Combination anticancer composition, use of the composition in the preparation of anticancer drugs

Abstract

The application discloses an anticancer composition of combined medication, and application of the composition in preparation of an anticancer drug. The combined medication composition provided by the application comprises a PARP inhibitor, Olaparib, and a calcium ion antagonist, Amlodipine maleate, and the two components have a synergistic effect, have higher sensitivity to gastric cancer compared with a single PARP inhibitor, and significantly improve the effective rate, thereby having a wide application prospect. The Amlodipine in the combined medication composition provided by the application enhances the curative effect of the PARP inhibitor, Olaparib, on gastric cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to anticancer compositions for combined drug use and the application of such compositions in the preparation of anticancer drugs. Background Technology

[0002] Gastric carcinoma (GC) is a highly aggressive and heterogeneous malignant tumor of the digestive tract, prevalent worldwide. Surgical resection remains the primary treatment for GC. However, early-stage GC often lacks obvious symptoms, leading to low diagnosis rates. Patients are typically diagnosed at an advanced stage, often with varying degrees of metastasis. Therefore, despite surgical intervention, GC patients still experience low survival rates and high recurrence rates. While advancements in radiotherapy, chemotherapy, and neoadjuvant therapy have significantly improved overall survival in recent years, the benefits of these treatments remain limited, and drug resistance and tolerance are frequently observed. Therefore, the development of new drugs for GC treatment remains crucial.

[0003] The poly(ADP-ribose) polymerase (PARP) family is a group of multifunctional protein post-translational modification enzymes involved in DNA damage repair. PARP1 plays a crucial role in various cellular processes, including chromatin structure, replication, transcription, energy metabolism, cell death, immunity, and inflammation, encompassing over 90% of the family's functions and holding a pivotal position in DNA repair. Based on the critical role of PARP1 in mediating DNA damage, a fundamental principle has been provided for developing PARP inhibitors (PARPi) to treat human malignancies.

[0004] In 2014, the first PARPi, olaparib, received approval from the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for maintenance therapy in advanced ovarian cancer with BRCA1 / 2 mutations in the DNA repair genes. Olaparib became the first PARPi approved for treating GC. With continued progress in basic research, olaparib as a monotherapy for GC also entered clinical trials. However, phase III trials evaluating the efficacy of olaparib failed to show a significant improvement in overall survival for GC patients. This demonstrates that the efficacy of PARPi as a monotherapy for GC is quite limited.

[0005] Patent application HK40036086A discloses a PARP inhibitor maintenance therapy in the treatment of gastric cancer, comprising administering a therapeutic or maintenance effective amount of a PARP inhibitor to a subject who has previously received chemotherapy. The PARP inhibitor is selected from olaparib, niraparib, rucaparib, or pamipanib.

[0006] The invention application with publication number WO2018099423A1 discloses the use of a combination of a VEGFR inhibitor and a PARP inhibitor in the preparation of a drug for treating gastric cancer. 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] Screening for other drugs that can be used in combination with PARPi to improve the treatment effect of gastric cancer remains of practical significance. Summary of the Invention

[0008] To address the aforementioned deficiencies in the prior art, this invention provides an anticancer composition for combined drug use and its application in the preparation of anticancer drugs.

[0009] Based on a "drug repurposing" development strategy, amlodipine maleate significantly enhances the sensitivity of PARP1 knockout cell lines through screening of the Food and Drug Administration (FDA) drug library. Furthermore, amlodipine, a calcium channel blocker and NF-κB pathway inhibitor, can be considered as a candidate for combination therapy, providing a novel strategy for GC patients to combine with PARP1.

[0010] The present invention first provides an anticancer composition for combination therapy, comprising a PARP inhibitor and a calcium channel blocker.

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

[0012]

[0013] Olaparib, chemically named 1-(cyclopropanoyl)-4-[5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoyl]piperazine, has the chemical formula C2. 24 H 23 FN4O3, CAS Registry Number 763113-22-0.

[0014] Preferably, the calcium ion antagonist is amlodipine maleate, with the following molecular structural formula:

[0015]

[0016] Amlodipine maleate is chemically named racemic-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylic acid 3-ethyl-5-methyl ester maleate, with the molecular formula C1. 24 H 29 ClN2O9, CAS number 88150-47-4.

[0017] The present invention further provides the use of the composition in the preparation of anticancer drugs, the composition comprising a PARP inhibitor and a calcium ion antagonist.

[0018] Preferably, the cancer type is stomach cancer, lung cancer, colon cancer, liver cancer, thyroid cancer, or esophageal cancer. More preferably, the cancer type is stomach cancer.

[0019] Preferably, the PARP inhibitor is olaparib.

[0020] Preferably, the calcium ion antagonist is amlodipine maleate.

[0021] The combination drug composition provided by this invention includes the PARP inhibitor olaparib and the calcium channel blocker amlodipine maleate. The two work synergistically and have a higher sensitivity to gastric cancer compared with single PARP inhibitors, significantly improving the efficacy rate and thus showing broad application prospects.

[0022] The combination drug composition provided by this invention contains amlodipine maleate, which enhances the efficacy of the PARP inhibitor olaparib against gastric cancer. Attached Figure Description

[0023] Figure 1 A flowchart for screening drugs to overcome PARPi resistance in the FDA drug library.

[0024] Figure 2 To validate Amlodipine maleate as a candidate drug for use in combination with Olaparib in the CCK8 experiment.

[0025] Figure 3 To evaluate the effect of olaparib combined with amlodipine maleate on GC cell proliferation for the purpose of colony formation assessment. Figure 3 In the image, A represents a photomicrograph; Figure 3 B in the figure represents the statistical result.

[0026] Figure 4 To evaluate the ability of olaparib combined with amlodipine maleate to promote apoptosis in GC cells using flow cytometry. Figure 4 In the graph, A represents the result of the flow cytometry test. Figure 4 B in the figure represents the statistical result.

[0027] Figure 5 The effect of olaparib combined with amlodipine maleate on the expression of Cleaved-Caspase3 and Cleaved-Caspase7 proteins in GC cells was evaluated by Western blot.

[0028] Figure 6 To evaluate the combined effects of olaparib and amlodipine maleate in vivo using a nude mouse axillary xenograft model. Figure 6 In this context, A represents the tumor volume in nude mice after three weeks of drug treatment. Figure 6 In this figure, B represents the tumor volume in nude mice after three weeks of drug treatment. Figure 6 In this context, C represents the tumor mass in nude mice after three weeks of drug treatment.

[0029] Figure 7 The tumor mass was subjected to H&E, Ki67, and TUNEL immunohistochemical assays. Among these, Figure 7 In the image, A represents an immunohistochemical staining image. Figure 7 In the figure, B represents the statistical results of Ki67 immunohistochemistry. Figure 7 C in the figure represents the statistical results of TUNEL immunohistochemistry.

[0030] Figure 8 H&E staining was performed to evaluate the toxic effects of the combination of Olaparib and Amlodipine maleate on organs in nude mice. Figure 8 In this context, A represents the change in the weight of nude mice measured and recorded every other day during drug treatment. Figure 8 B in the figure represents the H&E staining of the heart, liver, spleen, lungs, and kidneys of nude mice three weeks after drug treatment.

[0031] Figure 9 To evaluate the efficacy of combining Olaparib and Amlodipinemaleate in different tumors by assessing cell survival rates after application of combination therapy to different tumors. Figure 9 In this context, A represents A549 cells; Figure 9 B in the text represents AGS cells; Figure 9 C in the text represents HCT116 cells; Figure 9 D in the text represents HepG2 cells; Figure 9 E in the text refers to HTh-7 cells;Figure 9 F in the text represents KYSE150 cells. Detailed Implementation

[0032] Example 1: Screening FDA drug library for drugs to overcome PARPi resistance

[0033] To identify drugs that can be used in combination with Olaparib, we used the FDA drug database provided by MCE (containing over 2000 approved clinical drugs) for screening. These drugs cover areas such as oncology, cardiovascular, and immunology. Cell viability was measured using the CCK-8 assay after treatment of 293A WT and PARP1 KO cells with the drugs, and the difference between the two was calculated as [293A WT (viability) - PARP1 KO (viability)].

[0034] The filtering results are as follows Figure 1 As shown, the results indicate that amlodipine maleate was more sensitive to PARP1 KO cells than 293A WT cells when treated. Amlodipine maleate is a calcium channel blocker and an NF-κB pathway inhibitor. Amlodipine maleate is being considered as a candidate drug for combination therapy with olaparib, and further experimental studies are planned to validate its potential efficacy in GC treatment.

[0035] Example 2: Preparation of stock solutions of Olaparib and Amlodipine maleate

[0036] Accurately weigh appropriate amounts of Olaparib (purchased from MCE) and Amlodipine maleate (purchased from Targetmol), and use DMSO as solvent to prepare stock solutions with concentrations of 2.5 mg / mL and 0.2 mg / mL, respectively. Aliquot into 1.5 mL EP tubes, seal with sealing film, and store at -20°C.

[0037] Example 3: Detection of Cell Working Concentration

[0038] (1) Construction of PARP1 knockout plasmid: PARP1 sgRNA was designed. By combining scoring and editing efficiency, 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. Oligonucleotides for inserting sgRNA were prepared, where Oligo1 was the front primer for sgRNA1, sgRNA2, and sgRNA3, and Oligo2 was the back primer for sgRNA1, sgRNA2, and sgRNA3. The oligonucleotides were dissolved and resuspended to a final concentration of 100 μM. The sgRNA oligos were phosphorylated and annealed, as detailed in Table 2. The oligos were phosphorylated and annealed in a PCR instrument, as detailed in Table 3. The product was diluted with ddH2O at a ratio of 1:200 and stored 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, as detailed in Table 4. The digested products were collected by agarose gel electrophoresis and then subjected to agarose gel electrophoresis: 1× TAE was microwaved for 1 min until the agarose was completely dissolved. The liquid was then poured into the gel plate, and the loading comb was inserted. The gel was allowed to cool and solidify for approximately 20 min. The sample, gel buffer, and loading buffer were thoroughly mixed and set aside for loading. A 0.1% agarose gel was removed, the comb carefully removed, and the gel plate and electrophoresis apparatus were assembled. 1× ​​TAE electrophoresis buffer was added until the gel was just submerged. 5-15 μL of sample was loaded into each well, along with a DNA marker (DNA standard molecular weight) to indicate sample size. Electrophoresis was performed at 140V for approximately 30 min. The digested products were identified under UV light by comparing them with the DNA marker, and the gel was then excised and recovered. Based on the electrophoresis results, the size of the digested products and the integrity of the digested vector fragments were confirmed by comparing them with the DNA marker. The corresponding bands were then excised and recovered using a DNA gel recovery kit. The plasmid and vector fragment were ligated overnight at room temperature using T4 DNA ligase for recombinant plasmid ligation (see Table 5 for details). 10 μL of the recombinant plasmid was added to 40 μL of competent cells, incubated on ice for 30 min, then heat-shocked at 42°C for 90 s. Immediately afterward, the cells were transferred to ice and incubated for 3 min. 50 μL of LB medium was added, and the cells were incubated at 37°C and 200 rpm for 1 h on a shaker. The resistance of the recombinant plasmid was determined by the vector sequence. The corresponding resistant medium was selected, and the transformed liquid was evenly spread onto the medium and incubated upside down at 37°C overnight. The plates were placed in a clean bench, and individually grown single clones were picked and transferred to the corresponding resistant LB medium. Amplification was performed overnight at 37°C and 200 rpm on a shaker. Subsequently, plasmid extraction was performed, and the plasmid was purified and its concentration determined using an endotoxin-free plasmid mini-prep kit.

[0039] Table 1 PARP1 sgRNA primer sequences

[0040]

[0041] Table 2 Mixing ratio of sgRNA oligos phosphorylation and annealing systems

[0042]

[0043] Table 3 PCR Procedure for sgRNA Oligos Phosphorylation and Annealing System

[0044]

[0045] Table 4. Vector Enzyme Digestion System

[0046]

[0047] Table 5 Recombinant plasmid ligation system

[0048]

[0049] (2) CCK-8 assay: 293A WT (wild-type 293A cells) and PARP1 KO1 cells (gene knockout cells obtained by transfecting wild-type 293A cells with a constructed PARP1 knockout plasmid) were seeded at 100 μL / well in 96-well plates and cultured overnight. Using a pre-constructed clinical drug library (FDA drug library), Olaparib was serially diluted three-fold at a concentration of 1500 μM to prepare nine different concentrations of drug solution. Subsequently, 50 μL of drug solution was added to each well, so that the cells were treated with drug 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. Then, the CCK-8 reagent was mixed with DMEM culture medium at a ratio of 1:9 to prepare the working solution. Add 100 μL of working solution to each well and incubate at 37℃ for approximately 1.5 h. Detect the OD value of each well using a microplate reader at 450 nm and calculate the survival rate as follows: (Average OD value of experimental group - Average OD value of background) / (Average OD value of well group - Average OD value of background) × 100%. Use GraphPad software to perform fitting analysis on the experimental data and calculate the IC50 value of the drug.

[0050] The results are as follows Figure 2 As shown, this indicates that Amlodipine maleate is a potential candidate drug for use in combination with Olaparib.

[0051] (3) Colony formation: HGC27 and NUGC3 cells (two types of gastric cancer cells) were seeded in 6-well plates at 800 cells / well and 1000 cells / well, respectively. After 4 days of culture, the cells were treated with the corresponding drugs: appropriate amounts of olaparib and amlodipine maleate were accurately weighed and prepared into stock solutions with concentrations of 2.5 mg / mL and 0.2 mg / mL, respectively, using DMSO as the solvent. The solutions were dispensed into 1.5 mL EP tubes, sealed with sealing film, and finally stored at -20°C.

[0052] Set up the Ctrl group (control group, no drug added), the Olaparib group (Olaparib alone), the Amlodipinemaleate group (Amlodipine maleate alone), and the Combination group (combined drug group), add the corresponding stock solution to each group, and continue culturing.

[0053] Two weeks later, the colony formation status of each group of cells was observed using an inverted microscope, and the number and morphology of cell clones were recorded in preparation for sample collection. The culture medium was removed, and 1 mL of PBS solution was added to each well to wash the cells twice. 1 mL of paraformaldehyde solution was added to each well to fix the cells for 15 min to stabilize their morphology and structure. After fixation, the cells were washed twice more with PBS. 1 mL of 0.1% crystal violet staining solution was added to each well to stain the cells for 30 min. After staining, the cells were washed twice more with PBS to remove excess staining solution. The 6-well plates were placed in a dry, ventilated area to air dry, and then the cell clones were photographed using a scanner to record the experimental results.

[0054] The results showed that the combination therapy group exhibited stronger inhibitory effects on proliferation in both GC cell lines compared to the single therapy group. Figure 3 The difference was statistically significant (p<0.05). This result further confirms that Amlodipinemaleate can enhance the inhibitory effect of Olaparib on GC cell proliferation.

[0055] Example 4: Flow Cytometry Detection of Apoptosis

[0056] Cell preparation: Appropriate amounts of HGC27 and NUGC3 cells were seeded into six-well plates to ensure a cell density of approximately 60%-70% by the second day. The following groups were established: Ctrl group (control group, no drug), Olaparib group (Olaparib alone), Amlodipine maleate group (Amlodipine maleate alone), and Combination group (combined drug treatment). On the second day, the corresponding drugs (2.5 mg / mL Olaparib and 0.2 mg / mL Amlodipine maleate) were administered for 48 h. The following procedures were performed: Old culture medium was transferred to 15 mL centrifuge tubes. After trypsin digestion without EDTA in each well, cells were transferred to 15 mL centrifuge tubes and combined. The cells were centrifuged at 1000 g for 5 min, and the supernatant was discarded. The cells were resuspended in PBS, centrifuged again, and the supernatant was discarded. Cells were resuspended in PBS and counted. Approximately 80,000 cells were transferred to 1.5 mL EP tubes, centrifuged at 11000 g for 5 min, and the supernatant was discarded. Add 195 μL of Annexin V-FITC binding solution to the cells and gently resuspend them. 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 to resuspend the cells, filter the cell suspension through a cell filter membrane, and prepare for flow cytometry. Collect cell samples using a Beckman flow cytometer, adjust parameters such as flow cytometry compensation and voltage, collect 10,000 cells for each sample, perform data analysis, divide positive regions, and obtain the apoptosis rate of each group.

[0057] The results showed that the apoptosis rate increased after combined drug administration ( Figure 4 This indicates that Amlodipine maleate can enhance Olaparib-induced apoptosis in GC cells.

[0058] Example 5: Apoptosis Pathway Detection

[0059] Appropriate amounts of NUGC3 and HGC27 cells were seeded into six-well plates to ensure a cell density of approximately 60%-70% by the second day. Four groups were established: a Ctrl group (control group, no drug administered), an Olaparib group (Olaparib alone), an Amlodipine maleate group (Amlodipine maleate alone), and a Combination group (combined drug administration). On the second day, each group was treated with the corresponding drug (2.5 mg / mL Olaparib and 0.2 mg / mL Amlodipine maleate) for 48 h. Cells from each group were collected, proteins were extracted, and protein quantification was performed using the BCA method, followed by Western blotting.

[0060] 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-Caspase 7 and Cleaved-Caspase 3. Figure 5 This indicates that Olaparib combined with Amlodipine maleate exhibits a stronger induction of GC cell apoptosis.

[0061] Example 6: Subcutaneous tumor formation in nude mice

[0062] Four 4-week-old female nude mice were reared for one week, and each mouse was subcutaneously inoculated with 200 μL (cell suspension: matrix gel = 1:1) 5 × 10⁶ cells in the axilla. 6 A subcutaneous xenograft tumor model of GC in nude mice was established using individual cells. When the tumor grew to approximately 1 cm, it was dissected, removed, and cut into 1 mm sections. 3 Organ fragments, for future use.

[0063] Twenty four-week-old female nude mice were selected and fed for one week. Tissue fragments were then injected subcutaneously into the axillae of the mice using a tumor grafting needle. Approximately two weeks later, when the average tumor volume of the mice reached 80 mm... 3 Twenty nude mice were randomly divided into four groups: the Ctrl group (control group, no drug), the Olaparib group (Olaparib alone), the Amlodipine maleate group (Amlodipine maleate alone), and the Combination group (combined drug treatment). Intraperitoneal injection of the drugs was initiated. The drug stock solution was prepared fresh daily and diluted with PBS. Each nude mouse received an intraperitoneal injection of 25 mg / kg / day of Olaparib and 2 mg / kg / day of Amlodipine maleate, either alone or in combination. The Ctrl group received PBS. Tumor volume was recorded every other day, and the relative tumor volume V = (length × width) 2 ) / 2 mm3 Nude mice were euthanized by cervical dislocation after three weeks of continuous drug treatment. The tumor volume changes were plotted using GraphPad Prism, and the volume and weight of the euthanized mice were measured and photographed.

[0064] The results showed that, compared with the control group and the single-drug group, the combination therapy group exhibited significantly inhibited tumor growth rate and a significant reduction in tumor weight. Figure 6 This indicates that olaparib combined with amlodipine maleate can inhibit the growth of GC tumors in nude mice.

[0065] Example 7: Immunohistochemistry

[0066] H&E staining was performed on organ tissues (nude mice used in Example 6), and Ki67, Tunel, and H&E staining was performed on tumor blocks. The expression of Ki67 and Tunel staining was analyzed, and drug toxicity was evaluated. Primary GC lesions fixed in 10% neutral formalin and embedded in paraffin, as well as adjacent normal tissue specimens, were collected. Sections were dewaxed and washed with distilled water; antigen retrieval was performed followed by three PBS washes, each lasting 5 min; PARP1 antibody was added, and the cells were incubated overnight at 4°C, followed by three PBS washes, each lasting 5 min. Goat anti-mouse IgG H&L (biotin) was added to the tissue microarray at a 1:500 dilution and incubated for 30 min, followed by three PBS washes, each lasting 5 min. DAB staining and hematoxylin counterstaining of cell nuclei were then performed using a DAB chromogenic kit. Finally, the tissue microarray was dehydrated and blocked with a neutral gel.

[0067] The results showed that Ki67 expression was significantly inhibited after combination therapy, while Tunel expression was significantly increased. Figure 7 This indicates that combined drug therapy can significantly inhibit tumor proliferation and induce tumor cell apoptosis.

[0068] Example 8: Toxicity Impact Assessment

[0069] To evaluate the toxic effects of the combined use of olaparib and amlodipine maleate on nude mice, the changes in body weight of the mice after drug treatment in Example 5 were further investigated. During drug treatment, the weight of the nude mice was measured and recorded every other day. After data processing, a line graph of the weight changes of the nude mice was plotted using Graphpad. Simultaneously, upon euthanasia, the hearts, livers, spleens, lungs, and kidneys of the nude mice in each group were dissected for H&E staining to evaluate the toxic effects of the drugs on the organs of the nude mice.

[0070] The results show that ( Figure 8In the A group, compared to the control group, there were no significant changes in body weight in the Olaparib group, Amlodipine maleate group, and combination group. Compared to the control group, there were also no significant differences in H&E staining results for the heart, liver, spleen, lungs, and kidneys in the Olaparib group, Amlodipine maleate group, and combination group. Figure 8 (B) Furthermore, no mice in any group died during the experiment. Therefore, all the above experimental results indicate that the combined use of Olaparib and Amlodipine maleate has no significant toxic effects on nude mice.

[0071] Example 9: Evaluation of the efficacy of combination therapy in different tumors

[0072] This embodiment included a Ctrl group (control group, no drug administered), an Olaparib group (Olaparib alone), an Amlodipine maleate group (Amlodipine maleate alone), and a Combination group (combined drug administration). 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) were treated with the corresponding drugs, respectively. The results showed ( Figure 9 Compared with the control group and the single-drug group, the tumor cell survival rate of the combination therapy group (AF) was significantly reduced, indicating that the combination of Olaparib and Amlodipine maleate has significant efficacy in various tumors such as lung cancer, gastric cancer, colon cancer, liver cancer, thyroid cancer, and esophageal cancer.

Claims

1. An anticancer composition of combined drugs, characterized by, The combination includes a PARP inhibitor that is olaparib and a calcium ion antagonist that is amlodipine besylate.

2. Use of the composition for the manufacture of an anticancer medicament, characterized in that, The combination includes a PARP inhibitor that is olaparib and a calcium ion antagonist that is amlodipine besylate. The cancer type is gastric cancer, lung cancer, colon cancer, liver cancer, thyroid cancer, or esophageal cancer.

3. Use of the composition according to claim 2 for the preparation of an anticancer medicament, characterized in that, The cancer type is gastric cancer.

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