Drug-combined anticancer composition and application of composition in preparation of anticancer drugs

Abstract

The invention discloses a drug-combined anticancer composition and application of the composition in preparation of anticancer drugs. According to the present invention, the drug combination composition comprises the PARP inhibitor Olaparib and the calcium ion antagonist amlodipine maleate, the PARP inhibitor Olaparib and the calcium ion antagonist amlodipine maleate provide the synergistic effect, and compared with the single PARP inhibitor, the drug combination composition has characteristics of high stomach cancer sensitivity, significant gastric cancer efficiency improving, and wide application prospect. The Amlodipine in the drug combination composition provided by the invention enhances the curative effect of the PARP inhibitor Olaparib on the gastric cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to an anticancer composition for combined drug use and application of the composition in the preparation of anticancer drugs. Background Art

[0002] Gastric carcinoma (GC) is a common digestive tract malignancy worldwide that is highly invasive and heterogeneous. Currently, surgical resection remains the main treatment for GC. However, early GC often lacks obvious symptoms, resulting in a low diagnosis rate. Patients are usually diagnosed in the middle or late stages, accompanied by various degrees of metastasis. Therefore, despite surgical intervention, the survival rate of GC patients remains low and the recurrence rate is high. In recent years, with the advancement of technologies such as radiotherapy, chemotherapy, and neoadjuvant therapy, the overall survival rate of GC patients has been significantly improved, but the benefits of these treatments are still very limited, and drug resistance and resistance often occur. Therefore, the development of new drugs is still in urgent need of treatment for GC.

[0003] The poly(ADP-ribose) polymerase (PARP) family is a multifunctional protein post-translational modification enzyme involved in DNA damage repair. PARP1 plays a key role in numerous cellular processes, including chromatin structure, replication, transcription, energy metabolism, cell death, immunity, and inflammation. It encompasses over 90% of the family's functions and plays a crucial role in DNA repair. The key role of PARP1 in mediating DNA damage provides a rationale for the development of PARP inhibitors (PARPi) for the treatment of human malignancies.

[0004] In 2014, the first PARPi, olaparib, received approval from the US Food and Drug Administration (FDA) and the European Medicines Agency. Following its successful use as a maintenance treatment for advanced ovarian cancer harboring mutations in the DNA repair genes BRCA1 / 2, olaparib also became the first PARPi approved for the treatment of GC. With the continued advancement of preliminary basic research, olaparib's monotherapy for GC has entered the clinic. However, a Phase III trial evaluating the efficacy of olaparib failed to demonstrate a significant improvement in the overall survival rate of GC patients. This demonstrates the limited efficacy of PARPi as a monotherapy for GC.

[0005] The invention application with publication number HK40036086A discloses a PARP inhibitor maintenance therapy for the treatment of gastric cancer, comprising administering a therapeutically or maintenance-effective amount of a PARP inhibitor to a subject, wherein the subject has previously received chemotherapy. The PARP inhibitor is selected from olaparib, niraparib, rucaparib, or pamiparib.

[0006] The invention application with publication number WO2018099423A1 discloses the use of a VEGFR inhibitor in combination with a PARP inhibitor in the preparation of a drug for treating gastric cancer, wherein the PARP inhibitor is selected from Olaparib, Niraparib, Talazoparib, Veliparib, Rucaparib, CEP-8983 or BGB-290, and the VEGFR inhibitor is selected from VEGFR-2 inhibitors.

[0007] It is still of practical significance to screen other drugs that can be used in combination with PARPi to improve the treatment effect of gastric cancer. Summary of the Invention

[0008] In view of the above-mentioned deficiencies in the prior art, the present invention provides an anticancer composition for combined use and the use of the composition in the preparation of anticancer drugs.

[0009] Based on the "repurposing old drugs for new uses" R&D strategy, screening of the Food and Drug Administration (FDA) drug library revealed that amlodipine maleate significantly enhances the sensitivity of PARP1 knockout cell lines. Furthermore, amlodipine is a calcium antagonist and NF-κB pathway inhibitor, making it a candidate for combination therapy and providing a new strategy for GC patients in combination with PARPi.

[0010] The present invention first provides a combined anticancer composition comprising a PARP inhibitor and a calcium ion antagonist.

[0011] Preferably, the PARP inhibitor is olaparib, and the molecular structure is as follows:

[0012] Olaparib, chemical name 1-(cyclopropylcarbonyl)-4-[5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoyl]piperazine, chemical formula C 24 H 23 FN4O3, CAS registration number 763113-22-0.

[0013] Preferably, the calcium ion antagonist is amlodipine maleate, and the molecular structural formula is as follows:

[0014] The chemical name of amlodipine maleate is racemic-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylic acid 3-ethyl 5-methyl ester maleic acid dihydrochloride, molecular formula C 24 H 29 ClN2O9, CAS number is 88150-47-4.

[0015] The present invention further provides use of the composition in preparing anticancer drugs, wherein the composition comprises a PARP inhibitor and a calcium ion antagonist.

[0016] Preferably, the cancer type is gastric cancer, lung cancer, colon cancer, liver cancer, thyroid cancer or esophageal cancer. More preferably, the cancer type is gastric cancer.

[0017] Preferably, the PARP inhibitor is olaparib.

[0018] Preferably, the calcium ion antagonist is amlodipine maleate.

[0019] The combination drug composition provided by the present invention includes the PARP inhibitor Olaparib and the calcium ion antagonist Amlodipine maleate. The two exert a synergistic effect. Compared with a single PARP inhibitor, it has higher sensitivity to gastric cancer and significantly improved efficacy, thus having broad application prospects.

[0020] Amlodipine maleate in the combined pharmaceutical composition provided by the present invention enhances the therapeutic effect of the PARP inhibitor Olaparib on gastric cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Flowchart for screening drugs to overcome PARPi resistance for the FDA drug library.

[0022] Figure 2 CCK8 experiments validated Amlodipine maleate as a candidate drug for combination with Olaparib.

[0023] Figure 3 The effects of olaparib combined with amlodipine maleate on GC cell proliferation were evaluated for colony formation. Figure 3 A in the figure is a micrograph; Figure 3 B in is the statistical result.

[0024] Figure 4 Flow cytometry was used to evaluate the ability of Olaparib combined with Amlodipine maleate to promote GC cell apoptosis. Figure 4 A in the figure is the flow cytometry result graph; Figure 4 B in is the statistical result.

[0025] Figure 5 Western blot was used to evaluate the effect of Olaparib combined with Amlodipine maleate on the expression of Cleaved-Caspase3 and Cleaved-Caspase7 proteins in GC cells.

[0026] Figure 6 To evaluate the combined efficacy of Olaparib and Amlodipine maleate in vivo using a nude mouse axillary xenograft tumor model. Figure 6 A in the figure is the tumor volume of nude mice after three weeks of drug treatment; Figure 6 B in the figure is the tumor volume of nude mice after three weeks of drug treatment; Figure 6 C in the figure is the tumor mass of nude mice after three weeks of drug treatment.

[0027] Figure 7 The tumor was subjected to H&E, Ki67 and Tunel immunohistochemistry detection. Figure 7 A in the figure is a photo of immunohistochemistry detection; Figure 7 B in the figure is the statistical result of Ki67 immunohistochemistry; Figure 7 Figure C is the statistical result of Tunel immunohistochemistry.

[0028] Figure 8 H&E staining was used to evaluate the toxic effects of Olaparib and Amlodipine maleate on the organs of nude mice. Figure 8 A in the figure indicates that the weight changes of nude mice were weighed and recorded every other day during drug treatment; Figure 8 B is H&E staining of the heart, liver, spleen, lung and kidney of nude mice after three weeks of drug treatment.

[0029] Figure 9 To evaluate the efficacy of Olaparib combined with Amlodipinemaleate in different tumors by measuring the cell survival rate after combined use in different tumors. Figure 9 A in the figure represents A549 cells; Figure 9 B in the figure is AGS cell; Figure 9 C in the middle is HCT116 cells; Figure 9 D in the figure is HepG2 cells; Figure 9 E in the figure represents HTh-7 cells; Figure 9F in the figure indicates KYSE150 cells. DETAILED DESCRIPTION

[0030] Example 1: Screening of drugs to overcome PARPi resistance using the FDA drug library To identify drugs suitable for combination with olaparib, we screened the FDA drug library provided by MCE (comprising over 2,000 approved clinical drugs). These drugs cover areas such as anti-tumor, cardiovascular, and immunology. We measured cell viability in 293A WT and PARP1 KO cells after drug treatment using the CCK-8 assay, and calculated the difference between the two values: [293A WT (viability) - PARP1 KO (viability)].

[0031] Filter results such as Figure 1 Results showed that amlodipine maleate was more sensitive to PARP1 KO cells than 293A WT cells. Amlodipine maleate is a calcium antagonist and NF-κB pathway inhibitor. Amlodipine maleate is being considered as a candidate for combination with olaparib, and further experimental studies are planned to validate its potential efficacy in the treatment of GC.

[0032] Example 2: Preparation of Olaparib and Amlodipine maleate Stock Solutions

[0033] Accurately weigh appropriate amounts of olaparib (purchased from MCE) and amlodipine maleate (purchased from Targetmol) and prepare stock solutions at concentrations of 2.5 mg / mL and 0.2 mg / mL, respectively, using DMSO as the solvent. Aliquot into 1.5 mL EPDM tubes, seal with parafilm, and store in a -20°C freezer.

[0034] Example 3: Detection of cell working concentration

[0035] (1) Construction of PARP1 knockout plasmid: PARP1 sgRNA was designed. By combining the score and editing efficiency, a total of three PARP1 sgRNAs were screened from the whole genome sequence of human PARP1 and synthesized by Qingke Biotechnology. The primer sequences are detailed in Table 1. Prepare the insertion of sgRNA oligonucleotides (oligos), where Oligo1 is the front primer of sgRNA1, sgRNA2, and sgRNA3, and Oligo2 is the back primer of sgRNA1, sgRNA2, and sgRNA3. Dissolve and resuspend to a final concentration of 100 μM. Phosphorylate and anneal the sgRNA oligos, as detailed in Table 2. Oligos were phosphorylated and annealed in a PCR instrument, and the procedure is detailed in Table 3. The product was diluted with ddH2O at a ratio of 1:200 and placed on ice or at -20°C for later use. The lentiCRISPRv2 plasmid was treated with BsmBI restriction endonuclease, usually incubated at 55°C for 1 h. The system is detailed in Table 4. Collect the digested product for agarose gel electrophoresis: Heat 1× TAE in a microwave for 1 minute until the agarose is completely dissolved. Pour the solution onto a gel plate, insert a loading comb, and cool for approximately 20 minutes to solidify. Evenly mix the sample, Gelrad, and loading buffer, set aside, and load the sample. Remove the 0.1% agarose gel and carefully remove the comb. Place the gel plate and assemble the gel electrophoresis apparatus. Add 1× TAE electrophoresis buffer until just covering the gel. Load 5-15 μL of sample into each well. Simultaneously load a DNA marker (DNA molecular weight standard) to indicate sample size. Run the gel at 140 V for approximately 30 minutes. Confirm the digested product by comparing it to a DNA marker under UV light and excise the gel. Based on the electrophoresis results, compare the size of the digested product to the DNA marker and confirm the integrity of the digested vector fragment. Then excise the corresponding band and recover it using a DNA gel recovery kit. The plasmid and vector fragments were ligated using T4 DNA ligase overnight at room temperature to create a recombinant plasmid. See Table 5 for details. Add 10 μL of recombinant plasmid to 40 μL of competent cells, incubate on ice for 30 minutes, heat shock in a 42°C water bath for 90 seconds, and immediately transfer to ice for 3 minutes. Add 50 μL of LB medium and resuspend in a shaker at 37°C, 200 rpm, for 1 hour. Determine the resistance of the recombinant plasmid based on the vector sequence. Select a medium with the corresponding resistance. Spread the transformed liquid evenly onto the medium and incubate inverted at 37°C overnight. Place the plate in a clean hood, select a single clone, transfer it to LB medium with the corresponding resistance, and amplify overnight in a shaker at 37°C, 200 rpm. Plasmids were then extracted and purified using an endotoxin-free plasmid miniprep kit, and the concentration was determined.

[0036] Table 1 PARP1 sgRNA primer sequences

[0037] Table 2 sgRNA oligos phosphorylation and annealing system mixing ratio

[0038] Table 3 PCR program for sgRNA oligos phosphorylation and annealing system

[0039] Table 4 Vector enzyme digestion system

[0040] Table 5 Recombinant plasmid ligation system

[0041] (2) CCK-8 assay: 293A WT (wild-type 293A cells) and PARP1 KO1 cells (gene knockout cells obtained by transferring the constructed PARP1 knockout plasmid into wild-type 293A cells) were seeded into 96-well plates at 3000 cells / well in 100 μL and cultured overnight. Using the established clinical drug library (FDA drug library), according to the predetermined experimental design, Olaparib was diluted threefold at a concentration of 1500 μM to prepare 9 drug solutions of different concentrations. Subsequently, 50 μL of drug solution was added to each well, so that the cells were treated with drugs at final concentrations of 500, 166.7, 55.6, 18.5, 6.2, 2.0, 0.7, 0.23, and 0.08 μM. After 48 h, the old culture medium was discarded. Subsequently, the CCK-8 reagent was mixed with DMEM culture medium at a ratio of 1:9 to prepare a working solution. Add 100 μL of working solution to each well and incubate in a 37°C incubator for approximately 1.5 hours. Measure the OD value of each well at a wavelength of 450 nm using a microplate reader. Calculate the survival rate as (average OD value of the experimental group - average OD value of the background) / (average OD value of the well group - average OD value of the background) × 100%. GraphPad software was used to fit and analyze the experimental data and calculate the IC50 value of the drug.

[0042] The results are as follows Figure 2 This suggests that amlodipine maleate could be a candidate for combination with olaparib.

[0043] (3) Clone formation: HGC27 and NUGC3 cells (two gastric cancer cell lines) were seeded in 6-well plates at 800 cells / well and 1000 cells / well, respectively. After 4 days of culture, the corresponding drug treatment was given: Olaparib and Amlodipine maleate were accurately weighed and prepared into stock solutions of 2.5 mg / mL and 0.2 mg / mL, respectively, using DMSO as the solvent. The solutions were aliquoted into 1.5 mL EP tubes, sealed with parafilm, and stored in a -20°C refrigerator.

[0044] The Ctrl group (control group, no drug added), Olaparib group (Olaparib alone group), Amlodipinemaleate group (Amlodipine maleate alone group) and Combination group (combination drug group) were set up, and the corresponding reserve solution was added to each group and culture continued.

[0045] Two weeks later, use an inverted microscope to observe the clone formation status of each group of cells, record the number and morphology of cell clones, and prepare to collect samples. Remove the culture medium, add 1 mL of PBS solution to each well, and wash the cells twice. Add 1 mL of paraformaldehyde solution to each well and fix the cells for 15 minutes to fix the cell morphology and structure. After fixation, wash the cells twice again with PBS. Add 1 mL of 0.1% crystal violet staining solution to each well and stain the cells for 30 minutes. After staining, wash the cells twice again with PBS to remove excess staining solution. Place the 6-well plate in a dry and ventilated place to dry, then use a scanner to photograph the cell clones and record the experimental results.

[0046] The results showed that the combination group showed stronger proliferation inhibition effect in both GC cell lines compared with the single drug group ( Figure 3 ), and the difference was statistically significant (p<0.05). This result further confirmed that amlodipinemaleate can enhance the inhibitory effect of olaparib on GC cell proliferation.

[0047] Example 4: Flow cytometry apoptosis detection

[0048] Cell preparation: Appropriate numbers of HGC27 and NUGC3 cells were seeded in six-well plates, ensuring a cell density of approximately 60%-70% on the next day. Control (no drug), olaparib (olaparib alone), amlodipine maleate (amlodipine maleate alone), and combination (combination) groups were established. The next day, cells were treated with the corresponding drugs (2.5 mg / mL olaparib and 0.2 mg / mL amlodipine maleate) for 48 hours. The following procedures were performed: The old culture medium was transferred to a 15 mL centrifuge tube. After trypsinization in each well without EDTA, the cells were pipetted with the old culture medium and transferred to a 15 mL centrifuge tube for pooling. The cells were centrifuged at 1000 g for 5 minutes, and the supernatant was discarded. The cells were resuspended in PBS and centrifuged again, and the supernatant was discarded. The cells were resuspended in PBS and counted. Approximately 80,000 cells were transferred to a 1.5 mL EP tube and centrifuged at 11,000 g for 5 minutes, and the supernatant was discarded. Add 195 μL of Annexin V-FITC binding solution to the cells and gently resuspend the cells. Then add 5 μL of Annexin V-FITC and 10 μL of PI-PE and incubate at room temperature in the dark for 15-20 min. After incubation, add PBS, centrifuge at 1000 g for 5 min, discard the supernatant, add 400 μL of PBS and resuspend. After passing the cell suspension through a cell filter, prepare for the instrument. Use Beckman flow cytometer to collect cell samples, adjust flow compensation, voltage and other parameters, collect 10,000 cells for each sample, perform data analysis, divide the positive area, and obtain the cell apoptosis ratio of each group.

[0049] The results showed that the apoptosis rate increased after combined use ( Figure 4 ), which indicates that Amlodipine maleate can enhance the apoptosis of GC cells induced by Olaparib.

[0050] Example 5: Apoptosis pathway detection

[0051] Appropriate numbers of NUGC3 and HGC27 cells were seeded in six-well plates to ensure a cell density of approximately 60%-70% on the second day. A control group (control group, no drug added), an olaparib group (olaparib alone group), an amlodipinemaleate group (amlodipine maleate alone group), and a combination group (combination drug group) were set up. The cells were treated with the corresponding drugs (2.5 mg / mL olaparib and 0.2 mg / mL amlodipine maleate) for 48 hours on the second day. Cells in each group were harvested for protein extraction, quantified by the BCA assay, and analyzed by Western blotting.

[0052] The results showed that compared with the use of Olaparib and Amlodipine maleate alone, the combined use of the two drugs significantly increased the levels of Cleaved-Caspase7 and Cleaved-Caspase3 ( Figure 5 ), which indicated that Olaparib combined with Amlodipine maleate showed stronger induction of GC cell apoptosis.

[0053] Example 6: Subcutaneous tumor formation in nude mice

[0054] Four 4-week-old female nude mice were raised for one week, and 5×10 6 A nude mouse GC subcutaneous xenograft tumor model was established by inoculating cells. When the tumor mass grew to about 1 cm, it was dissected, removed, and cut into 1 mm 3 Tissue fragments, set aside.

[0055] Twenty four-week-old female nude mice were selected and raised for one week. Tissue fragments were inoculated into the subcutaneous tissue of the experimental nude mice's armpits through tumor implantation needles. About two weeks later, when the average tumor volume of the nude mice reached 80 mm 3 20 nude mice were randomly divided into 4 groups: Ctrl group (control group, no drug added), Olaparib group (Olaparib alone group), Amlodipine maleate group (Amlodipine maleate alone group), and Combination group (combination group). Intraperitoneal injection of drugs was started. The stock solution of drugs was prepared daily and diluted with PBS. Each nude mouse was intraperitoneally injected with 25 mg / kg / day of Olaparib and 2 mg / kg / day of Amlodipine maleate, either alone or in combination. The Ctrl group was given PBS. The tumor volume of the nude mice was recorded every other day, and the relative tumor volume (V) was calculated as (length × width) 2 ) / 2 mm3 After three weeks of continuous drug treatment, the nude mice were killed by cervical dislocation. The tumor volume change line graph was drawn using GraphPad Prism, and the volume weight of the nude mice after death was measured and photographed.

[0056] The results showed that the tumor growth rate in the combination group was significantly inhibited and the tumor weight was significantly reduced compared with the control group and the single drug group ( Figure 6 ), which showed that Olaparib combined with Amlodipine maleate could inhibit GC tumor growth in nude mice.

[0057] Example 7: Immunohistochemistry

[0058] Organ tissues (nude mice from the experiment in Example 6) were stained with H&E, and tumor masses were stained with Ki67, Tunel, and H&E. Ki67 and Tunel expression was analyzed and drug toxicity was assessed. GC primary lesions and adjacent normal tissue specimens were fixed in 10% neutral formalin and embedded in paraffin. Sections were deparaffinized and rinsed with distilled water. Antigen retrieval was performed, followed by three 5-minute washes with PBS. PARP1 antibody was added, incubated overnight at 4°C, and washed three times with PBS for 5 minutes each. Goat anti-mouse IgG H&L (biotin) was added to the tissue microarray at a dilution of 1:500 and incubated for 30 minutes. The microarray was then washed with PBS three times for 5 minutes each. DAB color development was performed using a DAB color development kit, and nuclei were counterstained with hematoxylin. The tissue microarray was dehydrated and blocked with neutral gelatin.

[0059] The results showed that after combined use, Ki67 expression was significantly inhibited, while Tunel expression was significantly increased ( Figure 7 ), which shows that the combined drug can significantly inhibit tumor proliferation and induce tumor cell apoptosis.

[0060] Example 8: Toxicity impact evaluation

[0061] To evaluate the toxic effects of olaparib and amlodipine maleate combined on nude mice, we further examined changes in nude mouse body weight following drug treatment in Example 5. During drug treatment, nude mice were weighed and recorded every other day. After data compilation, a line graph of weight changes was plotted using Graphpad. At the time of sacrifice, the hearts, livers, spleens, lungs, and kidneys of each group were dissected and stained with H&E to assess the toxic effects of the drugs on their internal organs.

[0062] The results show that ( Figure 8A in Figure 2 shows that there was no significant change in body weight in the Olaparib, Amlodipine maleate, and combination groups compared to the control group. There was also no significant difference in H&E staining results of the heart, liver, spleen, lung, and kidney in the Olaparib, Amlodipine maleate, and combination groups compared to the control group ( Figure 8 Furthermore, no nude mice in either group died during the experiment. Therefore, these experimental results demonstrate that the combination of olaparib and amlodipine maleate had no significant toxic effects on nude mice.

[0063] Example 9: Evaluation of the efficacy of combined drug therapy in different tumors

[0064] In this example, a Ctrl group (control group, no drug added), an Olaparib group (Olaparib alone group), an Amlodipine maleate group (Amlodipine maleate alone group), and a Combination group (combination group) were set up to treat A549 cells (lung cancer cell line), AGS cells (human gastric adenocarcinoma cell line), HCT116 cells (human colon cancer cell line), HepG2 cells (human liver cancer cell line), HTh-7 cells (human thyroid cancer cell line), and KYSE150 cells (human esophageal cancer cell line) with corresponding drugs. The results showed that ( Figure 9 AF in the combination group), the tumor cell survival rate was significantly reduced in the combination group compared with the control group and the single-drug group, indicating that the combination of Olaparib and Amlodipine maleate has significant therapeutic effects in various tumors such as lung cancer, gastric cancer, colon cancer, liver cancer, thyroid cancer, and esophageal cancer.

Claims

1. An anticancer composition for combined use, characterized in that: The invention comprises a PARP inhibitor and a calcium ion antagonist, wherein the PARP inhibitor is olaparib and the calcium ion antagonist is amlodipine maleate.

2. Use of the composition in the preparation of anticancer drugs, characterized in that: The composition comprises a PARP inhibitor and a calcium ion antagonist, wherein the PARP inhibitor is olaparib and the calcium ion antagonist is amlodipine maleate.

3. Use of the composition according to claim 2 in the preparation of anticancer drugs, characterized in that: The cancer type was stomach cancer, lung cancer, colon cancer, liver cancer, thyroid cancer, or esophageal cancer.

4. Use of the composition according to claim 3 in the preparation of anticancer drugs, characterized in that: The cancer type is gastric cancer.

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