Use of artesunate in the preparation of a medicament for enhancing a parp inhibitor drug

Abstract

This application discloses the use of artesunate in the preparation of drugs for enhancing the therapeutic effect of PARP inhibitors, belonging to the field of biomedical technology. The artesunate described in this application is a sesquiterpene lactone derivative extracted from the traditional Chinese medicine Artemisia annua. It has an inhibitory effect on the proliferation of BRCA wild-type ovarian cancer cells and low toxicity to normal human ovarian epithelial cells. This application's research found that artesunate can enhance the DNA damage and apoptosis induced by PARP inhibitors (such as olaparib, niraparib, and rucaparib), and improve the sensitivity of PARP inhibitors to BRCA wild-type ovarian cancer.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to the use of artesunate in the preparation of drugs for enhancing PARP inhibitors. Background Technology

[0002] Ovarian cancer is one of the deadliest malignant tumors of the female reproductive system. Surgery is currently the primary treatment for ovarian cancer, mainly involving comprehensive staging surgery and cytoreductive surgery. Chemotherapy is usually used as adjuvant therapy, employing platinum-based and taxane-based drugs. Radiotherapy is primarily used for postoperative adjuvant therapy or palliative care in advanced ovarian cancer, and its application is limited. Targeted therapy targets specific molecular abnormalities, such as BRCA-mutated ovarian cancer, using PARP (Poly ADP-Ribose Polymerase) inhibitors; lipid metabolism-related enzyme inhibitors; and protein-targeted therapy.

[0003] In PARP inhibitor therapy, PARP inhibitors are highly effective in treating ovarian cancer carrying BRCA gene mutations through their "synthetic lethality" effect. However, for approximately 80% of BRCA wild-type patients, primary or acquired resistance often develops due to the intact homologous recombination repair (HRR) capacity of tumor cells, limiting the efficacy of the treatment. Currently, there is a lack of highly selective drugs that can effectively reverse this resistance. Summary of the Invention

[0004] This application aims to solve one of the technical problems of the prior art, and to this end, provides an application of artesunate in the preparation of drugs for enhancing PARP inhibitors. Artesunate targets and inhibits the function of RAD51 protein and promotes the degradation of RAD51, blocking the homologous recombination repair pathway, thereby sensitizing PARP inhibitors such as olaparib and niraparib, for the development and application of drugs for the treatment of BRCA wild-type or homologous recombination repair-intact ovarian cancer.

[0005] The technical solution adopted in this invention is: A first aspect of this application provides the use of artesunate in the preparation of a medicament for enhancing the therapeutic effect of PARP inhibitors, said artesunate being a compound of formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof: (I).

[0006] The "pharmaceutically acceptable salt" refers to a conventional, non-toxic salt formed by reacting a compound of general formula (I) with an inorganic or organic acid. For example, the conventional non-toxic salt can be prepared by reacting a compound of general formula (I) with an inorganic or organic acid, wherein the inorganic acid includes hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, aminosulfonic acid, and phosphoric acid, and the organic acid includes citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pyric acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, and hydroxyethanesulfonic acid. Acids, etc.; or compounds of general formula (I) forming esters with propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, aspartic acid, or glutamic acid, and then forming sodium, potassium, calcium, aluminum, or ammonium salts with inorganic bases; or compounds of general formula (I) forming methylamine, ethylamine, or ethanolamine salts with organic bases; or compounds of general formula (I) forming esters with lysine, arginine, or ornithine, and then forming corresponding inorganic acid salts with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, or phosphoric acid, or corresponding organic acid salts with formic acid, acetic acid, picric acid, methanesulfonic acid, and ethanesulfonic acid.

[0007] More specifically, in the above technical solution, the drug has the following properties: increasing the expression of the DNA damage marker γ-H2AX.

[0008] More specifically, in the above technical solution, the drug is used to treat ovarian cancer.

[0009] More specifically, in the above technical solution, the ovarian cancer is BRCA wild-type ovarian cancer.

[0010] More specifically, in the above technical solution, the cancer cells of the BRCA wild-type ovarian cancer include Skov3 and / or A2780.

[0011] More specifically, in the above technical solution, the drug also includes the following characteristics: inducing apoptosis of the cancer cells and arresting the cancer cells in the S phase of the cell cycle.

[0012] More specifically, in the above technical solution, the dosage form of the drug includes: injection, tablet, capsule, reagent kit or patch.

[0013] It should be noted that those skilled in the art can prepare the above-mentioned pharmaceutical composition into various dosage forms according to specific circumstances. The preparation method is a well-known technology in the field, so it will not be described in detail in this application.

[0014] More specifically, in the above technical solution, the PARP inhibitor includes at least one of olaparib, niraparib, and rucaparib.

[0015] More specifically, in the above technical solution, preferably, the PARP inhibitor is niraparib.

[0016] A second aspect of this application provides the use of artesunate in the preparation of sensitizers for PARP inhibitors, said artesunate being a compound of formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof: (I).

[0017] Compared with the prior art, the embodiments of this application have the following beneficial effects: Artesunate exhibits a significant synergistic antitumor effect with PARP inhibitors. In vitro experiments showed that artesunate combined with olaparib or niraparib produced a significant synergistic effect (synergistic index CI < 0.9) and induced more severe DNA damage (increased γ-H2AX focal formation) and apoptosis. In a BRCA wild-type ovarian cancer xenograft model in nude mice, the tumor growth inhibition rate of the artesunate plus olaparib group was significantly better than that of the single-drug groups, and no significant toxic effects were observed on mouse body weight, liver and kidney function, or major organ pathology. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The image shows the synergistic antitumor proliferation effect of artesunate and niraparib in vitro (CCK-8 experiment, and the synergistic effect index CI value was calculated).

[0020] Figure 2 This image shows the synergistic antitumor proliferation effect of artesunate and niraparib in vitro (clonal formation experiment).

[0021] Figure 3 This figure shows the in vivo synergistic antitumor efficacy of artesunate combined with olaparib in a BRCA wild-type ovarian cancer xenograft model in nude mice. Detailed Implementation

[0022] To provide a clearer understanding of the technical content of this invention, the following embodiments are provided in conjunction with the accompanying drawings for detailed description. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0023] Artesunate, chemically named dihydroartemisinin-10α-succinic acid monoester, is a sesquiterpene lactone derivative extracted from the traditional Chinese medicine Artemisia annua, with the molecular formula C19H28O8. Artesunate was purchased from MedChemExpress Ltd. The powder was dissolved in DMSO to prepare a 50 mM stock solution, aliquoted, and stored at -20°C.

[0024] 1. Experimental Materials and Methods Cell lines: Human ovarian cancer cells A2780, Skov3 (BRCA wild type) and normal ovarian epithelial cells IOSE80 were all cryopreserved cells from the Central Laboratory of Jinan University.

[0025] Table 1. Antibodies required for the experiment and their suppliers: name company DMEM culture medium (1×) Jiangsu KeyGEN RAD51 anti-human monoclonal antibody Cell Signaling Technology γ-HA2X anti-human monoclonal antibody Cell Signaling Technology p-ATM anti-human monoclonal antibody Cell Signaling Technology Cleaved-Parp anti-human monoclonal antibody Cell Signaling Technology Cleaved-Caspase3 anti-human monoclonal antibody Cell Signaling Technology CCK8 reagent kit Biyuntian Biotechnology Annexin V-FITC Apoptosis Detection Kit Biyuntian Biotechnology Bovine serum albumin (BSA) Biyuntian Biotechnology Serum-free cell cryopreservation solution Suzhou Xinsaimei Biotechnology Co., Ltd. High-sensitivity ECL chemiluminescence reagent kit Suzhou Xinsaimei Biotechnology Co., Ltd. PAGE gel preparation kit Yamei siRNA Ruibo Biotechnology Co., Ltd. CCK8 reagent kit Biyuntian Biotechnology Annexin V-FITC Apoptosis Detection Kit Biyuntian Biotechnology Methods: This study evaluated the inhibitory effect of artesunate on the proliferation of ovarian cancer cells and its synergistic effect with PARP inhibitors using the CCK-8 assay, and calculated the combination index (CI). Long-term proliferation inhibition was detected using a colony formation assay. Cell apoptosis and cell cycle distribution were analyzed using flow cytometry. The regulation of homologous recombination repair and apoptosis pathway protein expression by artesunate was studied using Western blotting. Binding modes were predicted using molecular docking. Finally, a nude mouse xenograft model was constructed to verify the sensitizing effect and safety of artesunate on PARP inhibitors in vivo.

[0026] Cell proliferation inhibition assay (CCK8 assay) A2780, Skov3, and IOSE80 cells in logarithmic growth phase were digested with trypsin to prepare single-cell suspensions, and cell counts were performed. Cells were seeded at a density of 2000 cells per well in 96-well plates, with a volume of 100 μL per well. The culture plates were incubated at 37°C in a 5% CO2 incubator for 24 hours to allow cell adhesion.

[0027] The following experimental groups were set up: ① Blank control group (containing only culture medium); ② Artesunate monotherapy concentration gradient groups (0, 0.5, 1, 2, 4, 8, 16, 32 μM); ③ Olaparib monotherapy concentration gradient groups (0, 1, 2, 4, 8, 16, 32 μM); ④ Combination therapy group (fixed concentration of artesunate [IC50]).20 [Co-treatment with gradient concentrations of olaparib].

[0028] After drug treatment, the culture plates were returned to the incubator for another 48 hours. 10 μL of CCK8 solution was added to each well, and incubation continued for 2 hours. The absorbance of each well was measured at 450 nm using a microplate reader. Cell viability was calculated using software, and the half-maximal inhibitory concentration (IC50) of the drug was also calculated. 50 Drug interactions were analyzed using CompuSyn software, and the combination index (CI) was calculated.

[0029] Cloning experiment A2780 and Skov3 cells in logarithmic growth phase were digested, counted, and seeded at a low density of 500 cells per well in 6-well plates. The plates were gently shaken to ensure even cell distribution. The plates were then incubated overnight for cell adhesion.

[0030] The study included a control group, an artesunate monotherapy group, an olaparib monotherapy group, and a combination treatment group of artesunate and olaparib. The culture medium was replaced with fresh medium containing the corresponding drug every 3 days. Cells were cultured for 10-14 days until visible cell clones were observed.

[0031] Discard the culture medium and wash carefully twice with PBS. Add 1 mL of 4% paraformaldehyde solution to each well and fix for 30 minutes at room temperature. Discard the fixative, wash with PBS, and add 0.5% crystal violet staining solution to each well, staining for 30 minutes at room temperature. Rinse slowly with running water and air dry at room temperature. Photograph the stained clones and use image analysis software to count the number of clones with more than 50 cells.

[0032] Western blotting: Experimental antibodies: 1) Protein quantification After overnight adherent culture in culture plates, cells were treated with the appropriate concentration of drug or radiotherapy for 24 h. After 24 h, the culture medium was aspirated, and cells were washed with pre-chilled 1×PBS. Under low temperature conditions, RIPA strong lysis buffer was added, and the cell suspension was transferred to centrifuge tubes for lysis for 30 min. A high-speed refrigerated centrifuge was pre-started, and the temperature and speed were adjusted to 4℃ and 12000 rpm for 15 min. The protein supernatant was transferred to new microcentrifuge tubes, and the protein concentration was determined using a BCA kit. After protein quantification, the supernatant was diluted to 1× with SDS-PAGE protein loading buffer (5×) and heated in a 95℃ metal bath for approximately 10 min. The protein supernatant was aliquoted and stored at -80℃.

[0033] 2) Electrophoresis Prepare polyacrylamide gels of appropriate concentrations according to the molecular weight of the target protein, assemble the electrophoresis apparatus, and add 1× electrophoresis working solution. Then, load the Rainbow Protein Maker and the sample protein together for electrophoresis. Electrophoresis is performed at 80 V for 30 min, then increased to 120 V and continued until the indicator band is close to the bottom of the gel.

[0034] 3) Transfer membrane After electrophoresis, remove excess gel and place the membrane flat on transfer filter paper. Activate the PVDF membrane by soaking it in methanol, ensuring it adheres completely to the gel, and insert it into the transfer clamp with the gel side facing the black side of the clamp. Then, position the clamp with the black side facing the negative electrode of the transfer tank and the white side facing the positive electrode. Fill the transfer buffer and transfer the membrane at a constant current of 200 mA for 90 minutes. Fill the transfer tank with ice.

[0035] 4) Closed After the transfer is complete, immerse the membrane in 5% sealed milk, shake slowly at low speed, and incubate at room temperature for 1 hour.

[0036] 5) Antibody incubation and chemiluminescence imaging After blocking, wash away excess milk with 1×TBST buffer. Soak the primary antibody in a 1:1000 dilution buffer and incubate overnight at 4°C. After primary antibody incubation, recover the primary antibody and add 1×TBST washing buffer. Wash the membrane on a shaker for 8 minutes each time, for a total of 3 times. After washing, place the PVDF membrane in the corresponding secondary antibody solution and dilute... The concentration ratio was 1:7000, and the membrane was incubated on a shaker at room temperature for 1.5 h. Then, 1 × TBST was added to wash the membrane for 8 min each time, for a total of 3 times. After washing, a highly sensitive ECL chemiluminescent solution was applied to the membrane, and the membrane was exposed using a chemiluminescence imaging system to collect and analyze the experimental results.

[0037] Nude mouse xenograft model: (1) A2780 cells were subcutaneously inoculated into the right back of BALB / c-nu nude mice.

[0038] (2) When the tumor volume is approximately 100 mm 3 At that time, the patients were randomly divided into 4 groups: solvent control group, artesunate monotherapy group (50 mg / kg), olaparib monotherapy group (50 mg / kg), and combination therapy group.

[0039] (3) Administer the medication via intraperitoneal injection every 2 days for 2-3 weeks. Measure tumor volume and mouse weight periodically.

[0040] (4) At the end of the experiment, the mice were sacrificed, the tumors were removed and weighed, and the heart, liver, spleen, lungs and kidneys were analyzed by H&E staining.

[0041] Data processing and statistical analysis Data processing and analysis were performed using IBM SPSS Statistics 26 and GraphPad Prism 9. Student's t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. The Combination Index (CI) was calculated using CompuSyn software to assess the combined drug effect: CI < 0.9 indicated a synergistic effect, 0.9 < CI < 1.1 indicated an additive effect, and CI > 1.1 indicated an antagonistic effect. All experiments were independently repeated at least three times. Data are expressed as mean ± standard deviation. *P < 0.05 was considered statistically significant.

[0042] In the experiment of this study, such as Figure 1 The images show the synergistic inhibition of ovarian cancer cell proliferation by artesunate and PARP inhibitors. (A, B) CCK8 assay to detect cell viability of Skov3 cells treated with artesunate in combination with olaparib and niraparib. (C) CI value distribution of Skov3 cells treated with artesunate in combination with olaparib and niraparib. (D, E) CCK8 assay to detect cell viability of A2780 cells treated with artesunate in combination with olaparib and niraparib. (F) CI value distribution of A2780 cells treated with artesunate in combination with olaparib and niraparib.

[0043] like Figure 2 The results show the effects of artesunate and olaparib combined on the inhibition of A2780 and Skov3 cell proliferation using a colony formation assay. Quantitative statistical analysis of the colony formation assay using the combined effects of artesunate and olaparib is presented (A, B, C, D). Data are expressed as mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ns > 0.05.

[0044] like Figure 3 The image shows the inhibition of A2780 xenograft growth by artesunate combined with olaparib. We used 4×10 6A2780 cells were subcutaneously inoculated into BALB / c nude mice. When the tumor volume reached 100 mm3, the mice were divided into four groups: a blank control group, an artesunate (3 mg / kg) group, an olaparib (4 mg / kg) group, and a combination group (artesunate 3 mg / kg + olaparib 4 mg / kg). Intraperitoneal injection was administered for 24 consecutive days, with tumor volume and mouse weight measured every 3 days (B, D). We found that the combination group had a significant inhibitory effect on tumor volume, and the tumor volume in the combination group was significantly smaller than that in the other three groups (A, B). (E, F) Biochemical detection of the drug's toxic side effects on nude mice. Data are expressed as mean ± standard deviation (mean ± SD), n=6, *P<0.05, **P<0.01, ***P<0.001, ns>0.05. (G) HE-stained sections of heart, liver, spleen, lung, and kidney tissues from nude mice in each group at the experimental endpoint, image scale bar 100 μm.

[0045] The above experiments show that using low concentrations of artesunate significantly increases the sensitivity of BRCA wild-type ovarian cancer cells to PARP inhibitors, increasing the sensitivity of A2780 and SKOV3 ovarian cancer cells to olaparib by 60.6-fold and 6.6-fold, respectively, and to niraparib by 124.0-fold and 16.0-fold, respectively. Flow cytometry, Western blotting, and comet assays showed that the combined drug significantly increased the expression of the DNA damage marker γ-H2AX, induced apoptosis and S-phase arrest, and reversed the activation of the homologous recombination repair pathway induced by PARP inhibitors (the cell cycle is an important event regulating normal cell growth and division). The inventors also detected cell cycle distribution using PI staining and flow cytometry. In A2780 and SKOV3 cells, compared with the blank control group, due to DNA damage, DNA damage repair was initiated simultaneously, and cell cycle arrest in the olaparib monotherapy group mainly occurred in the G2 / M phase. Finally, in a nude mouse xenograft model, it was confirmed that artesunate combined with olaparib effectively inhibited tumor growth without showing significant systemic toxicity.

[0046] The embodiments described above are merely illustrative of several implementations of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Therefore, the scope of protection of this patent application should be determined by the appended claims.

Claims

1. Use of artesunate in the preparation of medicaments for enhancing the therapeutic effect of PARP inhibitors, wherein artesunate is a compound of formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof: (I)。 2. The application according to claim 1, characterized in that, The drug has the following properties: it increases the expression of the DNA damage marker γ-H2AX.

3. The application according to claim 2, characterized in that, The drug is used to treat ovarian cancer.

4. The application according to claim 3, characterized in that, The ovarian cancer mentioned is BRCA wild-type ovarian cancer.

5. The application according to claim 4, characterized in that, The cancer cells in the BRCA wild-type ovarian cancer include Skov3 and / or A2780.

6. The application according to claim 5, characterized in that, The drug also includes the following properties: inducing apoptosis in the cancer cells and arresting the cancer cells in the S phase of the cell cycle.

7. The application according to claim 1, characterized in that, The dosage forms of the drug include: injections, tablets, capsules, kits, or patches.

8. The application according to any one of claims 1-7, characterized in that, The PARP inhibitors include at least one of olaparib, niraparib, and rucaparib.

9. The application according to claim 8, characterized in that, Preferably, the PARP inhibitor is niraparib.

10. Use of artesunate in the preparation of sensitizers for PARP inhibitors, wherein artesunate is a compound of formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof: (I)。

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