Vorinostat-cholesterol prodrug-containing liposome as well as preparation method and application thereof

Abstract

The invention belongs to the field of new auxiliary materials and new dosage forms of pharmaceutical preparations, and relates to a vorinostat-cholesterol prodrug-containing liposome as well as a preparation method and application thereof, in particular to a vorinostat prodrug liposome and a doxorubicin-loaded vorinostat prodrug liposome as well as a preparation method and application thereof in preparation of a drug delivery system. The vorinostat-cholesterol prodrug disclosed by the invention is a disulfide bond bridged vorinostat-cholesterol prodrug, and the structure of the vorinostat-cholesterol prodrug is shown in the specification. The prodrug liposome membrane structure contains the disulfide bond bridged prodrug, and the disulfide bond can respond to high-expression oxidation-reduction conditions in a tumor microenvironment, specifically break and release vorinostat. Furthermore, breakage of the disulfide bond also initiates destruction of a liposome membrane structure and promotes release of adriamycin in an internal water phase, so that the synergistic anti-tumor effect of the vorinostat-cholesterol prodrug and adriamycin is exerted. # imgabs0 #

Description

Technical field:

[0001] The present invention belongs to the field of new excipients and new dosage forms for pharmaceutical preparations, and relates to liposomes containing vorinostat-cholesterol prodrugs, and a preparation method and application thereof, and specifically relates to vorinostat prodrug liposomes and vorinostat prodrug liposomes loaded with doxorubicin, their preparation methods, and applications in preparing drug delivery systems. Background technology:

[0002] Chemotherapy that targets DNA damage to induce apoptosis of tumor cells is one of the main strategies for treating cancer. Doxorubicin (DOX), as a widely used DNA damaging drug, can prevent the synthesis of DNA and RNA by inserting between DNA base pairs, while inhibiting topoisomerase II and interfering with the topological structure of DNA, ultimately leading to the inhibition of tumor cell proliferation and inducing apoptosis. Doxorubicin has been widely used to treat a variety of malignant tumors and solid tumors, including breast cancer, lung cancer, acute leukemia, etc. Among doxorubicin preparations, doxorubicin liposomes prepared by loading doxorubicin into the aqueous phase of liposomes by active drug loading are the most successful form. Doxorubicin liposomes can significantly improve the distribution of the drug in the body, increase its tumor targeting, and reduce the accumulation of the drug in normal tissues, especially significantly reducing the cardiac distribution of doxorubicin, greatly reducing the risk of cardiac toxicity caused by traditional doxorubicin treatment.

[0003] Although doxorubicin liposomes have been successful in clinical treatment, they still face two major challenges: (i) Doxorubicin forms rod-shaped crystals in the aqueous phase of liposomes through active drug loading, and the bilayer structure of liposomes has good stability, which means that doxorubicin in the aqueous phase can only be released through slow osmosis, resulting in an extremely slow release rate of doxorubicin, which significantly limits its anti-tumor effect; (ii) After causing DNA damage to tumor cells, doxorubicin will activate the DNA repair mechanisms of tumor cells, such as nucleotide excision repair and homologous recombination repair. Once these repair mechanisms repair DNA damage, tumor cells will restore their proliferation ability and may develop into drug-resistant cells. In view of the challenges of doxorubicin liposomes in drug release and DNA damage repair, the development of new doxorubicin drug preparations has important clinical significance. Summary of the invention:

[0004] The present invention aims to overcome the deficiencies in the prior art, proposes a disulfide-bridged vorinostat-cholesterol prodrug, and successfully constructs a vorinostat-cholesterol prodrug liposome. The prodrug liposome can further load doxorubicin hydrochloride in its internal aqueous phase by an active drug loading method. The disulfide bond can be specifically broken under the redox environment in tumor cells, thereby triggering the release of vorinostat, while destroying the bilayer membrane structure of the liposome, and promoting the accelerated release of doxorubicin. In addition, vorinostat, as an epigenetic drug, mainly plays a role by inhibiting histone deacetylase (HDAC), and then destroys the DNA damage response mechanism of tumor cells by interfering with transcription or protein binding, thereby restoring the sensitivity of tumor cells to DNA damage induced by doxorubicin, and finally achieving the synthetic lethal effect of tumor cells.

[0005] The present invention not only solves the problem of slow release rate of doxorubicin, but also aggravates the DNA damage effect through drug combination, providing a new idea for tumor DNA damage therapy.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a disulfide-bridged vorinostat-cholesterol prodrug or a pharmaceutically acceptable salt thereof:

[0008]

[0009] The method for synthesizing the vorinostat-cholesterol prodrug of the present invention comprises the following steps:

[0010]

[0011] Step 1: adding dithiodibutyric acid to acetic anhydride, reacting at room temperature until the solution is clear, and then removing the acetic anhydride by vacuum rotary evaporation to obtain dithiodibutyric anhydride;

[0012] Step 2: Add cholesterol and DMAP into a reaction vessel, dissolve them in an organic solvent, then add dithiodibutyric anhydride, react overnight at room temperature, and separate and purify the product to obtain the intermediate cholesterol-dithiodibutyric acid monoester:

[0013] Step 3: Cholesterol-dithiodibutyric acid ester, EDCI and DMAP are added to a reaction vessel, dissolved with an organic solvent, activated in an ice bath, and then vorinostat dissolved in an organic solvent is added. After reacting at room temperature overnight, the product is separated and purified to obtain the final product vorinostat-cholesterol prodrug.

[0014] The present invention also provides a vorinostat-cholesterol prodrug liposome Lipo-SAHA, wherein the vorinostat-cholesterol prodrug liposome comprises vorinostat-cholesterol prodrug and a lipid material, wherein the mass ratio of the vorinostat-cholesterol prodrug to the lipid material is 1:10-1:50, preferably 1:15-1:25; the lipid material comprises phospholipids, cholesterol, and PEGylated phospholipids, wherein the mass ratio of the phospholipids, cholesterol, and PEGylated phospholipids is 1-100:1-50:1-50, preferably 30-80:20-50:1-20, and more preferably 40-50:25-35:3-6.

[0015] The mass volume percentages of the components in the vorinostat-cholesterol prodrug liposome are as follows: vorinostat-cholesterol prodrug accounts for 3-10%, preferably 3-6%; lipid material accounts for 50%-80%, preferably 70-80%, and the rest is water.

[0016] Furthermore, the mass volume percentage of cholesterol in the vorinostat-cholesterol prodrug liposome is 25-35%.

[0017] The phospholipids are commonly used in the art, such as: phosphatidylcholine (PC), egg yolk phosphatidylcholine (EPC), soybean phosphatidylcholine (SPC), hydrogenated soybean phosphatidylcholine (HSPC), cephalin (PE), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylglycerol (DSPG), polyethylene glycol phospholipids (PEG-PE), phosphatidylserine (PS).

[0018] Different phospholipids have no significant effect on the properties of liposomes.

[0019] The PEGylated phospholipids are polyethylene glycol distearoylphosphatidylethanolamine (DSPE-PEG), polyethylene glycol monomethyl ether-phosphatidylethanolamine (mPEG-PE), polyethylene glycol phosphatidylcholine (PEG-PC), polyethylene glycol cholesterol (PEG-Chol), polyethylene glycol dipalmitoylphosphatidylethanolamine (DPPE-PEG), etc., as well as PEGylated phospholipids with targeted modification and functional modification of the above materials, and the preferred PEGylated phospholipid is DSPE-PEG. The molecular weight of the PEG is 1000-5000, preferably 1000, 2000 and 5000, and more preferably the molecular weight of PEG is 2000.

[0020] The present invention further provides a method for preparing the vorinostat-cholesterol prodrug liposomes, comprising the following steps:

[0021] (1) Weigh phospholipids, cholesterol, PEGylated phospholipids and vorinostat-cholesterol prodrug into an eggplant-shaped bottle, add a small amount of volatile organic solvent, and remove the organic solvent by vacuum rotary evaporation to form a uniform drug-containing lipid film at the bottom of the bottle.

[0022] (2) Add the inner aqueous phase for preheating, then add the inner aqueous phase dropwise while shaking the eggplant-shaped bottle. Continue shaking until the film at the bottom is completely dissolved and hydrated. Use the extrusion method to size the particles to obtain nano-sized liposomes, and then use agarose gel columns to exchange the outer aqueous phase to obtain vorinostat-cholesterol prodrug liposomes Lipo-SAHA.

[0023] Furthermore, in step (1), the mass ratio of phospholipid, cholesterol and PEGylated phospholipid is 1-100:1-50:1-50, preferably 30-80:20-50:1-20, and more preferably 40-50:25-35:3-6.

[0024] Furthermore, the organic solvent in step (1) is dichloromethane, chloroform or n-hexane, preferably dichloromethane.

[0025] Furthermore, in step (2), the inner aqueous phase is ammonium sulfate solution, citrate buffer, phosphate buffer and acetate buffer, preferably ammonium sulfate solution. The concentration of the inner aqueous phase is 100-500 mM, preferably 250-350 mM.

[0026] Furthermore, the external aqueous phase in step (2) is a histidine solution with a concentration of 100-500 mM, preferably 250-350 mM.

[0027] Furthermore, the hydration condition in step (2) is incubation at 50-70° C. for 10-200 min.

[0028] Furthermore, the extrusion method in step (2) is nitrogen pressurization, and the crude liposomes are passed through a polycarbonate filter membrane with a pore size of 50-1000 μm.

[0029] The present invention also provides vorinostat-doxorubicin dual-drug-loaded liposomes, wherein the vorinostat-doxorubicin dual-drug-loaded liposomes are vorinostat-cholesterol prodrugs loaded with doxorubicin hydrochloride. The vorinostat-cholesterol prodrugs are effectively embedded in the phospholipid bilayer structure of the liposomes, and doxorubicin hydrochloride is loaded in the inner water phase of the vorinostat-cholesterol prodrug liposomes by an active drug loading method to construct the vorinostat-doxorubicin dual-drug-loaded liposomes.

[0030] The vorinostat-doxorubicin dual-drug liposomes contain vorinostat-cholesterol prodrug, doxorubicin, and lipid materials. The molar ratio of doxorubicin to vorinostat-cholesterol prodrug is 10:1-1:2, preferably 4-5:1.

[0031] The mass ratio of the total mass of the vorinostat-cholesterol prodrug and doxorubicin to the lipid material is 1:3-1:50, preferably 1:6-15.

[0032] The mass volume percentages of the components in the vorinostat-doxorubicin dual-drug liposomes are as follows: vorinostat-cholesterol prodrug accounts for 3-10%, preferably 3-6%; doxorubicin accounts for 3-10%, preferably 3-6%; lipid material accounts for 50-80%, preferably 70-80%, and the rest is water.

[0033] The composition of the lipid material is the same as that of the lipid material in the vorinostat-cholesterol prodrug liposomes.

[0034] The present invention further provides a method for preparing vorinostat-doxorubicin dual-drug loaded liposomes, comprising the following steps:

[0035] Vorinostat-cholesterol prodrug liposomes were prepared according to the previous method.

[0036] Add doxorubicin hydrochloride dissolved in deionized water to the vorinostat-cholesterol prodrug liposomes prepared above, continue incubation for drug encapsulation, and then cool to obtain vorinostat-doxorubicin dual-drug liposomes Lipo-Co.

[0037] Furthermore, in the vorinostat-doxorubicin dual-drug loaded liposomes, the molar ratio of doxorubicin to vorinostat-cholesterol prodrug is 10:1-1:2, preferably 4-5:1.

[0038] Furthermore, the incubation condition of the above step is 50-70° C. for 10-200 min.

[0039] Furthermore, the present invention provides the use of the above-mentioned vorinostat-cholesterol prodrug liposomes and vorinostat-doxorubicin dual-drug liposomes, both liposomes have anti-tumor effects, among which the vorinostat-doxorubicin dual-drug liposomes exert a highly efficient synergistic anti-tumor effect of epigenetic regulation to aggravate genotoxicity.

[0040] Beneficial effects of the present invention:

[0041] (1) The present invention first designs and synthesizes a vorinostat-cholesterol prodrug, which can be embedded in the phospholipid bilayer membrane structure of the liposome. In addition, the prodrug liposome can also encapsulate doxorubicin hydrochloride by an active drug loading method to construct a vorinostat-doxorubicin dual-drug loaded liposome. In the vorinostat-cholesterol prodrug and vorinostat-doxorubicin dual-drug loaded liposomes of the present invention, the encapsulation rate of the vorinostat-cholesterol prodrug can reach more than 75%, and the encapsulation rate of doxorubicin can reach more than 98%. Compared with the traditional drug co-delivery nanoplatform, the dual-drug loaded liposomes of the present invention can not only better exert the effects of vorinostat and doxorubicin under certain proportion conditions, but also the combination of the two drugs can achieve the best synergistic effect, which is significantly better than the effect of using vorinostat and doxorubicin separately, and is also better than the sum of the effects of the two. The particle size of the two liposomes is between 100-150nm, PDI<0.2, and both are negatively charged.

[0042] (2) The prodrug liposome membrane structure of the present invention contains a disulfide-bridged prodrug, and the disulfide bond can respond to the highly expressed redox conditions in the tumor microenvironment, specifically cleave and release vorinostat. At the same time, the cleavage of the disulfide bond also triggers the destruction of the liposome membrane structure, promoting the release of doxorubicin in the inner water phase, thereby effectively solving the problem of slow release of doxorubicin liposome drugs.

[0043] (3) The vorinostat-doxorubicin dual-drug liposomes constructed by the present invention can achieve efficient combined delivery of doxorubicin and vorinostat, and the two have a significant synergistic effect in terms of mechanism of action. Doxorubicin exerts cytotoxicity by inducing DNA double-strand breaks, while vorinostat, as an epigenetic regulator, can inhibit the DNA repair mechanism of tumor cells, further aggravating DNA damage, thereby significantly enhancing the anti-tumor effect. The present invention not only solves the difficulties of doxorubicin liposomes in drug release and DNA damage repair, but also provides a promising new anti-tumor drug preparation product for clinical transformation and application. Description of the drawings:

[0044] Figure 1 It is the mass spectrum of vorinostat-cholesterol prodrug (SAHA-SS-CST) of Example 1 of the present invention.

[0045] Figure 2 This is the particle size distribution diagram of the prodrug liposomes of Example 3 of the present invention.

[0046] Figure 3 This is a transmission electron micrograph of the prodrug liposome of Example 3 of the present invention.

[0047] Figure 4 This is the particle size change curve of the prodrug liposomes of Example 4 of the present invention during the incubation process.

[0048] Figure 5 This is the in vitro drug release curve of the prodrug liposomes of Example 5 of the present invention.

[0049] Figure 6 This is a confocal immunofluorescence image of γH2AX in cells after treatment with the prodrug liposomes of Example 7 of the present invention.

[0050] Figure 7 This is a diagram showing the quantitative results of the fluorescence intensity of the confocal immunofluorescence image of γH2AX cells after treatment with the prodrug liposomes of Example 7 of the present invention.

[0051] P < 0.05 was considered to be significantly different (one-way analysis of variance).

[0052] Figure 8 This is a graph showing the changes in mouse body weight during the in vivo anti-tumor experiment of the prodrug liposomes of Example 8 of the present invention.

[0053] Fig. 9 This is a graph showing the changes in the mouse tumor growth curve in the in vivo anti-tumor experiment of the prodrug liposomes of Example 8 of the present invention.

[0054] P < 0.05 was considered to be significantly different (one-way analysis of variance).

[0055] Fig.10 This is a picture of mouse tumors in the in vivo anti-tumor experiment of the prodrug liposomes of Example 8 of the present invention.

[0056] Fig.11 This is a graph showing the tumor-bearing rate of mice in the in vivo anti-tumor experiment of the prodrug liposomes of Example 8 of the present invention.

[0057] P < 0.05 was considered to be significantly different (one-way analysis of variance).

[0058] Fig.12 These are the liver and kidney function indicators of mice treated with the prodrug liposomes of Example 9 of the present invention.

[0059] P < 0.05 was considered to be significantly different (one-way analysis of variance), n, s, no significant difference.

[0060] Fig.13 This is a staining diagram of key marker molecules in tumor tissue after prodrug liposome treatment according to Example 10 of the present invention.

[0061] Fig.14 This is a quantitative result diagram of TUNEL staining of tumors after prodrug liposome treatment in Example 10 of the present invention.

[0062] P < 0.05 was considered to be significantly different (one-way analysis of variance).

[0063] Fig.15This is a diagram showing the quantitative results of Ki67 immunofluorescence staining of tumors after prodrug liposome treatment according to Example 10 of the present invention.

[0064] P < 0.05 was considered to be significantly different (one-way analysis of variance).

[0065] Fig.16 This is a diagram showing the quantitative results of tumor γH2AX immunofluorescence staining after prodrug liposome treatment in Example 10 of the present invention.

[0066] P < 0.05 was considered to be significantly different (one-way analysis of variance). Specific implementation method:

[0067] The present invention is further described in detail below in conjunction with embodiments.

[0068] Example 1: Synthesis of Vorinostat-Cholesterol Prodrug

[0069] Weigh 2.38g of dithiodibutyric acid (10mmol), add 5mL of acetic anhydride, and stir at room temperature for 2h until completely clear. Remove acetic anhydride by rotary evaporation. Weigh 1.93g of cholesterol (5mmol) and 0.3g of DMAP, add 20mL of dichloromethane to dissolve, stir under ice bath, redissolve the product of the previous step in 5mL of dichloromethane, add dropwise to the reaction system, warm to room temperature and stir overnight, separate by silica gel column chromatography, and obtain the intermediate CST-SS-COOH. Weigh 1.52g of CST-SS-COOH (2.5mmol), 0.96g of EDCI (5mmol) and 0.15g of DMAP (1.25mmol), add 20mL of dichloromethane to dissolve, and stir and activate under ice bath for 2h. Then, 1 g of vorinostat (3.75 mmol) was weighed and added to the reaction system. The temperature was raised to room temperature and stirred for reaction overnight. The system was purified by preparative liquid phase separation to obtain the final product vorinostat-cholesterol prodrug SAHA-SS-CST.

[0070] Mass spectrometry and nuclear magnetic resonance spectroscopy were used to determine the structure of the prodrug in Example 1. The mass spectrometry results are as follows: Figure 1 The spectrum analysis results are as follows:

[0071] 1H NMR(600MHz,Chloroform-d)δ9.15(s,1H,Ar-NH-CO),7.52(d,J=8.0Hz,2H,ArH),7.32(s,3H,ArH),7.10(t,J=7.9Hz,1H,-CO-NH -O),5.37(d,J=5.0Hz,1H,-C=CH-),4.61(ddt,J=11.6,8.2,4.5Hz,1H,-CH-O-CO-),2.73(dt,J=23.3,7.1Hz,4H,-CH2-SS-),2.67 -2.59(m,4H,-CH2-CO-),2.44-2.21(m,9H,-CH2-),2.10(p,J=7.1Hz,2H,-CH2-),2.06-1.91(m,3H,-CH2-),1.91-1.66(m,5H,-C H2-),1.66-1.21(m,12H,-CH2-),1.20-0.93(m,13H,CST-CH2-),0.93-0.83(m,12H,-CH3),0.68(s,3H,-CH3).MS(ESI)m / z:Calcd forC 49 H 76 N2O6S2,852.5145; found,853.5208[M+H] + and 875.5050[M+Na] + .

[0072] Example 2: Screening of the optimal combination ratio of vorinostat and doxorubicin

[0073] In order to determine the optimal combination ratio of vorinostat (SAHA) and doxorubicin (DOX), this experiment selected mouse triple-negative breast cancer 4T1 cells, human triple-negative breast cancer MBA-MD-231 cells and mouse melanoma B16F10 cells as models, and investigated the mixed solutions of vorinostat and doxorubicin with different molar ratios. The specific operation is as follows: prepare the mixed solutions of Dox:SAHA=10:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:10, and measure the synergistic toxicity of the mixed solutions of different molar ratios on 4T1, MBA-MD-231 and B16F10. In addition, the toxicity of Dox and SAHA on 4T1, MBA-MD-231 and B16F10 cell models was measured as a reference. The half inhibitory concentration (IC 50 ) under the combined drug index (CI 50 ), the calculation formula is as follows:

[0074]

[0075] IC at each ratio 50 With CI 50 The values ​​are shown in Table 1. 50 If the value is <1, it means that there is a synergistic effect between the two drugs. 50 The smaller the value, the better the synergistic effect of the two drugs. The results showed that for 4T1, when the molar ratio of doxorubicin to vorinostat was 10:1-4:1 and 2:1, CI 50 When the value is <1, the two drugs have synergistic effect, and the synergistic effect is strongest when the ratio is 4:1. For MBA-MD-231, when the molar ratio of doxorubicin to vorinostat is 5:1-3:1 and 1:1-1:2, CI 50 When the molar ratio of doxorubicin to vorinostat is 5:1-4:1, the CI 50 When the value is <1, the two drugs have a synergistic effect, and the synergistic effect is strongest when the ratio is 4:1.

[0076] In summary, when the molar ratio of doxorubicin to vorinostat is 5:1-4:1, both drugs have good synergistic effects against the three tumor cells.

[0077] Table 1. The half inhibitory concentration (IC) of doxorubicin and vorinostat at different molar ratios 50 ) and the combination medication index (CI 50 )

[0078]

[0079]

[0080] a) Mixed solutions were prepared at different molar ratios (DOX:SAHA). b) Half inhibitory concentration (nM).

[0081] Example 3: Preparation and characterization of prodrug liposomes

[0082] Preparation of vorinostat-cholesterol prodrug liposomes: Weigh 44.8 mg HSPC, 29.5 mg cholesterol, 3.8 mg SAHA-SS-CST and 4 mg DSPE-PEG 2kIn a 250mL eggplant-shaped bottle, add a small amount of dichloromethane, dissolve it in a 37°C water bath, and then evaporate it under reduced pressure to form a uniform film at the bottom of the bottle. Place the eggplant-shaped bottle and the inner aqueous phase at 65°C, and add the inner aqueous phase ammonium sulfate solution while shaking the eggplant-shaped bottle. The concentration of the inner aqueous phase is 300mM. Continue shaking until the film at the bottom is completely dissolved. Add the crude liposomes to the liposome extruder and pass the crude liposomes through a 400μm polycarbonate filter. Take an appropriate amount of cross-linked agarose gel and add it to the chromatography column. Take an appropriate volume of liposomes and carefully load them. Use the outer aqueous phase histidine solution as the elution phase. The concentration of the histidine solution is 300mM. When the effluent appears opalescence, start collecting until the effluent has no opalescence and stop collecting. The volume is fixed to 10mL to obtain the vorinostat prodrug liposome Lipo-SAHA.

[0083] Preparation of vorinostat-doxorubicin dual-drug liposomes: 4 mL of the prodrug liposome Lipo-SAHA after replacing the external aqueous phase was measured and stirred in a 65°C water bath. 4 mg of doxorubicin hydrochloride was accurately weighed and dissolved in a small amount of deionized water, added to the stirring system for drug encapsulation to obtain vorinostat-doxorubicin dual-drug liposomes Lipo-Co.

[0084] Characterization of prodrug liposomes: The liposomes were diluted 10 times with deionized water, and the particle size, aggregation index (PDI) and potential were measured using a Malvern particle sizer. Morphological observation of prodrug liposomes: The liposomes were diluted 10 times with pure water to the sample volume, and then dripped onto the electron microscope copper grid and allowed to air dry naturally. 0.25% phosphotungstic acid was added to stain at room temperature in the dark, and the morphological characteristics of the nanoparticles were observed using a transmission electron microscope after standing and air drying.

[0085] Encapsulation efficiency and drug loading of prodrug liposomes: Doxorubicin uses a C18 column, the mobile phase is methanol: diammonium phosphate-acetate buffer (pH 3.0) = 60:40, and the detection wavelength is 480nm; vorinostat-cholesterol prodrug uses a C8 column, the mobile phase is pure acetonitrile, and the detection wavelength is 242nm. The encapsulation efficiency and drug loading of doxorubicin and vorinostat-cholesterol prodrug are calculated according to the following formula:

[0086] Encapsulation rate = drug content in the preparation / total amount of drug added × 100%

[0087] Drug loading = theoretical encapsulated drug amount / (theoretical encapsulated drug amount + lipid weight) × encapsulation rate × 100%

[0088] The particle size distribution of the prepared prodrug liposomes is as follows Figure 2 As shown in Figure 2, all three liposomes are clear and uniform preparations. Figure 3As shown in Table 2, both prodrug liposomes have a uniform spherical morphology. The particle size, dispersion index (PDI) and potential of the prodrug liposomes are shown in Table 2. The particle sizes of Lipo-SAHA and Lipo-Co are between 110-120nm, PDI < 0.15, and both are negatively charged. In Lipo-SAHA, the encapsulation efficiency of vorinostat-cholesterol prodrug is 82.4%, and the calculated drug loading is 3.63%. In Lipo-Co, the encapsulation efficiency of vorinostat-cholesterol prodrug is 79.7%, and the drug loading is 3.19%; the encapsulation efficiency of doxorubicin is 99.5%, and the drug loading is 8.77%.

[0089] Table 2. Characterization of prodrug liposomes

[0090]

[0091] a) Particle size (nm). b) Potential (mV).

[0092] Example 4: Serum stability study of prodrug liposomes

[0093] Commercially available doxorubicin liposomes (Lipo-DOX) were selected as controls. Take three 15 mL centrifuge tubes, add 8 mL PBS (pH 7.4) and 1 mL fetal bovine serum, and add 1 mL Lipo-DOX, Lipo-SAHA and Lipo-Co respectively. Place the centrifuge tube in a 37 ° C constant temperature oscillator for incubation, and take samples at 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h and 96 h, respectively. Use a Malvern particle size analyzer to measure the particle size changes of each nanoparticle to investigate the serum stability of the prodrug nanoparticles.

[0094] The serum stability results of liposomes are shown in Figure 4 As shown, no obvious particle size mutation was observed for Lipo-DOX, Lipo-SAHA and Lipo-Co, indicating that all three liposomes have good serum stability and can be used for intravenous injection.

[0095] Example 5: In vitro release study of prodrug liposomes

[0096] PBS (pH 7.4) containing 30% ethanol was used as the release medium, and a certain amount of dithiothreitol (DTT) was added to make the final concentration of DTT 0, 1 and 10 mM, respectively. 1 mL of Lipo-DOX, Lipo-SAHA and Lipo-Co was added to the dialysis bag and tied tightly, and the dialysis bag was placed in the release medium containing different concentrations of DTT, and incubated at 37°C with shaking. A certain volume of drug-containing release medium was drawn at 2h, 4h, 8h, 12h, 24h and 48h after the start of incubation (and the corresponding volume of fresh release medium was added), and the high performance liquid chromatography was used for detection (sampling n=3), and the content of vorinostat was measured at 242nm, and the content of doxorubicin was measured at 480nm.

[0097] The results of in vitro release Figure 5 As shown, the drug release of the three liposomes did not exceed 10% when incubated under PBS for 48 hours, indicating that there is no burst release of liposomes under normal physiological conditions. There was no significant difference in the release of doxorubicin in Lipo-DOX at different concentrations of DTT and none of them exceeded 10%, which means that Lipo-DOX has no tumor-specific drug release characteristics and can only rely on slow osmotic release of drugs. Liposomes Lipo-SAHA and Lipo-Co containing SAHA-SS-CST prodrugs both showed the ability of DTT concentration-dependent and time-dependent reduction response drug release, and the release rates of vorinostat at 24h and 48h were basically the same. Doxorubicin in Lipo-Co also has DTT concentration-dependent reduction response drug release characteristics, which means that Lipo-Co can maintain stability under physiological conditions and has good safety; it can also specifically and quickly release vorinostat and doxorubicin in the microenvironment of high tumor reduction, overcoming the problem of slow drug release of commercially available doxorubicin liposomes, thereby exerting better therapeutic effects.

[0098] Example 6: Cytotoxicity of Prodrug Liposomes

[0099] The MTT method was used to evaluate the in vitro antitumor ability and normal cell cytotoxicity of liposomes and solutions. 4T1, B16F10, MBA-MD-231 or NIH / 3T3 were seeded into 96-well plates at a density of 1000 per well. After overnight adherent growth, the culture medium was discarded and fresh complete culture medium containing liposomes or solutions with different drug concentration gradients was added. After 48 hours of culture, the culture medium was discarded and 1 mg mL -1 Place the fresh basal medium of thiazolyl blue in a constant temperature electric incubator at 37°C for 3.5-5h, discard the liquid and dry it with filter paper. Add a certain volume of DMSO, shake at room temperature in the dark for 10min, use an enzyme reader to detect the absorbance at 490nm and 570nm, and calculate the half inhibitory concentration (IC 50 ).

[0100] As shown in Table 3, compared with the mixed solution of doxorubicin / vorinostat, Lipo-Co showed slightly poorer cytotoxicity on tumor cell lines (IC 50 The value is about 2-4 times), which may be related to the process that Lipo-Co needs to disintegrate in the cell to release the parent drug before it can exert its efficacy. Compared with Lipo-DOX and Lipo-SAHA, the cytotoxicity of Lipo-Co on tumor cells is 2-3 times that of Lipo-DOX and Lipo-SAHA. On the one hand, the poor toxicity of Lipo-DOX shows the necessity of designing liposomes that are sensitive to stimulus response. On the other hand, the toxicity of Lipo-Co is far superior to that of Lipo-SAHA, which also shows the anti-tumor potential of the combined strategy of inducing DNA damage through DOX and inhibiting DNA damage repair with the epigenetic regulator SAHA. In addition, we measured the cytotoxicity of each preparation on the normal cell line NIH / 3T3 to examine the tumor selectivity and safety of each preparation. The toxicity of Lipo-Co to normal cells is only about 1 / 5 of that of Mixed sol.

[0101] Table 3. Cytotoxicity of each formulation

[0102]

[0103] a) Half inhibitory concentration (nM).

[0104] Example 7: Investigation of DNA damage induced by prodrug liposomes

[0105] 4T1 cells were seeded at a density of 100,000 per well into a 12-well plate containing a 20 mm cell crawler. After overnight growth and attachment, the culture medium was discarded and replaced with fresh culture medium containing no drug (Blank), mixed sol containing parent drug solution, Lipo-DOX, Lipo-SAHA and Lipo-Co (total drug molar concentration: 200 nM), placed in a carbon dioxide incubator for 48 hours, the culture medium was discarded, and washed 3 times with cold PBS (pH 7.4). 4% tissue cells were used for fixation and washed 3 times with cold PBS (pH 7.4). Anti-γH2AX rabbit primary antibody was added to the plate and incubated with cells overnight at 4°C. The primary antibody solution was aspirated and washed 3 times with cold PBS (pH 7.4). Cy3-labeled goat anti-rabbit secondary antibody was added and incubated with cells at room temperature in the dark for 1 hour. The secondary antibody solution was aspirated and washed 3 times with cold PBS (pH 7.4). Add diluted Hoechst 33342 dye solution and stain for 10 minutes in the dark, aspirate the dye solution, and wash 3 times with cold PBS (pH 7.4). Remove the slide and place it on an adhesive slide. Add anti-fluorescence attenuation blocking solution to seal the slide, and observe and take pictures using a laser confocal microscope. Use ImageJ to quantitatively analyze the red fluorescence channel of γH2AX, a marker of DNA double-strand break damage.

[0106] The results are as follows Figure 6 and Figure 7 As shown in the figure, Mixed sol and Lipo-Co treatment significantly upregulated the γH2AX level in tumor cells, and Mixed sol was slightly stronger than Lipo-Co. This may be because Lipo-Co takes time to release the drug, while Mixed sol can act directly after being taken into the cell. Lipo-DOX hardly induced DNA double-strand break damage in the cell model. Combined with the analysis of in vitro drug release experiments and cytotoxicity results, it may be that the excessive stability of Lipo-DOX in tumor cells caused it to release very little drug during the experimental period, and the experimental concentration was far less than its IC 50 Compared with the individual liposomes Lipo-DOX and Lipo-SAHA, Lipo-Co specifically responded in tumor cells, rapidly released a synergistic drug combination, induced stronger DNA double-strand break damage, and had stronger tumor inhibition and killing effects.

[0107] Example 8: In vivo anti-tumor experiment of prodrug liposomes

[0108] 4T1 cells were injected subcutaneously in the lower right back of female Balb / c mice. 3 The mice were divided into five groups: saline group, Mixed sol group, Lipo-DOX group (doxorubicin dose 4.4 μM kg -1) and Lipo-SAHA group (vorinostat dose 4.4 μM kg -1 ), Lipo-Co group (doxorubicin dose 3.3 μM kg-1, vorinostat dose 1.1 μM kg -1 ), the drug was given every 2 days for a total of 4 times. The volume of the mouse tumor (volume = short diameter × short diameter × long diameter / 2) and body weight were measured and recorded every other day. The mice were killed on the 12th day, the tumor was dissected, photographed, weighed, and the tumor-bearing rate was calculated (tumor-bearing rate = tumor weight / body weight × 100%).

[0109] The weight changes of mice Figure 8 As shown in Figure 2, no decrease in the body weight of mice was found after administration of all preparations. The efficacy results are shown in Figure 2. Fig. 9 , Fig.10 and Fig.11 As shown in the figure, although Mixed sol has the strongest inhibitory effect on the tumor cell model, the efficacy of Mixed sol in the animal model is significantly weaker than that of Lipo-Co. This may be due to the non-specific distribution and rapid metabolic clearance of the solution in the body, which seriously restricts its distribution and accumulation in the tumor, thus affecting its therapeutic effect; while Lipo-DOX is just the opposite. The inhibitory effect on the tumor cell model is very weak, but in the animal model, Lipo-DOX significantly slows down tumor growth. Lipo-DOX has excellent systemic circulation stability, which enables it to accumulate and release drugs in the tumor site for a long time, so it can show good therapeutic effects in the animal model. The vorinostat-doxorubicin dual-drug liposome Lipo-Co not only inhibits tumor growth, but also gradually reduces and disappears the tumor volume during the treatment process, showing the strongest tumor inhibition ability and the lowest tumor-bearing rate; when the cytotoxic drug doxorubicin is less than Lipo-DOX, Lipo-Co achieves a more significant anti-tumor effect than Lipo-DOX.

[0110] Example 9: Investigation of the therapeutic safety of prodrug liposomes

[0111] Before killing mice, 3 mice were randomly selected from each group, and the eyeballs were removed to collect blood, and then the mice were killed by cervical dislocation. The blood was collected in a quick-coagulation tube, and the serum on the upper layer of the gel was collected. According to the kit operation, the multifunctional microplate reader was used for determination, and the liver function indicators aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of the mice, and the renal function indicators blood urea nitrogen (BUN) and creatinine (CREA) of the mice were obtained according to the standard curve.

[0112] Quantitative results of liver function indicators such as Fig.12 As shown, there was no significant difference in the liver and kidney function indicators of mice among all groups, and all were within the normal range, which indicates that the prodrug liposomes have good safety during treatment and will not cause additional systemic toxicity.

[0113] Example 10: Characterization of key marker molecules in tumor tissues treated with prodrug liposomes

[0114] Preparation of tumor tissue sections: After the mouse was killed by dislocation, the tumor tissue was removed and fixed in 4% tissue cell fixative for preservation. When slicing, it was taken out, cut into small pieces, wrapped with gauze, and rinsed with running water to remove the fixative. The tissue block was placed in an alcohol solution with increasing concentrations for dehydration. The tissue block was then transparentized and immersed in xylene twice. The tissue block was immersed in paraffin wax 3 times, then embedded in an embedding machine, and the wax block was taken out and trimmed after the wax solidified. The trimmed wax block was fixed on a paraffin slicer and sliced ​​with a thickness of 4μm. After the slices were floated in the warm water of the spreader to flatten the tissue, they were picked up with a patch attached to the thin slice, placed in an oven to bake the slices, and stored at room temperature after the moisture was dried.

[0115] TUNEL staining: After the paraffin sections were dewaxed and hydrated to distilled water, proteinase K working solution was added to cover the tissue, incubated at 37°C, and rinsed 3 times with PBS (pH 7.4). After the sections were slightly dried, membrane permeabilization working solution was added to cover the tissue and incubated at room temperature for 20 minutes, and rinsed 3 times with PBS (pH 7.4). After the sections were slightly dried, 3% hydrogen peroxide solution was added, incubated for 15 minutes, and rinsed 3 times with PBS (pH 7.4). TUNEL working solution was prepared according to the number of sections, and the sections were placed flat in a wet box with water, incubated at 37°C for 2 hours, and rinsed 3 times with PBS (pH 7.4). After the sections were slightly dried, the prepared DAPI staining working solution was added, incubated at room temperature in the dark for 10 minutes to counterstain the cell nucleus, and rinsed 3 times with PBS (pH 7.4). After the slices were slightly dried, anti-fluorescence quenching sealing liquid was added to seal the slices, and the slices were photographed under a laser confocal microscope. The green fluorescence channel of the slices was quantified using ImageJ software.

[0116] Immunofluorescence staining: After paraffin sections were dewaxed and hydrated to distilled water, they were immersed in citric acid-sodium citrate solution (pH 6.8) and boiled for 10 minutes, cooled naturally to room temperature, and rinsed 3 times with PBS (pH 7.4). After the sections were slightly dried, 0.3% Triton X-100 in PBS (pH 7.4) was added to break the membrane at room temperature, and rinsed 3 times with PBS (pH 7.4). After the sections were slightly dried, 3% hydrogen peroxide solution was added, incubated for 15 minutes, and rinsed 3 times with PBS (pH 7.4). Rabbit monoclonal antibodies against γH2AX and Ki67 were added to the sections, placed flat in a wet box with water, incubated overnight at 4°C, and rinsed 3 times with PBS (pH 7.4). Cy3-labeled goat anti-rabbit secondary antibodies were added to the sections, incubated at room temperature in the dark for 1 hour, and rinsed 3 times with PBS (pH 7.4). After the slices were slightly dried, the prepared DAPI staining working solution was added, and the cells were incubated at room temperature in the dark for 10 minutes to counterstain the nuclei, and then rinsed three times with PBS (pH 7.4). After the slices were slightly dried, the anti-fluorescence quenching sealing solution was added to seal the slices, and the slices were photographed under a laser confocal microscope, and the red fluorescence channel of the slices was quantified using ImageJ software.

[0117] Staining of various key marker molecules such as Fig.13 As shown in the figure. The results of H&E staining and TUNEL staining showed that the cytoplasm volume was reduced, the nuclear structure was lysed or disappeared, the chromatin was shrunken and broken, and the cells were obviously marginalized in both the Lipo-Co and Lipo-DOX treatment groups, indicating that most cells in the tumor tissue underwent significant apoptosis, and the phenomenon of Lipo-Co was more obvious. The quantitative results of TUNEL staining are shown in the figure. Fig.14 As shown in the figure, most cells in the tumor tissue treated with Lipo-Co showed obvious green fluorescence, and the fluorescence intensity was significantly stronger than that of doxorubicin liposome Lipo-DOX. The results of H&E staining and TUNEL staining showed that the prodrug-doxorubicin co-loaded liposomes induced more cell apoptosis in tumor tissue, which also explains why Lipo-Co has the most significant tumor inhibitory effect. The quantitative staining results of Ki67 are shown in the figure. Fig.15 As shown in the figure, the Ki67 level in tumor tissues was significantly downregulated after Lipo-Co treatment, which was lower than that in the Lipo-DOX group, indicating that Lipo-Co treatment had the most significant inhibitory effect on the proliferation of tumor cells. Although Mixed sol was slightly stronger than Lipo-SAHA in inhibiting tumor growth, there was no significant difference between the two in TUNEL staining and Ki67 indicators. The quantitative results of γH2AX are shown in Fig.16 As shown, Lipo-Co has the strongest γH2AX fluorescence intensity, indicating that it induces the strongest DNA double-strand break damage.

[0118] The above results show that the vorinostat-doxorubicin dual-drug liposomes have successfully overcome the problem of slow drug release of commercially available doxorubicin liposomes. At the same time, compared with commercially available doxorubicin liposomes, they increase the synergistic mechanism of DNA damage through epigenetic regulation, achieving more efficient anti-tumor effects while reducing the dosage of toxic drugs, providing a new strategy for anti-tumor combination therapy.

Claims

1. Vorinostat-cholesterol prodrug or a pharmaceutically acceptable salt thereof:

2. Vorinostat-cholesterol prodrug liposomes, characterized in that It comprises the vorinostat-cholesterol prodrug according to claim 1 and a lipid material, wherein the mass ratio of the vorinostat-cholesterol prodrug to the lipid material is 1:10-1:50, preferably 1:15-1:25; and the lipid material comprises phospholipids, cholesterol, and PEGylated phospholipids.

3. The vorinostat-cholesterol prodrug liposome according to claim 2, characterized in that: The mass volume percentage of each component in the vorinostat-cholesterol prodrug liposome is as follows: vorinostat-cholesterol prodrug accounts for 3-10%, preferably 3-6%; lipid material accounts for 50%-80%, preferably 70-80%, and the rest is water.

4. A pharmaceutical composition, characterized in that It comprises the vorinostat-cholesterol prodrug according to claim 1 and doxorubicin, wherein the molar ratio of doxorubicin to vorinostat is 10:1-1:2, preferably 4-5:

1.

5. Vorinostat-doxorubicin dual drug-loaded liposomes, characterized in that: The dual-drug liposomes comprise the vorinostat-cholesterol prodrug according to claim 1, doxorubicin, and lipid materials, wherein the molar ratio of doxorubicin to vorinostat-cholesterol prodrug is 10:1-1:2, preferably 4-5:1; the lipid materials comprise phospholipids, cholesterol, and PEGylated phospholipids.

6. The vorinostat-doxorubicin dual drug-loaded liposome according to claim 5, characterized in that: The mass volume percentages of each component in the vorinostat-doxorubicin dual-drug liposome are as follows: vorinostat-cholesterol prodrug accounts for 3-10%, preferably 3-6%; doxorubicin accounts for 3-10%, preferably 3-6%; lipid material accounts for 50-80%, preferably 70-80%, and the rest is water.

7. The vorinostat-doxorubicin dual drug-loaded liposome according to claim 5, characterized in that: The mass ratio of the total mass of vorinostat-cholesterol prodrug and doxorubicin to the mass ratio of the lipid material is 1:3-1:50, preferably 1:6-15.

8. The vorinostat-cholesterol prodrug liposome according to claim 2 or the vorinostat-doxorubicin dual drug-loaded liposome according to claim 4, characterized in that: The mass ratio of the phospholipid, cholesterol and PEGylated phospholipid is 1-100:1-50:1-50, preferably 30-80:20-50:1-20, and more preferably 40-50:25-35:3-6.

9. The method for preparing the vorinostat-cholesterol prodrug liposome according to claim 4, characterized in that: (1) Weigh phospholipids, cholesterol, PEGylated phospholipids and vorinostat-cholesterol prodrug into an eggplant-shaped bottle, add a small amount of volatile organic solvent, and remove the organic solvent by vacuum rotary evaporation to form a uniform drug-containing lipid film at the bottom of the bottle; (2) adding the inner aqueous phase for preheating, then adding the inner aqueous phase dropwise while shaking the eggplant-shaped bottle, and continuously shaking until the thin film at the bottom is completely dissolved and hydrated, using an extrusion method to perform granulation to obtain nano-sized liposomes, and then using an agarose gel column to perform external aqueous phase exchange to obtain vorinostat-cholesterol prodrug liposomes; (3) adding doxorubicin hydrochloride dissolved in deionized water to the vorinostat-cholesterol prodrug liposomes prepared above, continuing to incubate for drug encapsulation, and then cooling to obtain vorinostat-doxorubicin dual-drug liposomes; In the step (2), the inner aqueous phase is ammonium sulfate solution, citrate buffer, phosphate buffer or acetate buffer, preferably ammonium sulfate solution; and the outer aqueous phase is histidine solution.

10. Use of the vorinostat-cholesterol prodrug or a pharmaceutically acceptable salt thereof according to claim 1, the vorinostat-cholesterol prodrug liposome according to claim 2 or 3, the pharmaceutical composition according to claim 4, or the vorinostat-doxorubicin dual-drug liposome according to claim 5 or 6 in the preparation of anti-tumor drugs.

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