fill them into hard capsules. Gemcitabine is usually administered by injection. If a fully oral regimen is considered, gemcitabine also needs to be prepared into a suitable oral dosage form or its oral prodrug.

[0076] Tablet: Granulate by mixing delatinib (or its suitable salt or prodrug form) with excipients (such as fillers, binders such as povidone, disintegrants, lubricants), and then press into tablets. Or use the direct powder compression technology. Similarly, special considerations are needed for the oral tablets of gemcitabine. More commonly, gemcitabine is administered by intravenous injection, and delatinib is used in combination as an oral drug (such as capsules or tablets). In this case, the "preparation" of the pharmaceutical composition of the present invention can be understood as providing a combined package of these two drugs, or providing delatinib for use in combination with gemcitabine to treat pancreatic cancer.

[0077] This drug is also supplemented with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include but are not limited to diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, solvents, solubilizers, emulsifiers, isotonicity regulators, buffers, pH regulators, stabilizers, antioxidants, preservatives, colorants, flavoring agents, etc. The choice of carrier depends on the administration route, dosage form, and physical and chemical properties of the active ingredient.

[0078] Example 5: Screening of the applicable population for the pharmaceutical composition, use of the pharmaceutical composition in the preparation of a medicament for treating pancreatic cancer, and the indication of the medicament can be further defined as patients with pancreatic ductal adenocarcinoma (PDAC) with high expression of FGFR3. The method for screening the applicable population includes: Immunohistochemistry (IHC): Take a pancreatic cancer tumor tissue sample from the patient and prepare it into paraffin-embedded sections. Perform immunohistochemical staining using a specific anti-FGFR3 antibody (such as rabbit anti-human FGFR3 polyclonal or monoclonal antibody). Score by observing the staining intensity and the percentage of positive cells. For example, a threshold can be set, such as H-score > X or the proportion of positive cells > Y% and the staining intensity ≥ 2+, to determine high expression of FGFR3. Figure 12 and Figure 13 The immunohistochemical staining of FGFR3 in clinical samples is shown, and it can be seen that the expression is higher in the drug-resistant group.

[0079] Gene sequencing: Extract DNA or RNA from the patient's tumor tissue sample. Detect the amplification, mutation, or mRNA expression level of the FGFR3 gene by next-generation sequencing (NGS) or other gene detection techniques (such as Sanger sequencing, ddPCR, qRT-PCR, etc.). The copy number variation (CNV) of the FGFR3 gene can be detected. If the copy number increase exceeds a certain threshold, it is considered high expression of FGFR3. Alternatively, detect the expression level of FGFR3 mRNA by RNA sequencing. If it is higher than the normal control or the set reference value, it is considered high expression of FGFR3. Figure 3 That is, the analysis of gene expression profile data shows that the expression of FGFR3 is increased in drug-resistant cells.

[0080] By screening PDAC patients with high expression of FGFR3 by the above method and administering the pharmaceutical composition described in the present invention for treatment, better therapeutic effects are expected to be obtained.

[0081] Example 6: Method of use and dosage regimen of the pharmaceutical composition, and the method includes the following steps: Detect the expression levels of FGFR2 / FGFR3 in the patient's tumor tissue: Use the immunohistochemistry (IHC) or gene sequencing methods described in Example 5 to detect the expression levels of FGFR2 and / or FGFR3 in the pancreatic cancer tumor sample of the patient. Focus on the expression level of FGFR3, and at the same time, the expression of FGFR2 can also be evaluated.

[0082] Administer a pharmaceutical composition to patients with high FGFR expression: For nude mice whose test results show high expression of FGFR2 and / or FGFR3 (especially high expression of FGFR3), administer a pharmaceutical composition containing derazantinib and gemcitabine. The dosing ratio of derazantinib to gemcitabine is 1:1 to 1:4 by mass, that is, the daily dose of derazantinib is 30 mg / kg, and the daily dose of gemcitabine is 30 mg / kg (ratio 1:1), or 60 mg / kg (ratio 1:2), or 120 mg / kg (ratio 1:4). Therefore, on the premise of meeting the dose range of Pharmaceutical Composition 1, such as derazantinib 25 mg / kg and gemcitabine 25 mg / kg (1:1); or derazantinib 25 mg / kg, and gemcitabine can be adjusted within this ratio range and meet the dose of 20 - 30 mg / kg.

[0083] In this method of use, the daily dose of derazantinib is 25 - 50 mg / kg, and the daily dose of gemcitabine is 20 - 30 mg / kg. The combined treatment cycle is set to 21 days. Derazantinib is administered orally once a day, and the dose is calculated according to the patient's body weight, ranging from 25 - 50 mg / kg. Gemcitabine is intravenously infused on the 1st and 8th days of each 21-day cycle, and the dose is calculated according to the patient's body surface area or body weight, and after conversion, its daily average dose or single-dose administration dose is within this range. Gemcitabine is administered at 25 mg / kg on the 1st and 8th days, and the specific dosing regimen and frequency are further optimized according to the clinical trial results. As Figures 27 - 30 shown in the animal experiment, the dosing dose of derazantinib is 20 mg / kg (gavage once a day), and the dosing dose of gemcitabine is 25 mg / kg (intraperitoneal injection twice a week). The treatment lasts for 21 days. The efficacy of the combined administration group is significantly higher than that of the single-drug group, showing good in vivo efficacy and safety, which provides a reference for clinical dose selection, but the actual clinical dose needs to follow the dose range defined in this method. The dosing frequency of derazantinib can be once every 3 days or twice a week.

[0084] Example 7: Verification of the in vivo efficacy of the pharmaceutical composition. To verify the anti-tumor effect of the pharmaceutical composition of the present invention in vivo, a subcutaneous xenograft tumor model of gemcitabine-resistant pancreatic cancer cells (such as AsPC-1 / GEM or BxPC-3 / GEM) in nude mice was established. After the tumor grew to a certain volume, the tumor-bearing mice were randomly divided into four groups: Control group (Control): Administer the vehicle.

[0085] Gemcitabine group (gemcitabine): Administer gemcitabine at 25 mg / kg twice a week.

[0086] Derazantinib group (derazantinib): Administer derazantinib at 20 mg / kg once a day.

[0087] Combined treatment group (G+D): Gemcitabine and deratinib were administered in combination at the above doses. The treatment lasted for 21 days. The results are as Figures 27 - 30 shown: Gross observation of tumors ( Figure 27 ) showed that the tumor volume in the combined treatment group was the smallest.

[0088] Tumor growth curve ( Figure 28 ) showed that the combined treatment group could most effectively inhibit tumor growth.

[0089] Tumor weight ( Figure 29 ) and tumor volume ( Figure 30 ) statistics showed that at the end of the treatment, both the tumor weight and volume in the combined treatment group were significantly smaller than those in other groups (for example, compared with the control group, P<0.001 or P<0.0001).

[0090] In summary, the present invention provides a pharmaceutical composition comprising deratinib and gemcitabine and its application method. By targeting and inhibiting the FGFR2 / FGFR3 signaling pathway, it effectively reduces the expression of FGFR protein in drug-resistant cells, synergistically enhances the chemosensitivity of gemcitabine, and potently induces apoptosis, providing a promising new strategy for the treatment of gemcitabine-resistant pancreatic cancer, especially for patients with high expression of FGFR3.

[0091] So far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the protection scope of the present invention.

Claims

1. A pharmaceutical composition for reversing gemcitabine resistance in pancreatic cancer, characterized in that, Comprising a pan-FGFR inhibitor and gemcitabine, wherein: The concentration of the pan-FGFR inhibitor is 25-50 mg / kg; The concentration of gemcitabine is 20-30 mg / kg; The composition reduces the expression of FGFR protein in drug-resistant pancreatic cancer cells by inhibiting the FGFR2 / FGFR3 signaling pathway and synergistically enhances the chemosensitivity of gemcitabine.

2. The pharmaceutical composition according to claim 1, wherein The pan-FGFR inhibitor is one of derazantinib, derazantinib salt, derazantinib solvate, and derazantinib prodrug.

3. The pharmaceutical composition according to claim 1, wherein The synergistic index CI of the composition is less than 1, determined by the CCK-8 method.

4. The pharmaceutical composition according to claim 1, wherein The composition achieves its therapeutic effect by inducing apoptosis in drug-resistant pancreatic cancer cells, with the apoptosis rate increased by more than 2 times compared to the single-drug group, and accompanied by the cleavage activation of caspase-3 and poly(ADP-ribose) polymerase (PARP).

5. Use of the pharmaceutical composition according to any one of claims 1-4 in the preparation of a drug for treating pancreatic cancer.

6. The application according to claim 5, characterized in that, The suitable population for the drug is patients with pancreatic ductal adenocarcinoma (PDAC) with high expression of FGFR3 screened by immunohistochemistry (IHC) or gene sequencing.

7. The application according to claim 5, wherein The drug is administered by intravenous injection or orally, and the dosage form is one of freeze-dried powder injection, capsule, and tablet.

8. A method for using a pharmaceutical composition, characterized in that, Comprising the following steps: Detect the expression level of FGFR2 / FGFR3 in the patient's tumor tissue; Administer the pharmaceutical composition according to claim 1 to patients with high FGFR expression, and the administration ratio of derazantinib to gemcitabine is 1:1 to 1:4 by mass ratio.

9. The method according to claim 8, wherein The daily dose of derazantinib is 25-50 mg / kg, and the daily dose of gemcitabine is 20-30 mg / kg, and the combined medication cycle is 21 days.

Images