GSH (glutathione) response type chidamide heterodimer nano prodrug as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicine, and discloses a GSH response type chidamine heterodimer nano prodrug and a preparation method and application thereof.The GSH response type chidamine heterodimer nano prodrug prepared through the method is high in loading rate, can be efficiently delivered to tumor cells, has the precise release capacity and can be used for preparing a drug for treating tumor tumors. The prodrug prepared by the method can efficiently deliver CDA and CPT to tumor cells, so that efficient delivery and on-demand release of the drug in the tumor cells are realized, the anti-tumor effect of the chemotherapeutic drug is improved, and a new direction and a new thought are provided for delivery of the chemotherapeutic drug.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and in particular to a GSH-responsive chidamide heterodimer nano-prodrug and a preparation method and application thereof. Background Art

[0002] Today, breast cancer has replaced lung cancer as the malignant tumor with the highest incidence rate in the world. It has become the main malignant tumor in some developed countries and regions, accounting for 7% to 10% of all malignant tumors in the body. The main treatment methods include surgery, chemotherapy, radiotherapy, targeted and endocrine therapy. Most breast cancer patients need chemotherapy, and most chemotherapy drugs are cytotoxic drugs, which have nonspecific or uncontrolled biodistribution, as well as rapid blood clearance, poor solubility, low bioavailability and poor pharmacokinetic properties, which lead to serious toxic side effects and other problems.

[0003] Chidamide (CDA) is a newly designed and synthesized histone deacetylase inhibitor (HDACi). Among solid tumors, Chidamide is widely used in a variety of tumors such as 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 invasion and migration capabilities. Compared with Chidamide monotherapy, Chidamide combined with chemotherapy shows obvious cytotoxicity to breast cancer cells in vitro and in vivo. Chidamide can significantly increase the histone H3 acylation level of drug-resistant cells and reduce the expression of HDAC1. Camptothecin (CPT) is a topoisomerase inhibitor and is currently the most widely used anti-tumor chemotherapy drug in clinical practice.

[0004] However, the two drugs currently have low targeting efficiency. After administration, only a small portion of the drug reaches the tumor site, and the majority of the remaining drugs are delivered to other tissues in the body, producing obvious toxic side effects. Therefore, how to effectively deliver cedabendine and camptothecin to breast cancer cells is the key to achieving safe and efficient treatment of breast cancer, and has great research value and significance.

[0005] In recent years, based on the unique properties of the tumor microenvironment (such as high concentration of glutathione (GSH), low pH), a series of functionalized polymer drug conjugates have been developed for tumor microenvironment-responsive drug delivery, and great progress has been made in drug targeted delivery, controlled release, and improved efficacy. People connect tumor-treating drugs to drug carriers through tumor microenvironment-responsive chemical bonds. The prepared tumor microenvironment-responsive nano drug delivery system is very sensitive to tumor tissue stimulation and can selectively release drugs at the tumor site, which can not only improve the efficacy of drugs, but also effectively reduce the toxic side effects of drugs on normal cells of the body. In the research of tumor microenvironment-responsive nano drug delivery systems, the construction of redox-responsive drug delivery systems is one of the research hotspots.

[0006] Based on this, the prodrug nano drug delivery system (prodrug-based nano-DDS) that integrates prodrug strategy and nanotechnology into one system has shown unique advantages. Prodrug refers to a compound that is inactive or less active in vitro after the drug is modified by chemical structure, and is converted and released into active drugs in vivo to exert its efficacy. The small molecule prodrug self-assembly nano drug delivery system released by tumor responsiveness has become a hot spot in the research of chemotherapy drug delivery in recent years due to its advantages such as no need for carriers, high drug loading, good stability, low toxic side effects, and rapid release of parent drugs in tumor sites. However, the prior art shows that the prodrugs with different structural modifications have different changes in drug properties, and the effects of the obtained prodrugs in vivo are also different. Therefore, in order to obtain prodrugs with optimal properties and make them self-assemble into nanoparticles, thereby improving efficacy and reducing toxicity, it is the direction that medical technicians are working hard to study. Summary of the invention

[0007] The purpose of the present invention is to provide a GSH-responsive chidamide heterodimer nanoprodrug and its preparation method and application in view of the above problems, to construct a GSH-responsive chidamide heterodimer prodrug (CDA-SS-CPT) through a disulfide bond, and to prepare CPT-SS-CDA, DSPE-PEG by a nanoprecipitation method. 2000 and DSPE-PEG-FA 2000 Co-assembly to prepare folic acid-modified DSPE-PEGylated heterodimer nano-prodrugs for the delivery of CDA and CPT, achieving efficient drug delivery and on-demand release in tumor cells, improving the anti-tumor effect of chemotherapy drugs, and providing new directions and new ideas for chemotherapy drug delivery.

[0008] In order to achieve its purpose, the present invention adopts the following technical solution:

[0009] The 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:

[0010]

[0011] The 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) reacting camptothecin with 2,2'-dithiodiacetic acid to obtain an intermediate product CPT-SS-OH;

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

[0014]

[0015] The camptothecin structural formula is as follows:

[0016]

[0017] The reaction equation is as follows:

[0018]

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

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

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

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

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

[0024] The 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 anti-tumor drugs.

[0025] Preferably, in the above-mentioned application technology solution, the tumor is breast cancer.

[0026] The beneficial effects of the present invention are:

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

[0028] 2. GSH-responsive cedabenbine heterodimer prodrug compounds improve the therapeutic effect 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the GSH-responsive chidamide heterodimer prodrug in Example 1 of the present application;

[0031] Figure 2 is a particle size distribution diagram of the nanoprodrug in Example 2 of the present application, wherein "Diameter" means "diameter", "Intensity" means "intensity", and "PDI" means "polydispersity index";

[0032] Figure 3 is a transmission electron microscopy image of the nanoprodrug in Example 2 of the present application;

[0033] Figure 4 is a curve diagram of the cumulative release of CDA of the nano prodrug prepared in Example 2 of the present application under different conditions, wherein "Time" means "time" and "Accumulated release of CDA" means "accumulated release of CDA";

[0034] Figure 5is a curve diagram of the cumulative release of camptothecin of the nano prodrug prepared in Example 2 of the present application under different conditions, wherein "Time" means "time" and "Accumulated release of CPT" means "accumulated release of camptothecin";

[0035] Figure 6 This is a graph showing the toxicity of the nanoprodrug prepared in Example 2 of the present application to breast cancer cells, wherein "Concentration (μM)" means "concentration (micromoles per liter)" and "Cell viability" means "cell viability";

[0036] Figure 7 It is a synergistic index diagram of chidamide and camptothecin in the nano prodrug prepared in Example 2 of the present application, wherein "Fraction affected" means "partial effect (also known as inhibition rate)" and "Combination Index" means "combination index"). DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with examples and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Those skilled in the art will make modifications or equivalent substitutions based on the understanding of the technical scheme of the present invention, without departing from the spirit and scope of the technical scheme of the present invention, and all should be included in the protection scope of the present invention. The experimental methods in the following embodiments, unless otherwise specified, are conventional methods, and other unspecified specific conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments that do not indicate the manufacturer are all conventional products that can be purchased commercially.

[0038] The abbreviations and Chinese meanings of the reagents in the examples 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 the reaction between camptothecin and 2,2'-dithiodiacetic acid (CPT-SS-OH), 4-dimethylaminopyridine (DMAP), triphosgene (Triphosgene), dichloromethane (DCM), N,N-dimethylformamide (DMF), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000 ), distearoylphosphatidylethanolamine-polyethylene glycol-folic acid (DSPE-PEG2000 -FA), dithiothreitol (DTT).

[0040] 1. Implementation

[0041] Example 1

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

[0043] (1) Camptothecin is reacted with 2,2'-dithiodiacetic acid to obtain an intermediate product CPT-SS-OH: Specifically, 0.5 mmol camptothecin and 1.5 mmol DMAP are weighed in a vacuum-dried 100 mL reaction tube, vacuum-treated, and 10 mL ultra-dry tetrahydrofuran is added under a nitrogen atmosphere and stirred to dissolve; the reaction tube is placed in a 0°C ice-water bath, triphosgene (1.0 mmol) is dissolved in 5 ml ultra-dry tetrahydrofuran, and slowly added dropwise to the reaction mixture. After the addition is completed, the reactant is returned to room temperature and reacted for 30 minutes; 3 mmol 2-hydroxyethyl disulfide is dissolved in 5 ml DCM was added dropwise to the reaction tube and reacted at room temperature for 12 hours; after the reaction was completed, the mixture was extracted with dichloromethane three times, the organic phases were combined, the dichloromethane was removed by spin drying, ethyl acetate was added, the mixture was washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered; the solvent was removed by rotary evaporation, the crude product was rapidly separated and purified by column chromatography using dichloromethane / methanol as eluent to obtain a light yellow solid, which was 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, and the tube was treated with a vacuum line. Under a nitrogen atmosphere, 10 mL of ultra-dry dichloromethane was added and stirred to dissolve; the reaction tube was placed in a 0°C ice water bath, triphosgene (0.114 mmol) was dissolved in 3 mL of ultra-dry dichloromethane, and the solution was slowly added dropwise to the reaction tube. , return to room temperature and react for 2 hours; 0.336mmol CDA is dissolved in 5mL ultra-dry DMF, added to the reaction tube under nitrogen atmosphere, and reacted at room temperature for 24 hours; after the reaction is completed, it is extracted with chloroform three times, the organic phases are combined, chloroform is removed by spin drying, ethyl acetate is added, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered; the solvent is removed by rotary evaporator, and the crude product is rapidly separated and purified by column chromatography using dichloromethane / methanol as eluent to obtain a white solid, which is a GSH-responsive chidamide heterodimer prodrug. The synthetic route of step (2) is as follows:

[0046]

[0047] The obtained product is a GSH-responsive chidamide heterodimer prodrug, Figure 1 It is the nuclear magnetic resonance hydrogen spectrum of the GSH-responsive chidamide heterodimer prodrug, and its structural formula is:

[0048]

[0049] Example 2

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

[0051] Table 1 Components and dosages of FDSC NPs prepared by nanoprecipitation method

[0052]

[0053] 2. Performance Characterization Experiment

[0054] 1. FDSC NPs Particle Size Measurement

[0055] The particle size of the preparation prepared in Example 2 was measured using a dynamic light scattering particle size analyzer (NanoBrook 90 Plus PALS). The measurement results are as follows: Figure 2 As shown in the figure, it can be seen that the average particle size of the prepared nanoformulation is about 126nm and the PDI is 0.2.

[0056] 2. FDSC NPs appearance inspection

[0057] The preparation prepared in Example 2 was diluted and added dropwise to a copper mesh covered with a carbon film for 2 minutes. The remaining liquid was then absorbed with filter paper. After evaporation, 5% phosphotungstic acid was added dropwise for negative staining for 5 minutes. The remaining liquid was absorbed with filter paper, and the mixture was dried naturally. The appearance was observed using a transmission electron microscope.

[0058] Specific results such as Figure 3As shown in the figure, it can be seen from the transmission electron microscopy results that FDSC NPs are spherical in shape with a relatively uniform particle size distribution of about 100 nm, which is close to and slightly smaller than the particle size measured by the particle size analyzer before. This is because there is a thicker hydration layer on the surface of the nanoparticles measured by dynamic light, while the hydration layer disappears on the dried nanoparticles measured by transmission electron microscopy, making the size of a single nanoparticle slightly smaller than the size after hydration.

[0059] 3. Investigation of the in vitro release behavior of FDSC NPs

[0060] The preparation 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, respectively, and the release of chidamide and camptothecin was detected by HPLC.

[0061] The results are as follows Figure 4 and Figure 5 As shown in the figure, in the absence of DTT, the cumulative release of CDA and CPT in FDSC NPs within 24 hours was less than 5%; the cumulative release percentages of CDA and CPT increased with the increase of DTT culture 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 the two were released almost simultaneously.

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

[0063] 1. 4T1 cells (breast cancer mouse cell line) were cultured at 5×10 3 The cells were grown into a 96-well plate at a cell density of 10 cells and cultured overnight at 37°C, 5% CO2; then, the culture medium was removed and replaced with new culture medium diluted to the required concentrations of CDA, CPT, CDA / CPT, DSC, DSC NPs and FDSC NPs; after another 48 hours of culture, 10 μL of CCK-8 solution was added to each well and cultured for 1 hour and 30 minutes; finally, the absorbance of each well was detected at 450 nm using a multi-function microplate reader (Thermo Scientific VarioskanLUX, USA).

[0064] The statistical results of the cell viability of breast cancer cells by each group of preparations are as follows Figure 6As shown in the figure, each preparation group showed concentration-dependent cytotoxicity to 4T1 cells, and FDSC NPs showed better therapeutic effects compared with other drug groups. This may mean that the active targeting of the ligand FA enhances the tumor targeting of FDSC NPs, thereby enhancing the cytotoxicity of FDSCNPs to 4T1 cells, proving that FDSC NPs can effectively kill breast cancer cells.

[0065] 2. The above results were further evaluated using the Chou and Talalay method to further evaluate the synergy index (CI).

[0066] We investigated the synergistic effect of CDA and CPT released from FDSC NPs based on the combination index (CI) method. The statistical results of the CI values ​​of the combined application of CDA and CPT are shown in Figure 7 As shown in the figure, FDSC NPs showed a synergistic effect on 4T1 cells and significantly improved the therapeutic potential, which indicates that FDSC NPs released CDA and CPT in response to GSH in the tumor microenvironment, and the two worked together to kill breast cancer cells.

[0067] 5. Results

[0068] In summary, the present invention provides a GSH-responsive chidamide heterodimer nanoprodrug and a preparation method and application thereof, wherein a GSH-responsive chidamide heterodimer prodrug (CDA-SS-CPT) is constructed by disulfide bonds, and CPT-SS-CDA, DSPE-PEG 2000 and DSPE-PEG 2000 -FA co-assembly to prepare folic acid-modified DSPE-PEGylated heterodimer nanoprodrugs for the delivery of CDA and CPT. The preparation process is simple to operate, the raw materials are easily available, and the cost is low. Performance verification experiments show that the prepared nanoparticles have uniform particle size. In vitro release behavior investigation experiments show that when FDSC NPs are incubated with 5mM DTT for 24 hours, the cumulative release percentages of CDA and CPT are 70.91% and 80.99%, respectively, and the release ratio of the two free drugs is close to 1:1, indicating that the two are released almost simultaneously. Combined with in vitro pharmacodynamic experiments, it is proved that the nanoprodrug prepared by this method can effectively kill breast cancer cells, and the combined application of CDA and CPT produces obvious synergistic effects. Therefore, the prodrug prepared by this method can efficiently deliver CDA and CPT to tumor cells, achieve efficient delivery and on-demand release of drugs in tumor cells, and improve the anti-tumor effect of chemotherapeutic drugs, thereby providing a new direction and new ideas for the delivery of chemotherapeutic drugs.

Claims

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

2. The method for preparing the GSH-responsive chidamide heterodimer prodrug according to claim 1, characterized in that: The steps include: (1) reacting camptothecin with 2,2'-dithiodiacetic acid to obtain an intermediate product CPT-SS-OH; (2) reacting the obtained intermediate product CPT-SS-OH with chidamide to obtain the chidamide heterodimer prodrug CDA-SS-CPT; Wherein, the structural formula of the cedamide is as follows: The camptothecin structural formula is as follows: The reaction equation is as follows: The obtained product is a GSH-responsive chidamide heterodimer prodrug.

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

3.

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

1.

5. A method for preparing a GSH-responsive chidamide heterodimer nanoprodrug, characterized in that: The method comprises the following steps: mixing the GSH-responsive chidamide heterodimer prodrug described in formula I of claim 1, DSPE-PEG 2000 and DSPE-PEG 2000 -FA was dissolved in an organic solvent and mixed evenly. The mixed solution was slowly added dropwise into deionized water under stirring conditions, and then the organic solvent was dialyzed using a dialysis bag to obtain a 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 -FA weight ratio is 5:1:

1.

7. Use of the GSH-responsive chidamide heterodimer nanoprodrug prepared according to the preparation method of claim 5 or 6 in the preparation of anti-tumor drugs.

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

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