A GSH-responsive chidamide heterodimer nanoprodrug, its preparation method and application

Abstract

This invention relates to the field of pharmaceutical technology, and discloses a GSH-responsive chidamide heterodimer nanoprodrug, its preparation method, and its application. The GSH-responsive chidamide heterodimer nanoprodrug prepared by this method has a high loading rate, can be efficiently delivered to tumor cells, has the ability to release precisely, and improves the therapeutic effect of breast cancer through synergistic chemotherapy. The prodrug prepared by this method can efficiently deliver CDA and CPT to tumor cells, realize efficient delivery and on-demand release of drugs in tumor cells, and improve the anti-tumor effect of chemotherapy drugs, thus providing a new direction and new ideas for chemotherapy drug delivery.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical technology, specifically to a GSH-responsive chidamide heterodimer nanoprodrug, its preparation method, and its application. Background Technology

[0002] Today, breast cancer has surpassed lung cancer to become the most common malignant tumor worldwide, and in some developed countries and regions, it has become the main malignant tumor, accounting for 7% to 10% of all malignant tumors. The main treatment methods include surgery, chemotherapy, radiotherapy, targeted therapy, and endocrine therapy. Most breast cancer patients need chemotherapy, but most chemotherapy drugs are cytotoxic drugs, which have disadvantages such as non-specific or uncontrolled biological distribution, rapid blood clearance, poor solubility, low bioavailability, and poor pharmacokinetic properties, resulting in serious toxic side effects.

[0003] Chidamide (CDA) is a newly designed and synthesized histone deacetylase inhibitor (HDACi). In solid tumors, chidamide is widely used for various cancers, including breast cancer, colon cancer, lymphoma, and non-small cell lung cancer. Basic research shows that HDAC inhibitors can significantly inhibit the growth of breast cancer cells, induce apoptosis, and reduce their invasive and migratory abilities. Compared with chidamide monotherapy, chidamide combined with chemotherapy exhibits significant cytotoxicity against breast cancer cells in vitro and in vivo. Chidamide can significantly increase histone H3 acylation levels and reduce HDAC1 expression in drug-resistant cells. Camptothecin (CPT) is a topoisomerase inhibitor and is currently the most widely used antitumor chemotherapy drug in clinical practice.

[0004] However, both drugs currently exhibit low targeting efficiency; only a small portion reaches the tumor site after administration, while the majority is delivered to other tissues, resulting in significant toxic side effects. Therefore, efficiently delivering the combined chidamide and camptothecin to breast cancer cells is crucial for achieving safe and effective treatment of breast cancer and holds significant research value and importance.

[0005] In recent years, based on the unique properties of the tumor microenvironment (such as high concentrations of glutathione (GSH) and low pH), a series of functionalized polymer drug conjugates have been developed for tumor microenvironment-responsive drug delivery, achieving significant progress in targeted drug delivery, controlled release, and improved efficacy. By linking anti-tumor drugs to drug carriers through tumor microenvironment-responsive chemical bonds, the resulting tumor microenvironment-responsive nanoparticle drug delivery systems are highly sensitive to tumor tissue stimulation and can selectively release drugs at the tumor site. This not only improves drug efficacy but also effectively reduces the toxic side effects of drugs on normal cells. In the research of tumor microenvironment-responsive nanoparticle drug delivery systems, the construction of redox-responsive drug delivery systems is one of the research hotspots.

[0006] Based on this, prodrug-based nano-DDS systems, which integrate prodrug strategies and nanotechnology into a single system, have demonstrated unique advantages. Prodrugs are compounds that, after chemical modification, are inactive or have low inactivity in vitro but transform and release active drugs in vivo to exert their therapeutic effects. Tumor-responsive small-molecule prodrug self-assembly nano-DDS systems have become a hot topic in chemotherapy drug delivery research in recent years due to their advantages such as no need for carriers, high drug loading capacity, good stability, low toxicity, and rapid release of the parent drug from the tumor site. However, current technologies show that prodrugs with different structural modifications alter the drug properties differently, resulting in varying in vivo effects. Therefore, obtaining prodrugs with optimal properties and enabling their self-assembly into nanoparticles to improve efficacy and reduce toxicity is a direction that pharmaceutical engineers are actively researching. Summary of the Invention

[0007] The purpose of this invention is to address the aforementioned problems by providing a GSH-responsive chidamide heterodimer nanoprodrug, its preparation method, and its applications. The invention constructs a GSH-responsive chidamide heterodimer prodrug (CDA-SS-CPT) via disulfide bonds, and then uses a nanoprecipitation method to combine CPT-SS-CDA and DSPE-PEG. 2000 and DSPE-PEG-FA 2000 By co-assembling folic acid-modified DSPE-PEGylated heterodimer nanoprodrugs, which are used to deliver CDA and CPT, the efficient delivery and on-demand release of drugs in tumor cells are achieved, thereby improving the anti-tumor effect of chemotherapy drugs and providing a new direction and idea for chemotherapy drug delivery.

[0008] To achieve its objective, the present invention employs the following technical solution:

[0009] A first aspect of the present invention provides a GSH-responsive chidamide heterodimer prodrug, wherein the structural formula of the GSH-responsive chidamide heterodimer prodrug is as follows:

[0010]

[0011] A second aspect of the present invention provides a method for preparing the above-mentioned GSH-responsive chidamide heterodimer prodrug, comprising the following steps:

[0012] (1) Camptothecin was reacted with 2,2'-dithiodiacetic acid to obtain the intermediate product CPT-SS-OH;

[0013] (2) The obtained intermediate product CPT-SS-OH was reacted with chidamide to obtain the GSH-responsive chidamide heterodimer prodrug CDA-SS-CPT; wherein, the structural formula of the chidamide is as follows:

[0014]

[0015] The structural formula of camptothecin is as follows:

[0016]

[0017] The reaction equation is as follows:

[0018]

[0019] The resulting product is a GSH-responsive chidamide heterodimer prodrug.

[0020] Preferably, in the above preparation method, in step (1), the molar ratio of camptothecin to 2,2'-dithiodiacetic acid is 0.5:3.

[0021] Preferably, in the above preparation method, in step (2), the molar ratio of the intermediate product CPT-SS-OH to chidamide is 1:1.

[0022] A third aspect of the present invention provides a method for preparing a GSH-responsive chidamide heterodimer nanoprodrug, comprising the following steps: mixing the GSH-responsive chidamide heterodimer prodrug of Formula I with DSPE-PEG... 2000 and DSPE-PEG 2000 -FA was dissolved in an organic solvent and mixed thoroughly. Under stirring conditions, the mixture was slowly added dropwise to deionized water. The organic solvent was then dialyzed using a dialysis bag to obtain GSH-responsive chidamide heterodimer nanoprodrug.

[0023] Preferably, in the above preparation method, the GSH-responsive chidamide heterodimer prodrug and DSPE-PEG... 2000 and DSPE-PEG 2000 The weight ratio of -FA is 5:1:1.

[0024] A fourth aspect of the present invention provides the use of the GSH-responsive chidamide heterodimer nanoprodrug prepared by the above preparation method in the preparation of antitumor drugs.

[0025] Preferably, in the above application technical solution, the tumor is breast cancer.

[0026] The beneficial effects of this invention are:

[0027] 1. The GSH-responsive chidamide heterodimer prodrug compound of the present invention, wherein a GSH-responsive chidamide heterodimer prodrug is constructed via disulfide bonds, and is then combined with DSPE-PEG. 2000 and DSPE-PEG 2000 -FA self-assembles folic acid-modified chidamide heterodimer prodrugs (FDSC NPs) via nanoprecipitation, achieving targeted drug delivery and GSH-responsive release. It has a high loading rate, can efficiently deliver to tumor cells, and has the ability to release precisely.

[0028] 2. GSH-responsive chidamide heterodimer prodrug compounds improved the treatment efficacy of breast cancer through synergistic chemotherapy.

[0029] 3. The preparation method of the present invention is simple to operate, the raw materials are easy to obtain, the cost is low, and it is easy to promote. Attached Figure Description

[0030] Figure 1 This is the 1H NMR spectrum of the GSH-responsive chidamide heterodimer prodrug in Example 1 of this application;

[0031] Figure 2 This is a particle size distribution diagram of the nano-prodrug in Example 2 of this application, where "Diameter" means "diameter", "Intensity" means "strength", and "PDI" means "polydispersity index".

[0032] Figure 3 This is a transmission electron microscope image of the nanoprodrug in Example 2 of this application;

[0033] Figure 4 This is a graph showing the cumulative release of daidzepine under different conditions of the nano-prodrug prepared in Example 2 of this application. In this graph, "Time" means "time" and "Accumulated relesease of CDA" means "cumulative release of daidzepine".

[0034] Figure 5This is a graph showing the cumulative release of camptothecin under different conditions of the nano-prodrug prepared in Example 2 of this application. In this graph, "Time" means "time" and "Accumulated release of CPT" means "cumulative release of camptothecin".

[0035] Figure 6 This is a toxicity graph of the nano-prodrug prepared in Example 2 of this application on breast cancer cells, where "Concentration (μM)" means "concentration (micromoles per liter)" and "Cell viability" means "cell viability".

[0036] Figure 7 This is a synergistic index diagram of the present invention's chidamide and camptothecin in the nano-prodrug prepared in Example 2 of this application, where "Fraction affected" means "partial effect (also known as inhibition rate)" and "Combination Index" means "combination index". Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Modifications or equivalent substitutions made by those skilled in the art based on their understanding of the technical solutions of this invention, without departing from the spirit and scope of the invention, should be covered within the protection scope of this invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Other unspecified specific conditions should be performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0038] The abbreviations and Chinese definitions of the reagents in the embodiments of this application are as follows:

[0039] GSH-responsive chidamide heterodimer prodrug (CDA-SS-CPT, DSC), chidamide (CDA), camptothecin (CPT), GSH-responsive chidamide heterodimer nanoprodrug (DSC NPs), folic acid-modified GSH-responsive chidamide heterodimer nanoprodrug (FDSC NPs), intermediate product of camptothecin reaction with 2,2'-dithiodiacetic acid (CPT-SS-OH), 4-dimethylaminopyridine (DMAP), triphosgene, dichloromethane (DCM), N,N-dimethylformamide (DMF), distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG) 2000 ), distearate phosphatidylethanolamine-polyethylene glycol-folic acid (DSPE-PEG)2000 -FA), dithiothreitol (DTT).

[0040] I. Implementation Examples

[0041] Example 1

[0042] A method for preparing a GSH-responsive chidamide heterodimer prodrug includes the following steps:

[0043] (1) Camptothecin was reacted with 2,2'-dithiodiacetic acid to obtain the intermediate product CPT-SS-OH: Specifically, 0.5 mmol of camptothecin and 1.5 mmol of DMAP were weighed into a vacuum-dried 100 mL reaction tube, treated under a vacuum line, and 10 mL of ultra-dry tetrahydrofuran was added under a nitrogen atmosphere and stirred to dissolve. The reaction tube was placed in an ice-water bath at 0 °C, and triphosgene (1.0 mmol) was dissolved in 5 mL of ultra-dry tetrahydrofuran and slowly added dropwise to the reaction mixture. After the addition was complete, the reactants were brought back to room temperature and reacted for 30 minutes. 3 mmol of 2-hydroxyethyl disulfide was dissolved in 5 mL of... In DCM, the solution was added dropwise to the reaction tube and reacted at room temperature for 12 hours. After the reaction was complete, the solution was extracted three times with dichloromethane, the organic phases were combined, the dichloromethane was removed by rotary evaporation, ethyl acetate was added, and the solution was washed with water, saturated brine, dried over anhydrous sodium sulfate, and filtered. The solvent was removed by rotary evaporation, and the crude product was rapidly separated and purified by column chromatography using dichloromethane / methanol as the eluent to obtain the pale yellow solid, which is the intermediate product CPT-SS-OH. The synthetic route of step (1) is as follows:

[0044]

[0045] (2) The obtained intermediate product CPT-SS-OH was reacted with chidamide to obtain the GSH-responsive chidamide heterodimer prodrug CDA-SS-CPT: Specifically, 0.336 mmol of CPT-SS-OH and 1.008 mmol of DMAP were weighed into a vacuum-dried 100 mL reaction tube, treated under a vacuum line, and 10 mL of ultra-dry dichloromethane was added under a nitrogen atmosphere and stirred to dissolve. The reaction tube was placed in an ice-water bath at 0 °C, and 0.114 mmol of triphosgene was dissolved in 3 mL of ultra-dry dichloromethane and slowly added dropwise to the reaction tube. After the addition was complete... The reaction was brought back to room temperature for 2 hours; 0.336 mmol of CDA was dissolved in 5 mL of ultra-dry DMF, added to the reaction tube under nitrogen atmosphere, and reacted at room temperature for 24 hours; after the reaction was completed, the mixture was extracted three times with chloroform, the organic phases were combined, the chloroform was removed by rotary evaporation, ethyl acetate was added, the mixture was washed with water, washed with saturated brine, dried with anhydrous sodium sulfate, and filtered; the solvent was removed by rotary evaporation, and the crude product was rapidly separated and purified by column chromatography using dichloromethane / methanol as eluent to obtain a white solid, which is the GSH-responsive chidamide heterodimer prodrug. The synthetic route of step (2) is as follows:

[0046]

[0047] The resulting product is a GSH-responsive chidamide heterodimer prodrug, with attached... Figure 1 The 1H NMR spectrum of the GSH-responsive chidamide heterodimer prodrug is shown below. Its structural formula is as follows:

[0048]

[0049] Example 2

[0050] A method for preparing a GSH-responsive chidamide heterodimer nanoprodrug (FDSC NPs) includes the following steps: preparation by nanoprecipitation method, specifically, weighing 5 mg of CDA-SS-CPT obtained in Example 1 and 1 mg of DSPE-PEG. 2000 and 1mg DSPE-PEG 2000 - FA was dissolved in 1 mL of DMF and dissolved by vortexing and centrifugation. This was used as the organic phase. 1 mL of the organic phase was added dropwise to the aqueous phase under vigorous magnetic stirring. After stirring evenly, the residual organic solvent was removed by dialysis to obtain FDSC NPs. The specific amounts of the components are shown in Table 1.

[0051] Table 1. Components and dosages used in the preparation of FDSC NPs by nanoprecipitation method.

[0052]

[0053] II. Performance Characterization Experiment

[0054] 1. FDSC NPs particle size measurement

[0055] The formulation prepared in Example 2 was used to measure the particle size using a dynamic light scattering particle size analyzer (NanoBrook90PlusPALS). The measurement results are as follows: Figure 2 As shown in the figure, the average particle size of the prepared nano-formulation is about 126 nm and the PDI is 0.2.

[0056] 2. FDSC NPs visual inspection

[0057] After diluting the preparation prepared in Example 2, it was dropped onto a copper grid covered with a carbon film and timed for 2 minutes. Then, the residual liquid was blotted with filter paper and allowed to evaporate. After that, 5% phosphotungstic acid was added for negative staining for 5 minutes. The residual liquid was blotted with filter paper and allowed to dry naturally. Its appearance was observed using a transmission electron microscope.

[0058] Specific results are as follows Figure 3As shown in the transmission electron microscopy results, FDSC NPs are spherical with a relatively uniform particle size distribution of around 100 nm, which is close to but smaller than the particle size measured by the particle size analyzer. This is because the surface of the nanoparticles measured by dynamic light has a thick hydration layer, while the hydration layer of the dried nanoparticles measured by transmission electron microscopy disappears, making the size of the individual nanoparticles slightly smaller than that after hydration.

[0059] III. Investigation of the in vitro release behavior of FDSC NPs

[0060] The formulation prepared in Example 2 was added to DTT solutions of different concentrations (0, 1, 5 mM) and shaken in a shaker at 37°C for release. Samples were taken at 0, 0.5, 1, 8, 12, and 24 h, and the release of chidamide and camptothecin was detected by high performance liquid chromatography.

[0061] The results are as follows Figure 4 and Figure 5 As shown in the figure, without DTT, the cumulative release of CDA and CPT from FDSC NPs within 24 hours was less than 5%; the cumulative release percentage of CDA and CPT increased with the increase of DTT medium; when FDSC NPs were incubated with 5mM DTT for 24 hours, the cumulative release percentages of CDA and CPT were 70.91% and 80.99%, respectively; the release ratio of the two free drugs was close to 1:1, indicating that they were released almost simultaneously.

[0062] IV. In vitro pharmacodynamic evaluation of FDSC NPs

[0063] 1. 4T1 cells (a mouse cell line for breast cancer) were injected at a rate of 5 × 10⁻⁶ cells / year. 3 Cells were grown at a density of 100 cells per well into 96-well plates and cultured overnight at 37°C and 5% CO2. Subsequently, the culture medium was removed and replaced with fresh medium diluted to the required concentrations of CDA, CPT, CDA / CPT, DSC, DSC NPs, and FDSC NPs. After culturing for another 48 hours, 10 μL of CCK-8 solution was added to each well and incubated for 1 hour and 30 minutes. Finally, the absorbance of each well was measured at 450 nm using a multi-functional microplate reader (Thermo Scientific Varioskan LUX, USA).

[0064] The statistical results of cell viability values ​​of each formulation against breast cancer cells are as follows: Figure 6As shown in the figure, all formulations exhibited concentration-dependent cytotoxicity against 4T1 cells. Compared with other drug groups, FDSC NPs showed superior therapeutic efficacy. This may mean that the active targeting of the ligand FA enhanced the tumor targeting of FDSC NPs, thereby enhancing the cytotoxicity of FDSC NPs against 4T1 cells, thus demonstrating that FDSC NPs can effectively kill breast cancer cells.

[0065] 2. The Chou and Talalay methods were used to further evaluate the synergy index (CI) based on the aforementioned results.

[0066] We investigated the synergistic effect of CDA and CPT released from FDSC NPs using the combined index (CI) method. The statistical results of the CI values ​​for the combined application of CDA and CPT are as follows: Figure 7 As shown in the figure, FDSC NPs exhibit a synergistic effect on 4T1 cells, significantly enhancing their therapeutic potential. This indicates that FDSC NPs release CDA and CPT in response to GSH in the tumor microenvironment, and the two work together to kill breast cancer cells.

[0067] V. Results

[0068] In summary, this invention provides a GSH-responsive chidamide heterodimer prodrug nanoparticle, its preparation method, and its application. A GSH-responsive chidamide heterodimer prodrug (CDA-SS-CPT) is constructed via disulfide bonds, and CPT-SS-CDA and DSPE-PEG are precipitated using a nanoprecipitation method. 2000 and DSPE-PEG 2000 -FA co-assembly was used to prepare folic acid-modified DSPE-PEGylated heterodimer nanoprodrugs for the delivery of CDA and CPT. The preparation process is simple, the raw materials are readily available, and the cost is low. Performance verification experiments showed that the prepared nanoparticles had uniform particle size. In vitro release behavior experiments showed that when FDSC NPs were incubated with 5 mM DTT for 24 hours, the cumulative release percentages of CDA and CPT were 70.91% and 80.99%, respectively, and the release ratio of the two free drugs was close to 1:1, indicating that they were released almost simultaneously. Combined with in vitro pharmacodynamic experiments, it was demonstrated that the nanoprodrugs prepared by this method can effectively kill breast cancer cells, and the combined application of CDA and CPT produced a significant synergistic effect. Therefore, the prodrugs prepared by this method can efficiently deliver CDA and CPT to tumor cells, achieving efficient delivery and on-demand release of drugs in tumor cells, improving the anti-tumor effect of chemotherapy drugs, and providing a new direction and idea for chemotherapy drug delivery.

Claims

1. A GSH-responsive chidamide heterodimer prodrug, characterized in that: The structural formula of the GSH-responsive chidamide heterodimer prodrug is: Formula I.

2. The method for preparing the GSH-responsive chidamide heterodimer prodrug according to claim 1, characterized in that: Includes the following steps: (1) Camptothecin was reacted with 2,2'-dithiodiethanol to give the intermediate product CPT-SS-OH; (2) The obtained intermediate product CPT-SS-OH was reacted with daidramamine to obtain the daidramamine heterodimer prodrug CDA-SS-CPT; The structural formula of the daidzepine is as follows: Formula II; The structural formula of camptothecin is as follows: Formula III; The reaction equation is as follows: ； The resulting product is a GSH-responsive chidamide heterodimer prodrug.

3. The preparation method according to claim 2, characterized in that: In step (1), the molar ratio of camptothecin to 2,2'-dithiodiethanol is 0.5:

3.

4. The preparation method according to claim 2, characterized in that: In step (2), the molar ratio of the intermediate product CPT-SS-OH to damide is 1:

1.

5. A method for preparing a GSH-responsive chidamide heterodimer nanoprodrug, characterized in that: The process includes the following steps: mixing the GSH-responsive chidamide heterodimer prodrug of formula I in claim 1 with DSPE-PEG. 2000 and DSPE-PEG 2000 -FA was dissolved in an organic solvent and mixed thoroughly. Under stirring conditions, the mixture was slowly added dropwise to deionized water. The organic solvent was then dialyzed using a dialysis bag to obtain GSH-responsive chidamide heterodimer nanoprodrug.

6. The preparation method according to claim 5, characterized in that: The GSH-responsive chidamide heterodimer prodrug, DSPE-PEG 2000 and DSPE-PEG 2000 The weight ratio of -FA is 5:1:

1.

7. The application of the GSH-responsive chidamide heterodimer nanoprodrug prepared by the preparation method according to claim 5 or 6 in the preparation of antitumor drugs.

8. The application according to claim 7, wherein the tumor is breast cancer.

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