A metformin-enhanced PARP inhibitor, its preparation method and application

Abstract

This invention discloses a metformin-enhanced PARP inhibitor, its preparation method, and its application, belonging to the field of biomedical technology. Specifically, it relates to a metformin-enhanced PARP inhibitor comprising metformin and olaparib, with a molar ratio of olaparib to metformin of 1:200-600. This invention reshapes the energy metabolism state of different BRCA1-mutant subtypes of triple-negative breast cancer cells through metformin, significantly enhancing the inhibitory effect of olaparib on these cancer cells under drug-resistant conditions, and effectively promoting apoptosis and inhibiting cell migration, thereby achieving a synergistic enhancement of the efficacy of PARP inhibitors.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a metformin-enhanced PARP inhibitor, its preparation method, and its application. Background Technology

[0002] Triple-negative breast cancer (TNBC) is one of the most challenging subtypes of breast cancer to treat. It is characterized by the lack of expression of estrogen receptors, progesterone receptors, and human epidermal growth factor receptors, thus limiting effective targeted therapies. This disease exhibits high heterogeneity and aggressiveness, resulting in poor prognosis and a high likelihood of drug resistance and recurrence. Currently, TNBC treatment primarily relies on chemotherapy and immune checkpoint inhibitors. However, while chemotherapy can slow disease progression in the short term, long-term use often leads to drug resistance.

[0003] In recent years, synthetic lethality strategies have become a research hotspot. These strategies induce death in cancer cells through specific gene mutations or loss of function, and are particularly suitable for treating breast cancer susceptibility gene 1 (BRCA1)-mutant TNBC. Olaparib, a poly(ADP-ribose) polymerase inhibitor (PARPi), is a representative drug of synthetic lethality strategies and has been approved for the treatment of BRCA1-mutant breast cancer, becoming an important treatment option for TNBC patients. However, despite the significant initial efficacy of olaparib in BRCA1-mutant breast cancer, resistance often develops with continued treatment in BRCA1-mutant TNBC.

[0004] The resistance mechanism of BRCA1-mutant TNBC is not only related to the functional recovery of BRCA1, but also closely related to changes in the metabolic state of tumor cells, DNA repair capacity, and oxidative stress in the tumor microenvironment. Therefore, it is necessary to further explore new combination therapy strategies, especially metabolic regulation of DNA damage repair, to overcome PARPi resistance and broaden its therapeutic application.

[0005] Currently, the efficacy of olaparib in treating BRCA1-mutant TNBC is limited by several factors. First, resistance to PARPi exists in clinical use, making its therapeutic effect difficult to sustain. Second, the therapeutic effect of olaparib depends on the DNA repair mechanisms of tumor cells, and factors such as altered metabolic state of tumor cells, activation of glycolysis pathways, and lactate accumulation may interfere with the efficacy of PARPi by affecting the activity of DNA repair pathways. Furthermore, although studies have shown that metformin, as an antidiabetic drug, has significant antitumor effects in TNBC, its clinical application in BRCA1-mutant TNBC still faces many challenges, especially regarding how to combine it with PARPi through metabolic pathways; related research remains insufficient. Therefore, developing new treatment strategies and drugs, especially low-toxicity and broad-spectrum therapies, is of significant clinical importance. Summary of the Invention

[0006] The purpose of this invention is to provide a metformin-enhanced PARP inhibitor, its preparation method, and its application. This invention not only significantly enhances the inhibitory, apoptosis-promoting, and anti-migration effects of PARP inhibitors on drug-resistant BRCA1-mutant triple-negative breast cancer cells and improves the energy metabolism status of drug-resistant BRCA1-mutant triple-negative breast cancer cells, but also encapsulates the drug components in a drug nanocarrier, significantly increasing the drug loading rate of the prepared PARP inhibitor, reducing the initial burst release and cumulative release of the drug, thereby prolonging the drug's effect.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0008] A metformin-enhanced PARP inhibitor comprising metformin and olaparib; wherein the molar ratio of olaparib to metformin is 1:200-600.

[0009] This invention reshapes the energy metabolism of drug-resistant BRCA1-mutant triple-negative breast cancer cells using metformin, inhibits mitochondrial oxidative phosphorylation and glycolytic reprogramming, significantly reduces cellular oxygen consumption, ATP production rate, and maximum respiration capacity, while increasing proton leakage levels. Combining metformin with olaparib further weakens the energy metabolism of drug-resistant BRCA1-mutant triple-negative breast cancer cells, leading to decreased adaptability to PARP inhibitors, thereby further enhancing the inhibitory, apoptosis-promoting, and anti-migration effects on drug-resistant BRCA1-mutant triple-negative breast cancer cells.

[0010] Preferably, the PARP inhibitor includes a drug nanocarrier and metformin and olaparib encapsulated in the drug nanocarrier; the drug nanocarrier is a composite system formed by ultrasonic dispersion of magnetite magnetic nanoparticles, modified acylhydrazine-polyethylene glycol and polyvinyl alcohol solution in dimethyl sulfoxide; the modified acylhydrazine-polyethylene glycol is an isotretinoin-modified acylhydrazine-polyethylene glycol derivative.

[0011] More preferably, the method for preparing modified acylhydrazine-polyethylene glycol includes reacting acylhydrazine-polyethylene glycol-hydroxyl and isotretinoin under the action of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to obtain modified acylhydrazine-polyethylene glycol.

[0012] More preferably, the mass ratio of hydrazide-polyethylene glycol-hydroxyl to isotretinoin is 1:0.2-1.

[0013] More preferably, the mass ratio of hydrazide-polyethylene glycol-hydroxyl and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:0.5-1.

[0014] More preferably, the mass ratio of hydrazide-polyethylene glycol-hydroxyl and N-hydroxysuccinimide is 1:0.1-0.5.

[0015] More preferably, the mass ratio of the magnetite nanoparticles to the modified hydrazide-polyethylene glycol is 1:1-3.

[0016] More preferably, the mass concentration of the polyvinyl alcohol solution is 1-5%.

[0017] More preferably, the preparation method of PARP inhibitor includes adding metformin and olaparib to a drug nanocarrier, sonicating for 20-60 seconds, adding ultrapure water and stirring for 8-24 hours, purifying by magnetic bead separation, and freeze-drying to obtain PARP inhibitor.

[0018] More preferably, the modified acylhydrazine-polyethylene glycol is an acylhydrazine-polyethylene glycol derivative modified with at least one of isotretinoin, isotretinoin derivatives and isopropyl glycidyl ether.

[0019] More preferably, the modified acylhydrazide-polyethylene glycol is an isotretinoin-modified acylhydrazide-polyethylene glycol derivative. This invention uses isotretinoin to modify the hydroxyl group of acylhydrazide-polyethylene glycol, which may enhance the interaction between the drug nanocarrier and drug molecules. This not only enables efficient drug encapsulation and significantly improves the drug loading rate, but also effectively delays drug diffusion and release efficiency, reduces initial burst release and cumulative release, thereby effectively regulating the drug release process while improving drug encapsulation capability.

[0020] More preferably, the modified hydrazide-polyethylene glycol is an hydrazide-polyethylene glycol derivative modified with an isotretinoin derivative, and the isotretinoin derivative is an isotretinoin derivative modified with methyl 3-aminocrotonic acid. This invention uses isotretinoin modified with methyl 3-aminocrotonic acid, which may significantly enhance the intermolecular stability between the drug and drug nanocarrier molecules through hydrophobic interactions, allowing the drug to be more effectively encapsulated within the drug nanocarrier, while effectively delaying the rapid release process of the drug, reducing the cumulative release amount of the drug, and prolonging the drug's effect.

[0021] More preferably, the modified acylhydrazide-polyethylene glycol is an acylhydrazide-polyethylene glycol derivative modified with isotretinoin and isopropyl glycidyl ether, and the isotretinoin derivative is an isotretinoin derivative modified with methyl 3-aminocrotonate. This invention further uses isopropyl glycidyl ether to modify the hydroxyl group of the acylhydrazide-polyethylene glycol, which helps to further enhance the structural stability of the prepared modified acylhydrazide-polyethylene glycol, thereby significantly improving its drug encapsulation ability, while reducing the initial burst release and cumulative release of the drug during delivery, achieving sustained drug release.

[0022] More preferably, the preparation method of the isotretinoin derivative is as follows:

[0023] Isoretinoic acid and methyl 3-aminocrotonate were dissolved in dichloromethane, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine were added and mixed thoroughly. The mixture was reacted under nitrogen protection for 8-24 h. After the reaction, the mixture was washed 1-3 times with dilute hydrochloric acid and then 1-3 times with saturated sodium bicarbonate solution. The organic phase was retained, filtered, concentrated by rotary evaporation, purified by column chromatography, and then tetrahydrofuran was added and stirred thoroughly. Sodium hydroxide solution was added and the mixture was reacted for 8-24 h. After the reaction, the pH was adjusted to 2-3, the organic phase was extracted with ethyl acetate and collected, concentrated by rotary evaporation, and dried to obtain the isotretinoin derivative. The ratio of isotretinoin to dichloromethane is 1 g: 20-100 mL; the ratio of isotretinoin to methyl 3-aminocrotonate is 1:0.2-1; the mass ratio of isotretinoin to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1:0.5-1; the mass ratio of isotretinoin to 4-dimethylaminopyridine is 1:0.02-0.1; the concentration of dilute hydrochloric acid is 0.1-1 mol / L; the volume ratio of dichloromethane to tetrahydrofuran is 1:0.5-2; the concentration of sodium hydroxide solution is 0.1-1 mol / L, and the volume ratio of sodium hydroxide solution to tetrahydrofuran is 1:0.5-2.

[0024] More preferably, the preparation method of modified hydrazide-polyethylene glycol is as follows:

[0025] The modified acylhydrazide-polyethylene glycol-hydroxy group was dissolved in acetic acid solution and treated with nitrogen gas for 20-40 min. Isoretinoic acid solution or isotretinoin derivative solution was added at 0-10℃. The pH was then adjusted to 4-6, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added and reacted for 20-40 min. The reaction was continued at room temperature for 8-24 h. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain the modified acylhydrazide-polyethylene glycol.

[0026] More preferably, the mass concentration of the acetic acid solution is 1-2%.

[0027] More preferably, the ratio of hydrazide-polyethylene glycol-hydroxyl and acetic acid solution is 1g:50-200mL.

[0028] More preferably, the isotretinoin solution is prepared by ultrasonically stirring isotretinoin and anhydrous ethanol at 30-45℃ to obtain the isotretinoin solution, wherein the ratio of isotretinoin to anhydrous ethanol is 1g:30-90mL.

[0029] More preferably, the isotretinoin derivative solution is prepared by ultrasonically stirring isotretinoin and anhydrous ethanol at 30-45℃ to obtain the isotretinoin derivative solution, wherein the ratio of isotretinoin derivative to anhydrous ethanol is 1g:30-90mL.

[0030] More preferably, the mass ratio of hydrazide-polyethylene glycol-hydroxyl to isotretinoin is 1:0.2-1.

[0031] More preferably, the mass ratio of hydrazide-polyethylene glycol-hydroxyl to isotretinoin derivative is 1:0.2-1.

[0032] More preferably, the mass ratio of hydrazide-polyethylene glycol-hydroxyl and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:0.5-1.

[0033] More preferably, the mass ratio of hydrazide-polyethylene glycol-hydroxyl and N-hydroxysuccinimide is 1:0.1-0.5.

[0034] More preferably, the preparation method of modified hydrazide-polyethylene glycol is as follows:

[0035] The modified acylhydrazide-polyethylene glycol-hydroxy group was dissolved in acetic acid solution and treated with nitrogen for 20-40 min. An isotretinoin derivative solution was added at 0-10℃, and the pH was adjusted to 4-6. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added and reacted for 20-40 min. The reaction was continued at room temperature for 8-24 h. After the reaction, the mixture was dialyzed and freeze-dried. Dimethyl sulfoxide was added at 50-70℃ and stirred for 0.5-2 h. Triethylamine and isopropyl glycidyl ether were added, and the mixture was stirred at 70-90℃ for 8-24 h. After the reaction, the mixture was cooled to room temperature, filtered, washed 1-5 times with acetone, and then dialyzed and freeze-dried to obtain the modified acylhydrazide-polyethylene glycol.

[0036] More preferably, the mass concentration of the acetic acid solution is 1-2%.

[0037] More preferably, the ratio of hydrazide-polyethylene glycol-hydroxyl and acetic acid solution is 1g:50-200mL.

[0038] More preferably, the isotretinoin derivative solution is prepared by ultrasonically stirring isotretinoin and anhydrous ethanol at 30-45℃ to obtain the isotretinoin derivative solution, wherein the ratio of isotretinoin derivative to anhydrous ethanol is 1g:30-90mL.

[0039] More preferably, the mass ratio of hydrazide-polyethylene glycol-hydroxyl to isotretinoin derivative is 1:0.2-1.

[0040] More preferably, the mass ratio of hydrazide-polyethylene glycol-hydroxyl and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:0.5-1.

[0041] More preferably, the mass ratio of hydrazide-polyethylene glycol-hydroxyl and N-hydroxysuccinimide is 1:0.1-0.5.

[0042] More preferably, the ratio of hydrazide-polyethylene glycol-hydroxyl to dimethyl sulfoxide is 1g:10-30mL.

[0043] More preferably, the mass ratio of hydrazide-polyethylene glycol-hydroxyl to triethylamine is 1:2-10.

[0044] More preferably, the mass ratio of hydrazide-polyethylene glycol-hydroxyl to isopropyl glycidyl ether is 1:1-5.

[0045] More preferably, the method for preparing drug nanocarriers includes,

[0046] Magnetic nanoparticles of iron oxide were ultrasonically dispersed in dimethyl sulfoxide, and a modified hydrazide-polyethylene glycol and polyvinyl alcohol solution was added and ultrasonically stirred for 0.5-2 hours to obtain a drug nanocarrier.

[0047] More preferably, the ratio of iron oxide magnetic nanoparticles to dimethyl sulfoxide is 1g:20-100mL.

[0048] More preferably, the mass ratio of the magnetite nanoparticles to the modified hydrazide-polyethylene glycol is 1:1-3.

[0049] More preferably, the mass concentration of the polyvinyl alcohol solution is 1-5%.

[0050] More preferably, the volume ratio of dimethyl sulfoxide to polyvinyl alcohol is 1:0.5-2.

[0051] More preferably, the preparation method of PARP inhibitors is as follows:

[0052] The preparation method of PARP inhibitors includes adding metformin and olaparib to a drug nanocarrier, sonicating for 20-60 seconds, adding ultrapure water and stirring for 8-24 hours, purifying by magnetic bead separation, and freeze-drying to obtain PARP inhibitors.

[0053] More preferably, the ratio of olaparib to ultrapure water is 1 μmol: 0.1-1 L.

[0054] More preferably, the molar ratio of olaparib to metformin is 1:200-600.

[0055] More preferably, the volume ratio of the drug nanocarrier to ultrapure water is 1:50-200.

[0056] This invention also discloses the application of the above-mentioned PARP inhibitor in the preparation of anti-breast cancer drugs.

[0057] This invention, by combining metformin and olaparib as PARP inhibitors, has the following beneficial effects: Compared to using metformin or olaparib alone, the combined use of metformin and olaparib as PARP inhibitors helps improve the energy metabolism status of BRCA1-mutant triple-negative breast cancer cells, reduces cellular oxygen consumption, ATP production rate, and maximum respiration capacity, and increases proton leakage level; at the same time, it significantly enhances the inhibitory, apoptosis-promoting, and anti-migration effects on drug-resistant BRCA1-mutant breast cancer cells, achieving a synergistic improvement in the efficacy of PARP inhibitors.

[0058] This invention utilizes a drug nanocarrier to load metformin and olaparib. The drug nanocarrier is a composite system formed by ultrasonic dispersion of magnetite nanoparticles, modified acylhydrazine-polyethylene glycol, and polyvinyl alcohol solution in dimethyl sulfoxide. The modified acylhydrazine-polyethylene glycol is an acylhydrazine-polyethylene glycol derivative modified with isopropyl glycidyl ether, and the isotretinoin derivative is an isotretinoin derivative modified with methyl 3-aminocrotonate. Therefore, it has the following beneficial effects: after being encapsulated by the drug nanocarrier, the loading rate of the obtained PARP inhibitor is significantly increased to 81.61-93.42%, and the cumulative release at 96 hours in vitro is significantly reduced to 26.18-39.88%, effectively prolonging the drug action time. Attached Figure Description

[0059] Figure 1 The overall effect of PARP inhibitors on the growth of BRCA1-mutated breast cancer cells with different drug resistance.

[0060] Figure 2 The effect of PARP inhibitors on the growth of drug-resistant HCC1937 cell lines.

[0061] Figure 3 The effect of PARP inhibitors on the growth of drug-resistant MDA-MB-436 cell lines.

[0062] Figure 4 The effect of PARP inhibitors on the growth of drug-resistant SUM1315MO2 cell lines.

[0063] Figure 5 The results show the apoptosis of the drug-resistant HCC1937 cell line.

[0064] Figure 6 The results show the apoptosis of the drug-resistant MDA-MB-436 cell line.

[0065] Figure 7 The results show the apoptosis of the drug-resistant SUM1315MO2 cell line.

[0066] Figure 8 Cell migration of the drug-resistant MDA-MB-436 cell line.

[0067] Figure 9 Cell migration of the drug-resistant SUM1315MO2 cell line.

[0068] Figure 10 Cell migration of drug-resistant HCC1937 cell line.

[0069] Figure 11 The overall result shows the relative scratch area of ​​BRCA1-mutated breast cancer cells with different drug resistance.

[0070] Figure 12 The relative scratch area is for the drug-resistant MDA-MB-436 cell line.

[0071] Figure 13 The relative scratch area is for the drug-resistant SUM1315MO2 cell line.

[0072] Figure 14 The relative scratch area is for the drug-resistant HCC1937 cell line.

[0073] Figure 15 Oxygen consumption rate of the drug-resistant HCC1937 cell line.

[0074] Figure 16 Oxygen consumption rate of the drug-resistant SUM1315MO2 cell line.

[0075] Figure 17 Oxygen consumption rate of the drug-resistant MDA-MB-436 cell line.

[0076] Figure 18 This is the basic respiration of BRCA1-mutated breast cancer cells with different drug resistance.

[0077] Figure 19 The maximum respiration of BRCA1-mutated breast cancer cells with different drug resistance.

[0078] Figure 20 This is a proton leak in BRCA1-mutated breast cancer cells with different drug resistance.

[0079] Figure 21 ATP production in BRCA1 mutant breast cancer cells with different drug resistance.

[0080] Figure 22 The ATP production rate of the drug-resistant HCC1937 cell line.

[0081] Figure 23 The ATP production rate of the drug-resistant SUM1315MO2 cell line.

[0082] Figure 24 The ATP production rate of the drug-resistant MDA-MB-436 cell line.

[0083] Figure 25 This represents the cumulative release of olaparib.

[0084] Figure 26 The image shows the infrared spectrum of the modified hydrazide-polyethylene glycol. Detailed Implementation

[0085] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0086] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0087] Example 1:

[0088] PARP inhibitors, including metformin.

[0089] Example 2:

[0090] PARP inhibitors, including olaparib.

[0091] Example 3:

[0092] PARP inhibitors include metformin and olaparib. The molar ratio of olaparib to metformin is 1:400.

[0093] Example 4:

[0094] PARP inhibitors include drug nanocarriers and metformin and olaparib encapsulated within the drug nanocarriers. The molar ratio of olaparib to metformin is 1:400. The drug nanocarrier is a magnetically modified acylhydrazine-polyethylene glycol-polyvinyl alcohol composite nanocarrier, where the modified acylhydrazine-polyethylene glycol is an isotretinoin-modified acylhydrazine-polyethylene glycol derivative.

[0095] The preparation method of modified acylhydrazide-polyethylene glycol includes,

[0096] The modified acylhydrazide-polyethylene glycol-hydroxy group was dissolved in acetic acid solution and treated with nitrogen gas for 30 min. Isoretinoic acid solution was added at 4 °C, and then the pH was adjusted to 5. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added and reacted for 30 min. The reaction was continued at room temperature for 12 h. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain the modified acylhydrazide-polyethylene glycol. The acetic acid solution has a mass concentration of 1%, and the ratio of acylhydrazine-polyethylene glycol-hydroxy to acetic acid solution is 1 g: 100 mL. The isotretinoin solution is prepared by ultrasonically stirring isotretinoin and anhydrous ethanol at 40 °C to obtain the isotretinoin solution, and the ratio of isotretinoin to anhydrous ethanol is 1 g: 60 mL. The mass ratio of acylhydrazine-polyethylene glycol-hydroxy to isotretinoin is 1:0.5. The mass ratio of acylhydrazine-polyethylene glycol-hydroxy to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:0.6. The mass ratio of acylhydrazine-polyethylene glycol-hydroxy to N-hydroxysuccinimide is 1:0.3.

[0097] Methods for preparing drug nanocarriers include,

[0098] Magnetic nanoparticles of iron oxide (Fe3O4) were ultrasonically dispersed in dimethyl sulfoxide (DMSO), and then a modified hydrazide-polyethylene glycol (PEG) and polyvinyl alcohol (PVA) solution was added and ultrasonically stirred for 1 h to obtain a drug nanocarrier. The molar ratio of Fe3O4 magnetic nanoparticles to DMSO was 1 g:50 mL; the mass ratio of Fe3O4 magnetic nanoparticles to modified hydrazide-PEG was 1:1.5; the mass concentration of the PVA solution was 2%; and the volume ratio of DMSO to PVA was 1:1.

[0099] Methods for preparing PARP inhibitors include,

[0100] Metformin and olaparib were added to a drug nanocarrier, sonicated for 30 seconds, and then stirred with ultrapure water for 12 hours. After purification by magnetic bead separation and freeze-drying, a PARP inhibitor was obtained. The volume ratio of olaparib to ultrapure water was 1 μmol: 0.2 L; the molar ratio of olaparib to metformin was 1:400; and the volume ratio of drug nanocarrier to ultrapure water was 1:100.

[0101] Example 5:

[0102] PARP inhibitors include drug nanocarriers and metformin and olaparib encapsulated within the drug nanocarriers. The molar ratio of olaparib to metformin is 1:400. The drug nanocarrier is a magnetically modified acylhydrazine-polyethylene glycol-polyvinyl alcohol composite nanocarrier, where the modified acylhydrazine-polyethylene glycol is an isotretinoin derivative-modified acylhydrazine-polyethylene glycol derivative.

[0103] Methods for preparing isotretinoin derivatives include,

[0104] Isoretinoic acid and methyl 3-aminocrotonate were dissolved in dichloromethane, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine were added and mixed thoroughly. The mixture was reacted under nitrogen protection for 12 h. After the reaction, the mixture was washed twice with dilute hydrochloric acid and then twice with saturated sodium bicarbonate solution. The organic phase was retained, filtered, concentrated by rotary evaporation, purified by column chromatography, and then tetrahydrofuran was added and stirred thoroughly. Sodium hydroxide solution was added and the mixture was reacted for 12 h. After the reaction, the pH was adjusted to 2, the organic phase was extracted with ethyl acetate and collected, concentrated by rotary evaporation, and dried to obtain the isotretinoin derivative. The ratio of isotretinoin to dichloromethane is 1 g: 50 mL; the ratio of isotretinoin to methyl 3-aminocrotonate is 1:0.5; the mass ratio of isotretinoin to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1:0.9; the mass ratio of isotretinoin to 4-dimethylaminopyridine is 1:0.05; the concentration of dilute hydrochloric acid is 1 mol / L; the volume ratio of dichloromethane to tetrahydrofuran is 1:1; the concentration of sodium hydroxide solution is 1 mol / L, and the volume ratio of sodium hydroxide solution to tetrahydrofuran is 1:1.

[0105] The preparation method of modified hydrazide-polyethylene glycol is the same as that of Example 4, except that isotretinoin is replaced with the isotretinoin derivative prepared in this example, and all other conditions are the same as those of Example 4.

[0106] The preparation method of the drug nanocarrier is the same as that in Example 4, except that the modified hydrazide-polyethylene glycol is replaced with the modified hydrazide-polyethylene glycol prepared in this example.

[0107] The preparation method of PARP inhibitor is the same as that in Example 4, except that the drug nanocarrier is replaced with the drug nanocarrier prepared in this example, and all other conditions are the same as in Example 4.

[0108] Example 6:

[0109] PARP inhibitors include drug nanocarriers and metformin and olaparib encapsulated within these nanocarriers. The molar ratio of olaparib to metformin is 1:400. The drug nanocarrier is a magnetically modified acylhydrazine-polyethylene glycol-polyvinyl alcohol composite nanocarrier, wherein the modified acylhydrazine-polyethylene glycol is an isotretinoin derivative and an isopropyl glycidyl ether-modified acylhydrazine-polyethylene glycol derivative.

[0110] The preparation method of isotretinoin derivatives is the same as in Example 5.

[0111] The preparation method of modified acylhydrazide-polyethylene glycol includes,

[0112] The modified hydrazide-polyethylene glycol-hydroxy group was dissolved in acetic acid solution and treated with nitrogen gas for 30 min. Isoretinoic acid solution was added at 4 °C, and the pH was adjusted to 5. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added and reacted for 30 min. The reaction was continued at room temperature for 12 h. After the reaction was completed, the mixture was dialyzed and freeze-dried. After the treatment, dimethyl sulfoxide was added at 60 °C and stirred for 1 h. Triethylamine and isopropyl glycidyl ether were added and stirred at 80 °C for 12 h. After the reaction was completed, the mixture was cooled to room temperature, filtered, washed three times with acetone, dialyzed and freeze-dried to obtain the modified hydrazide-polyethylene glycol. The acetic acid solution had a mass concentration of 1%, and the ratio of acylhydrazine-polyethylene glycol-hydroxyl to acetic acid solution was 1 g:100 mL. The isotretinoin solution was prepared by ultrasonically stirring isotretinoin and anhydrous ethanol at 40°C, with an isotretinoin to anhydrous ethanol ratio of 1 g:60 mL. The mass ratio of acylhydrazine-polyethylene glycol-hydroxyl to isotretinoin was 1:0.5. The acylhydrazine-polyethylene glycol-hydroxyl and 1 g acetic acid solution... The mass ratio of ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:0.6; the mass ratio of acylhydrazine-polyethylene glycol-hydroxyl to N-hydroxysuccinimide is 1:0.3; the mass ratio of acylhydrazine-polyethylene glycol-hydroxyl to dimethyl sulfoxide is 1g:20mL; the mass ratio of acylhydrazine-polyethylene glycol-hydroxyl to triethylamine is 1:5; and the mass ratio of acylhydrazine-polyethylene glycol-hydroxyl to isopropyl glycidyl ether is 1:3.

[0113] The preparation method of the drug nanocarrier is the same as that in Example 4, except that the modified hydrazide-polyethylene glycol is replaced with the modified hydrazide-polyethylene glycol prepared in this example.

[0114] The preparation method of PARP inhibitor is the same as that in Example 4, except that the drug nanocarrier is replaced with the drug nanocarrier prepared in this example, and all other conditions are the same as in Example 4.

[0115] Comparative Example 1:

[0116] PARP inhibitors include drug nanocarriers and metformin and olaparib encapsulated within these nanocarriers. The molar ratio of olaparib to metformin is 1:400. The drug nanocarriers are magnetic polylactic acid-glycolic acid-polyethyleneimine-polyvinyl alcohol composite nanocarriers.

[0117] The preparation method of the drug nanocarrier is the same as that in Example 4, except that the modified hydrazide-polyethylene glycol is replaced with polylactic acid-glycolic acid-polyethyleneimine.

[0118] The preparation method of PARP inhibitor is the same as that in Example 4, except that the drug nanocarrier is replaced with the drug nanocarrier prepared in this example, and all other conditions are the same as in Example 4.

[0119] Comparative Example 2:

[0120] PARP inhibitors include drug nanocarriers and metformin and olaparib encapsulated within the drug nanocarriers. The molar ratio of olaparib to metformin is 1:400. The drug nanocarriers are magnetic polyethylene glycol / polyvinyl alcohol composite nanocarriers.

[0121] The preparation method of the drug nanocarrier is the same as that in Example 4, except that the modified hydrazide-polyethylene glycol is replaced with polyethylene glycol.

[0122] The preparation method of PARP inhibitor is the same as that in Example 4, except that the drug nanocarrier is replaced with the drug nanocarrier prepared in this example, and all other conditions are the same as in Example 4.

[0123] Comparative Example 3:

[0124] PARP inhibitors include drug nanocarriers and metformin and olaparib encapsulated within these nanocarriers. The molar ratio of olaparib to metformin is 1:400. The drug nanocarrier is a magnetic hydrazide-polyethylene glycol-polyvinyl alcohol composite nanocarrier.

[0125] The preparation method of the drug nanocarrier is the same as that in Example 4, except that the modified hydrazide-polyethylene glycol is replaced with hydrazide-polyethylene glycol-hydroxyl.

[0126] The preparation method of PARP inhibitor is the same as that in Example 4, except that the drug nanocarrier is replaced with the drug nanocarrier prepared in this example, and all other conditions are the same as in Example 4.

[0127] Comparative Example 4:

[0128] PARP inhibitors include drug nanocarriers and metformin and olaparib encapsulated within the drug nanocarriers. The molar ratio of olaparib to metformin is 1:400. The drug nanocarrier is a magnetic hydrazide-polyethylene glycol-hydroxy mixture-polyvinyl alcohol composite nanocarrier. The hydrazide-polyethylene glycol-hydroxy mixture is a mixture of hydrazide-polyethylene glycol-hydroxy, isotretinoin, and methyl 3-aminocrotonate, wherein the mass ratio of hydrazide-polyethylene glycol-hydroxy and isotretinoin is 1:1, and the mass ratio of hydrazide-polyethylene glycol-hydroxy and methyl 3-aminocrotonate is 1:1.

[0129] The preparation method of the drug nanocarrier is the same as that in Example 4, except that the modified hydrazide-polyethylene glycol is replaced with a mixture of hydrazide-polyethylene glycol-hydroxy, isotretinoin and methyl 3-aminocrotonate, wherein the mass ratio of hydrazide-polyethylene glycol-hydroxy and isotretinoin is 1:1 and the mass ratio of hydrazide-polyethylene glycol-hydroxy and methyl 3-aminocrotonate is 1:1, and the other conditions are the same as in Example 4.

[0130] The preparation method of PARP inhibitor is the same as that in Example 4, except that the drug nanocarrier is replaced with the drug nanocarrier prepared in this example, and all other conditions are the same as in Example 4.

[0131] Comparative Example 5:

[0132] PARP inhibitors include drug nanocarriers and metformin and olaparib encapsulated within the drug nanocarriers. The molar ratio of olaparib to metformin is 1:400. The drug nanocarrier is a magnetically modified acylhydrazine-polyethylene glycol-polyvinyl alcohol composite nanocarrier, where the modified acylhydrazine-polyethylene glycol is an isopropyl glycidyl ether-modified acylhydrazine-polyethylene glycol derivative.

[0133] The preparation method of modified acylhydrazide-polyethylene glycol includes,

[0134] The modified acylhydrazine-polyethylene glycol-hydroxy group was dissolved in acetic acid solution, treated with nitrogen gas for 30 min, and then stirred with dimethyl sulfoxide at 60 °C for 1 h. Triethylamine and isopropyl glycidyl ether were then added, and the reaction was stirred at 80 °C for 12 h. After the reaction was completed, the mixture was cooled to room temperature, filtered, washed three times with acetone, dialyzed, and freeze-dried to obtain the modified acylhydrazine-polyethylene glycol. The mass ratio of acylhydrazine-polyethylene glycol-hydroxy group to dimethyl sulfoxide was 1 g:20 mL; the mass ratio of acylhydrazine-polyethylene glycol-hydroxy group to triethylamine was 1:5; and the mass ratio of acylhydrazine-polyethylene glycol-hydroxy group to isopropyl glycidyl ether was 1:3.

[0135] The preparation method of the drug nanocarrier is the same as that in Example 4, except that the modified hydrazide-polyethylene glycol is replaced with the modified hydrazide-polyethylene glycol prepared in this example.

[0136] The preparation method of PARP inhibitor is the same as that in Example 4, except that the drug nanocarrier is replaced with the drug nanocarrier prepared in this example, and all other conditions are the same as in Example 4.

[0137] Experimental example:

[0138] 1. Constructing three subtypes of drug-resistant BRCA1-mutated breast cancer cells

[0139] Drug-resistant BRCA1-mutated breast cancer cells were constructed for three types of BRCA1-mutated breast cancer cells: HCC1937, MDA-MB-436, and SUM1315MO2. These BRCA1-mutated cells included drug-resistant HCC1937, MDA-MB-436, and SUM1315MO2 cell lines. The construction methods are as follows:

[0140] S1. BRCA1 mutant breast cancer cells were cultured in complete medium until the cells adhered. The complete medium was then replaced with complete medium containing 0.5 μmol / L olaparib. The complete medium containing 0.5 μmol / L olaparib was replaced every 2 days. When the surviving cell colonies grew to cover about 60-70% of the bottom of the culture dish, they were digested with trypsin and transferred to complete medium without the drug to allow them to recover and expand, thus obtaining BRCA1 mutant breast cancer cells adapted to the 0.5 μmol / L olaparib drug concentration.

[0141] S2. BRCA1 mutant breast cancer cells adapted to a 0.5 μmol / L olaparib concentration were cultured in complete medium until the cells adhered. The complete medium was then replaced with complete medium containing 1 μmol / L olaparib. When the surviving cell colonies grew to cover about 60-70% of the bottom of the culture dish, they were digested with trypsin and transferred to complete medium without the drug to allow them to recover and expand, thus obtaining BRCA1 mutant breast cancer cells adapted to a 1 μmol / L olaparib concentration.

[0142] S3. BRCA1 mutant breast cancer cells adapted to a 1 μmol / L olaparib drug concentration were cultured in complete medium until the cells adhered. The complete medium was then replaced with complete medium containing 2 μmol / L olaparib. When the surviving cell colonies grew to cover about 60-70% of the bottom of the culture dish, they were digested with trypsin and transferred to complete medium without the drug to allow them to recover and expand, thus obtaining BRCA1 mutant breast cancer cells adapted to a 2 μmol / L olaparib drug concentration.

[0143] Cell death may occur in complete culture media containing 1 μmol / L olaparib and 2 μmol / L olaparib. To obtain resistant cell lines, it is necessary to reculture and wait for new resistant cell colonies to appear, eventually obtaining drug-resistant BRCA1 mutant breast cancer cells that grow stably at a concentration of 2 μmol / L olaparib.

[0144] 2. Preparation of PARP inhibitor working solution

[0145] Preparation of the PARP inhibitor working solution in Example 1: The PARP inhibitor of Example 1 was dissolved in sterile phosphate buffer to obtain a stock solution with a final concentration of 200 mmol / L. The solution was filtered through a 0.22 μm filter membrane. Complete culture medium was added to the stock solution to further dilute the PARP inhibitor of Example 1 to a concentration of 2 mmol / L, thus obtaining the PARP inhibitor working solution of Example 1.

[0146] Preparation of PARP inhibitor working solution in Example 2: Dissolve the PARP inhibitor of Example 2 in dimethyl sulfoxide to a final concentration of 10 mmol / L. Then add complete culture medium to the mother liquor to further dilute the PARP inhibitor of Example 2 to a concentration of 5 μmol / L, thus obtaining the PARP inhibitor working solution of Example 2.

[0147] Preparation of the PARP inhibitor working solution in Example 3: Olaparib was dissolved in dimethyl sulfoxide to obtain an olaparib stock solution with a final concentration of 10 mmol / L; metformin was dissolved in sterile phosphate buffer to obtain a final concentration of 200 mmol / L, and filtered through a 0.22 μm filter membrane to obtain a metformin stock solution. 0.5 mL of olaparib stock solution and 10 mL of metformin stock solution were mixed and added to 989.5 mL of complete culture medium to obtain the PARP inhibitor working solution of Example 3. The PARP inhibitor solution of Example 3 was a complete culture medium containing 2 mmol / L metformin and 5 μmol / L olaparib.

[0148] Preparation of control solvent: Dimethyl sulfoxide was diluted with complete culture medium to a mass concentration of 0.05% to obtain the control solvent.

[0149] 3. Effects of PARP inhibitors on the proliferation and apoptosis of drug-resistant BRCA1-mutated breast cancer cells

[0150] Using drug-resistant HCC1937, MDA-MB-436, and SUM1315MO2 cell lines, a 2×2 factorial design was employed to evaluate the effects of the PARP inhibitors described in Examples 1-3 on the proliferation and apoptosis-inducing effects on drug-resistant BRCA1-mutant breast cancer cells. Quantitative analysis was performed by flow cytometry. The specific steps are as follows: After digestion, drug-resistant BRCA1-mutant breast cancer cells were cultured at 5×10⁻⁶ cells / cells. 4Cells were seeded at cell / well density and cultured. After plating, the cells were cultured at 37°C and 5% CO2 for 24 hours to synchronize the cell cycle and achieve complete cell adhesion. A control group and three experimental groups were set up. The control group received the control solvent, experimental group 1 received the PARP inhibitor working solution from Example 1, experimental group 2 received the PARP inhibitor working solution from Example 2, and experimental group 3 received the PARP inhibitor working solution from Example 3. Cells in each group were cultured at 37°C and 5% CO2 for 48 hours. After culture, cell proliferation was measured using an MTT assay kit, and apoptosis was measured using flow cytometry.

[0151] The results are as follows Figures 1 to 4 As can be seen, compared with experimental groups 1 and 2, experimental group 3 showed the lowest OD values ​​in all three types of drug-resistant BRCA1-mutant breast cancer cells, indicating that the PARP inhibitor in Example 3 of this invention has the most significant inhibitory effect on drug-resistant BRCA1-mutant breast cancer cells. Compared with the use of metformin or olaparib alone, the combination of the two drugs has the best inhibitory effect on drug-resistant BRCA1-mutant breast cancer cells.

[0152] The results are as follows Figures 5 to 7 It can be seen that, compared with experimental groups 1 and 2, experimental group 3 showed the best effect in promoting apoptosis in all three types of drug-resistant BRCA1 mutant breast cancer cells. In experimental groups 1-3, metformin all showed an effect in promoting apoptosis, but the effect on different drug-resistant BRCA1 mutant breast cancer cell subtypes was inconsistent. This indicates that the PARP inhibitor in Example 3 of this invention has the most significant effect in promoting apoptosis in drug-resistant BRCA1 mutant breast cancer cells.

[0153] 4. Effects of PARP inhibitors on the migration of drug-resistant BRCA1-mutated breast cancer cells

[0154] The core application of the cell scratch assay is to evaluate the inhibitory or promoting effect of drugs on cell migration, thereby indirectly reflecting their potential anti-tumor metastasis or healing-promoting efficacy. By evaluating and comparing the migration ability of three drug-resistant BRCA1-mutant breast cancer cell subtypes to different drug treatments at 0 and 24 hours after drug administration, this study compared the effects of PARP inhibitors on the migration of drug-resistant BRCA1-mutant breast cancer cells in Examples 1-3. The specific steps are as follows: Drug-resistant BRCA1-mutant breast cancer cells were seeded in 24-well plates, and 500 μL of complete culture medium containing 10% fetal bovine serum was added. The cells were gently shaken and cultured at 37°C and 5% CO2 for 24 hours. A control group and three experimental groups were set up. Each group was scratched using a scratcher, with the scratcher perpendicular to the bottom of the well and following the guide of a ruler, making a straight cut across the cell monolayer in one stroke. After scratching, the cells were gently washed twice with sterile phosphate buffer to completely remove the scratched cell debris. After aspirating the sterile phosphate buffer, each group was immediately treated with the drug. The control group received 500 μL of the control solvent, experimental group 1 received 500 μL of the PARP inhibitor working solution from Example 1, experimental group 2 received 500 μL of the PARP inhibitor working solution from Example 2, and experimental group 3 received 500 μL of the PARP inhibitor working solution from Example 3. After treatment, images were taken at 0 hours using a live-cell imaging microscope, denoted as T0. Cells in each group were cultured at 37°C and 5% CO2 for 24 hours. The exact same location marked at T0 was quickly located and photographed, denoted as T24h. The scratch healing area, scratch healing rate, and relative migration inhibition rate were calculated using the following formulas: Scratch healing area = T0 area - T24h area; Scratch healing rate (%) = (T0 area - T24h area) / T0 area × 100%; Relative scratch area (%) = (1 - (experimental group healing area / control group healing area)) × 100%.

[0155] The results are as follows Figures 8 to 14 It can be seen that, compared with experimental groups 1 and 2, experimental group 3 showed the best inhibitory effect on cell migration in all three types of drug-resistant BRCA1 mutant breast cancer cells. In experimental groups 1-3, metformin showed an inhibitory effect on cell migration, but the effect on different drug-resistant BRCA1 mutant breast cancer cell subtypes was inconsistent. This indicates that the PARP inhibitor in Example 3 of this invention has the most significant inhibitory effect on cell migration of drug-resistant BRCA1 mutant breast cancer cells.

[0156] 5. Effects of PARP inhibitors on mitochondrial metabolic regulation in drug-resistant BRCA1-mutated breast cancer cells

[0157] Mitochondrial stress testing is the "gold standard" technique for assessing the effects of drugs on cellular energy metabolism, and it analyzes mitochondrial function by real-time monitoring of cellular oxygen consumption. Using a cell metabolism analyzer, real-time monitoring of oxygen consumption (OCR) was performed on three subtypes of drug-resistant BRCA1-mutant breast cancer cells. The specific steps are as follows: Drug-resistant BRCA1-mutant breast cancer cells were seeded in 96-well cell culture microplates, gently shaken, and cultured at 37°C and 5% CO2 for 24 hours. A control group and three experimental groups were set up. After confirming good cell adhesion in each group, drug treatment was immediately administered. The control group received 100 μL of control solvent preheated to 37°C; experimental group 1 received 100 μL of PARP inhibitor working solution from Example 1 preheated to 37°C; experimental group 2 received 100 μL of PARP inhibitor working solution from Example 2 preheated to 37°C; and experimental group 3 received 100 μL of PARP inhibitor working solution from Example 3 preheated to 37°C. After drug treatment, the cells were cultured for another 24 hours. Subsequently, the culture medium of each group was aspirated and washed twice with test medium preheated to 37°C to completely remove the drug. Finally, 175 μL of test medium preheated to 37°C was added, and the mixture was equilibrated at 37°C for 60 min under CO2-free conditions. The test medium was Seahorse XF DMEM basal medium containing 1 mol / L glucose, 2 mmol / L glutamine, and 100 mmol / L, with a pH of 7.4. After treatment, oxygen consumption was measured using a cell metabolism analyzer. Basal respiration, ATP production, proton leakage, and maximum respiration indices were calculated using the following formulas: Basal respiration = (last baseline measurement OCR) = (average OCR after Rot / AntiA); ATP production = (last baseline measurement OCR) = (lowest OCR after Oligomycin); Proton leakage = (lowest OCR after Oligomycin) = (average OCR after Rot / AntiA); Maximum respiration = (highest OCR after FCCP) = (average OCR after Rot / AntiA).

[0158] The results are as follows Figures 15 to 21 As shown, mitochondrial stress experiments revealed that metformin or olaparib alone could reduce the OCR value related to mitochondrial energy metabolism, increase proton leakage, reduce ATP production, and decrease maximum respiratory capacity in three subtypes of drug-resistant BRCA1-mutant breast cancer cells. Compared with experimental groups 1 and 2, experimental group 3 showed the most significant effect in all three subtypes of drug-resistant BRCA1-mutant breast cancer cells.

[0159] 6. Effects of PARP inhibitors on ATP production rate in drug-resistant BRCA1-mutated breast cancer cells

[0160] The effects of the PARP inhibitors described in Examples 1-3 on the ATP production rate of drug-resistant BRCA1-mutant breast cancer were evaluated using drug-resistant HCC1937, drug-resistant MDA-MB-436, and drug-resistant SUM1315MO2 cell lines, respectively. The specific steps are as follows: After digesting the drug-resistant BRCA1-mutant breast cancer cells, they were subjected to a 5×10⁻⁶... 4 Cells were seeded at cell / well density and cultured. After plating, the cells were cultured at 37°C and 5% CO2 for 24 hours to synchronize the cell cycle and achieve complete cell adhesion. A control group and three experimental groups were set up. The control group was supplemented with the control solvent. Experimental group 1 was supplemented with the PARP inhibitor working solution from Example 1; experimental group 2 was supplemented with the PARP inhibitor working solution from Example 2; and experimental group 3 was supplemented with the PARP inhibitor working solution from Example 3. Cells in each group were cultured at 37°C and 5% CO2 for 48 hours. After the culture period, the ATP production rate was measured using an ATP rate assay kit.

[0161] The results are as follows Figures 22 to 24 As shown, metformin affected the ATP production rate of three types of BRCA1-mutated breast cancer cells. This indicates that metformin can act on cellular glycolytic metabolism, reducing the ability of breast cancer cells to re-edit energy metabolism; at the same time, it inhibits the ability of breast cancer cells to adjust energy metabolism, thus weakening the ability of breast cancer cells to respond to olaparib, thereby improving the efficacy of olaparib.

[0162] 7. Drug loading rate

[0163] The content of olaparib in the PARP inhibitors prepared in Examples 4-6 and Comparative Examples 1-5 was determined at 254 nm using ultraviolet spectrophotometry, and the encapsulation rate was calculated according to the following formula: Encapsulation rate (%) = mass of olaparib in PARP inhibitor / total mass of PARP inhibitor × 100%.

[0164] Table 1 Packet Load Rate

[0165]

[0166] The results are shown in Table 1. Compared with Comparative Examples 1-2, Examples 4-6 and Comparative Examples 3-5 all showed significantly improved encapsulation rates. This is because, in the preparation of the drug nanocarrier, Comparative Example 1 used polylactic acid-glycolic acid-polyethyleneimine, Comparative Example 2 used polyethylene glycol, while Examples 4-6 and Comparative Examples 3-5 used acylhydrazide-polyethylene glycol-hydroxy or the modified acylhydrazide-polyethylene glycol prepared in this invention. This result indicates that, compared with the commonly used drug carriers polylactic acid-glycolic acid-polyethyleneimine and polyethylene glycol, the use of acylhydrazide-polyethylene glycol-hydroxy or the modified acylhydrazide-polyethylene glycol prepared in this invention helps to improve the encapsulation rate of olaparib in the PARP inhibitor .

[0167] Compared to Comparative Example 3, the loading rates of Examples 4-6 were significantly improved. This is because Examples 4-6 used modified acylhydrazine-polyethylene glycol in the preparation of the drug nanocarriers, while Comparative Example 3 used acylhydrazine-polyethylene glycol-hydroxyl. This result indicates that the drug nanocarriers prepared using modified acylhydrazine-polyethylene glycol can effectively improve the drug loading rate compared to unmodified acylhydrazine-polyethylene glycol-hydroxyl.

[0168] Compared to Comparative Example 4, the loading rates of Examples 4-5 were significantly improved. This is because in the preparation process of the modified hydrazide-polyethylene glycol, Example 4 used isotretinoin to modify the hydroxyl group of the hydrazide-polyethylene glycol; Example 5 first reacted isotretinoin and methyl 3-aminocrotonate to obtain an isotretinoin derivative, and then used the isotretinoin derivative to modify the hydroxyl group of the hydrazide-polyethylene glycol; while Comparative Example 4 only used a mixture of hydrazide-polyethylene glycol-hydroxyl group, isotretinoin, and methyl 3-aminocrotonate, without modifying the hydroxyl group of the hydrazide-polyethylene glycol. This result indicates that the modified hydrazide-polyethylene glycol obtained by modification with isotretinoin or isotretinoin derivatives can effectively improve the drug loading rate when used to prepare drug nanocarriers. The loading rate of Example 5 was higher than that of Example 4, showing that the drug nanocarrier prepared by modifying the hydrazide-polyethylene glycol with isotretinoin derivatives is more effective in improving the loading rate.

[0169] Compared to Example 5 and Comparative Example 6, the loading capacity of Example 6 was further improved because, in the preparation of the modified hydrazide-polyethylene glycol, Example 6 further modified the hydrazide-polyethylene glycol-hydroxyl group with isopropyl glycidyl ether after modifying it with an isotretinoin derivative; Example 5 did not use isopropyl glycidyl ether modification, and Comparative Example 6 did not use isotretinoin derivative modification. This result indicates that, compared to modifying the hydrazide-polyethylene glycol-hydroxyl group with isotretinoin derivative or isopropyl glycidyl ether alone, the modified hydrazide-polyethylene glycol obtained by simultaneously using both isotretinoin derivative and isopropyl glycidyl ether can further improve the loading capacity of the prepared drug nanocarrier.

[0170] 8. In vitro release rate

[0171] The in vitro release rates of the PARP inhibitors in Examples 2-6 and Comparative Examples 1-5 were determined using the following steps: A mixture of anhydrous ethanol and phosphate buffer (1:4 volume ratio) was used as the release medium. 0.1 g of PARP inhibitor was added to 100 mL of the release medium and mixed. The entire liquid was then transferred to a dialysis bag, which was sealed and placed in a beaker containing 800 mL of the release medium. The mixture was incubated at 37°C in a shaker for 96 h. Samples of 0.5 mL were taken at 0, 0.5, 1, 2, 4, 6, 8, 12, 18, 24, 48, 72, and 96 h. The olaparib content was measured at 254 nm using a UV spectrophotometer. An equal volume of release medium was added immediately after each sampling. The cumulative release of olaparib was calculated.

[0172] The results are as follows Figure 25 As shown, the PARP inhibitor in Example 2 was free olaparib, which was rapidly released within the first 18 hours, with a cumulative release of 98.76%. Encapsulating olaparib in a drug nanocarrier significantly regulated its release behavior. Specifically, in Comparative Example 1, olaparib encapsulated in a magnetic polylactic acid-glycolic acid-polyethyleneimine-polyvinyl alcohol composite nanocarrier showed a cumulative release of 48.37% at 18 hours and 56.74% at 96 hours; in Comparative Example 3, olaparib encapsulated in a magnetic hydrazide-polyethylene glycol-hydroxy-polyvinyl alcohol composite nanocarrier showed a cumulative release of 37.68% at 18 hours and 45.31% at 96 hours. This result indicates that, compared to free olaparib, encapsulating olaparib in a drug nanocarrier can effectively delay its release, and that the magnetic hydrazide-polyethylene glycol-hydroxy-polyvinyl alcohol composite nanocarrier has better sustained-release performance than the magnetic polylactic acid-glycolic acid-polyethyleneimine-polyvinyl alcohol composite nanocarrier, which can effectively prolong the duration of drug action.

[0173] Table 2. Cumulative release (%) at 96 hours

[0174]

[0175] The results are shown in Table 1. Compared with Comparative Examples 1-2, Examples 4-6 and Comparative Examples 3-5 all showed significantly reduced cumulative release. This is because, in the preparation of the drug nanocarrier, Comparative Example 1 used polylactic acid-glycolic acid-polyethyleneimine, Comparative Example 2 used polyethylene glycol, while Examples 4-6 and Comparative Examples 3-5 used acylhydrazide-polyethylene glycol-hydroxy or the modified acylhydrazide-polyethylene glycol prepared in this invention. This result indicates that, compared with the commonly used drug carriers polylactic acid-glycolic acid-polyethyleneimine and polyethylene glycol, the use of acylhydrazide-polyethylene glycol-hydroxy or the modified acylhydrazide-polyethylene glycol prepared in this invention helps to improve the sustained-release performance of the drug nanocarrier, reduce the cumulative release of olaparib, and effectively prolong the duration of drug action.

[0176] Compared to Comparative Example 3, the cumulative release amounts in Examples 4-6 were significantly reduced. This is because, in the preparation of the drug nanocarriers, Examples 4-6 used modified acylhydrazine-polyethylene glycol, while Comparative Example 3 used acylhydrazine-polyethylene glycol-hydroxyl. This result indicates that, compared to unmodified acylhydrazine-polyethylene glycol-hydroxyl, the drug nanocarriers prepared using modified acylhydrazine-polyethylene glycol can effectively reduce the cumulative drug release amount.

[0177] Compared to Comparative Example 4, the cumulative release amounts in Examples 4-5 were significantly reduced. This is because in the preparation of the modified hydrazide-polyethylene glycol, Example 4 used isotretinoin to modify the hydroxyl group of the hydrazide-polyethylene glycol; Example 5 first reacted isotretinoin and methyl 3-aminocrotonate to obtain an isotretinoin derivative, and then used the isotretinoin derivative to modify the hydroxyl group of the hydrazide-polyethylene glycol; while Comparative Example 4 only used a mixture of hydrazide-polyethylene glycol-hydroxyl group, isotretinoin, and methyl 3-aminocrotonate, without modifying the hydroxyl group of the hydrazide-polyethylene glycol. This result indicates that the modified hydrazide-polyethylene glycol obtained by modification with isotretinoin or isotretinoin derivatives can effectively reduce the cumulative release amount of the drug when used to prepare drug nanocarriers. The cumulative release amount in Example 5 was lower than that in Example 4, indicating that the drug nanocarrier prepared by modifying the hydrazide-polyethylene glycol with isotretinoin derivatives has better sustained-release performance.

[0178] Compared to Example 5 and Comparative Example 6, the cumulative release amount in Example 6 was further reduced. This is because, in the preparation of the modified hydrazide-polyethylene glycol, Example 6 further modified the hydrazide-polyethylene glycol-hydroxyl group with isopropyl glycidyl ether after modifying it with an isotretinoin derivative; Example 5 did not use isopropyl glycidyl ether modification, and Comparative Example 6 did not use isotretinoin derivative modification. This result indicates that, compared to modifying the hydrazide-polyethylene glycol-hydroxyl group with isotretinoin derivative or isopropyl glycidyl ether alone, the modified hydrazide-polyethylene glycol obtained by simultaneously using both isotretinoin derivative and isopropyl glycidyl ether can further reduce the cumulative drug release amount and improve the sustained-release performance of the prepared drug nanocarrier.

[0179] 9. Material Characterization

[0180] The modified acylhydrazide-polyethylene glycol prepared in Example 6 was characterized by Fourier transform infrared spectroscopy.

[0181] The results are as follows Figure 26 As shown, 3400cm -1 The characteristic peak of NH appears nearby, at 1250 cm⁻¹. -1 The presence of a COC absorption peak nearby indicates that the hydroxyl group on the hydrazide-polyethylene glycol-hydroxyl group reacts with isopropyl glycidyl ether; 2930 cm⁻¹ -1 Characteristic peaks of CH appear nearby; 1660 cm⁻¹ -1 An absorption peak appears near C=O, at 1630 cm⁻¹. -1 A characteristic peak for C=C appears nearby, at 1725~1700cm. -1 The disappearance of the nearby carboxyl characteristic peak indicates that the acylhydrazine-polyethylene glycol-hydroxy group undergoes an amide reaction with the isotretinoin derivative.

[0182] The conventional operations in the operation steps of this invention are well known to those skilled in the art and will not be described in detail here.

[0183] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any changes and modifications made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A metformin-enhanced PARP inhibitor, characterized in that, The invention includes a drug nanocarrier and metformin and olaparib encapsulated in the drug nanocarrier; the molar ratio of olaparib to metformin is 1:200-600. The drug nanocarrier is a composite system formed by ultrasonic dispersion of magnetic nanoparticles of iron oxide, modified acylhydrazine-polyethylene glycol and polyvinyl alcohol solution in dimethyl sulfoxide; The modified acylhydrazine-polyethylene glycol is an acylhydrazine-polyethylene glycol derivative modified with isotretinoin and isopropyl glycidyl ether, wherein the isotretinoin derivative is an isotretinoin derivative modified with methyl 3-aminocrotonate; the preparation method of the modified acylhydrazine-polyethylene glycol includes: dissolving acylhydrazine-polyethylene glycol-hydroxy in acetic acid solution, treating with nitrogen gas for 20-40 min, adding isotretinoin derivative solution at 0-10℃, then adjusting the pH to 4-6, and adding 1-ethyl-3-(3-dimethylaminopropyl)carbon The modified hydrazide-polyethylene glycol was obtained by reacting diimine hydrochloride and N-hydroxysuccinimide for 20-40 min, followed by 8-24 h at room temperature. After the reaction was completed, the mixture was dialyzed and freeze-dried. After the freeze-drying, dimethyl sulfoxide was added and stirred for 0.5-2 h at 50-70 °C. Triethylamine and isopropyl glycidyl ether were then added and stirred for 8-24 h at 70-90 °C. After the reaction was completed, the mixture was cooled to room temperature, filtered, washed 1-5 times with acetone, dialyzed, and freeze-dried. The preparation method of the isotretinoin derivative includes: dissolving isotretinoin and methyl 3-aminocrotonate in dichloromethane, adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine, mixing evenly, and reacting under nitrogen protection for 8-24 h. After the reaction, the mixture is washed 1-3 times with dilute hydrochloric acid, and then washed 1-3 times with saturated sodium bicarbonate solution. The organic phase is retained, filtered, concentrated by rotary evaporation, purified by column chromatography, and then tetrahydrofuran is added and stirred evenly. Sodium hydroxide solution is added and reacted for 8-24 h. After the reaction, the pH is adjusted to 2-3, the organic phase is extracted with ethyl acetate and collected, concentrated by rotary evaporation, and dried to obtain the isotretinoin derivative.

2. The metformin-enhanced PARP inhibitor according to claim 1, characterized in that, The mass ratio of the acylhydrazine-polyethylene glycol-hydroxyl and isotretinoin derivative is 1:0.2-1.

3. The metformin-enhanced PARP inhibitor according to claim 1, characterized in that, The mass ratio of the acylhydrazine-polyethylene glycol-hydroxyl and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:0.5-1.

4. The metformin-enhanced PARP inhibitor according to claim 1, characterized in that, The mass ratio of the hydrazide-polyethylene glycol-hydroxyl and N-hydroxysuccinimide is 1:0.1-0.

5.

5. The metformin-enhanced PARP inhibitor according to claim 1, characterized in that, The mass ratio of the magnetic nanoparticles of iron oxide to the modified hydrazide-polyethylene glycol is 1:1-3.

6. The metformin-enhanced PARP inhibitor according to claim 1, characterized in that, The mass concentration of the polyvinyl alcohol solution is 1-5%.

7. The metformin-enhanced PARP inhibitor according to claim 1, characterized in that, The preparation method of the PARP inhibitor includes adding metformin and olaparib to a drug nanocarrier, sonicating for 20-60 seconds, adding ultrapure water and stirring for 8-24 hours, purifying by magnetic bead separation, and freeze-drying to obtain the PARP inhibitor.

8. The use of the PARP inhibitor according to any one of claims 1-7 in the preparation of an anti-breast cancer drug.

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