A liposome of a carbamate prodrug, its preparation method and application

Abstract

This invention discloses a cabazitaxel prodrug liposome, its preparation method, and its application, belonging to the field of novel excipients and dosage forms for pharmaceutical formulations. This invention designs and synthesizes a cabazitaxel prodrug with a structure similar to phospholipids, linking cabazitaxel to a branched-chain fatty alcohol with a similar structure to phospholipids. Furthermore, tumor microenvironment-sensitive bridging disulfide bonds are introduced, and cabazitaxel is passively loaded onto the liposome membrane. The prepared prodrug liposomes have small particle size, uniform distribution, high encapsulation efficiency, and high stability. The preparation method is simple and highly feasible. The intelligent responsive prodrug liposome strategy designed in this invention can improve the delivery efficiency of chemotherapeutic drugs, potentially increasing the tolerable dose of chemotherapeutic drugs and enhancing their antitumor effects, providing a new direction and approach for chemotherapeutic drug delivery.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to a carbazide predrug liposome, its preparation method, and its application. Background Technology

[0002] Currently, cancer treatment strategies mainly include surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. Chemotherapy is a crucial method in cancer treatment, especially important for tumors that cannot be surgically removed or have metastasized. Taxanes are among the most commonly used chemotherapy drugs in clinical practice and have been widely used to treat cancers such as lung cancer, breast cancer, and ovarian cancer. Cabazitaxel (CTX), a second-generation taxane, exhibits stronger cytotoxicity against tumor cells. Furthermore, CTX reduces the sensitivity of P-glycoprotein, which is beneficial in overcoming tumor resistance. Studies have shown that cabazitaxel not only inhibits the growth of prostate cancer cells by affecting microtubule function but also inhibits androgen receptor (AR) function, AR expression, and AR-related heat shock proteins. In prostate cancer cells, cabazitaxel is 10 times more effective than docetaxel. In vivo experiments have shown that cabazitaxel exhibits more effective anti-tumor effects in both docetaxel and paclitaxel-resistant tumor models.

[0003] Jevtana was the first cabazitaxel formulation developed by Sanofi-Aventis. In 2010, Jevtana was approved by the FDA for the treatment of prostate cancer that had developed resistance to docetaxel. However, due to the extremely poor solubility of cabazitaxel in water, the Jevtana formulation must use Tween 80 and ethanol as solubilizers, which may lead to hemolysis, allergic reactions, and other side effects, further reducing the tolerable dose of cabazitaxel. Even with the help of solubilizers to improve drug solubility, the resulting solution has poor stability and is prone to precipitation after dilution, resulting in undesirable drug formulation properties that affect usability. Simultaneously, the pharmacokinetic properties of the solution are poor, with a short half-life, non-targeted distribution in vivo, and poor delivery efficiency. Most importantly, non-specific distribution of cabazitaxel can cause serious adverse reactions, including gastrointestinal disturbances, allergic reactions, renal failure, and neutropenia. Clinical trial results indicate that the maximum tolerated dose of cabazitaxel is 25 mg / m². 2 The efficacy of cabazitaxel is significantly lower than that of the other two classes of taxane antitumor drugs. These drawbacks limit the clinical application of cabazitaxel. Furthermore, since Jevtana received FDA approval in 2010, the number of existing cabazitaxel delivery systems is relatively small compared to paclitaxel and docetaxel; therefore, developing efficient cabazitaxel delivery strategies is of great importance.

[0004] Nanotechnology can enhance the efficacy of anticancer treatments by increasing drug distribution, imparting drug targeting, and reducing exposure. Among them, liposomes are the earliest nanotechnology to be used clinically. They are spherical particles containing single or multiple vesicles composed of a phospholipid bilayer membrane. They have advantages such as good biocompatibility, biodegradability, high delivery efficiency, and reduced side effects, and have been widely used as drug delivery carriers.

[0005] Due to the unique structure of the phospholipid bilayer in liposomes, hydrophilic drugs can be encapsulated in a water core, while lipophilic drugs can be encapsulated within the lipid bilayer. Cabazitaxel is a lipophilic drug and therefore can be encapsulated within the liposome bilayer. However, cabazitaxel has poor affinity for phospholipids, resulting in low encapsulation efficiency and poor stability. Summary of the Invention

[0006] The purpose of this invention is to overcome the aforementioned deficiencies in the existing technology and provide a cabazitaxel prodrug liposome, its preparation method, and its application. This invention designs and synthesizes a cabazitaxel prodrug with a structure similar to phospholipids, links cabazitaxel to a branched-chain fatty alcohol with a structure similar to phospholipids, and further introduces tumor microenvironment-sensitive bridging disulfide bonds. Cabazitaxel prodrug liposomes are prepared by passively loading cabazitaxel onto the bilayer membrane of the liposome. The intelligent responsive prodrug liposome strategy designed in this invention can improve the delivery efficiency of chemotherapeutic drugs, potentially increasing the tolerable dose of chemotherapeutic drugs and enhancing their antitumor effects, providing a new direction and approach for chemotherapeutic drug delivery.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] A carbacino pre-race drug, the structural formula of which is as follows:

[0009]

[0010] This invention also discloses a method for synthesizing the above-mentioned carbamazepine pre-race drug, comprising the following steps:

[0011] (1) A dicarboxylic acid is dehydrated by acetic anhydride to obtain a dicarboxylic acid anhydride; the dicarboxylic acid is 2,2'-dithiodiacetic acid;

[0012] (2) The fatty alcohol undergoes a ring-opening esterification reaction with the diacid anhydride under the action of a catalyst to obtain an intermediate product; the fatty alcohol is octyl lauryl alcohol;

[0013] (3) The intermediate product undergoes an esterification reaction with cabazitaxel under the action of a catalyst to obtain the cabazitaxel prodrug; the reaction equation is as follows:

[0014]

[0015] The present invention also discloses a carbappa pre-race drug liposome, wherein the carbappa pre-race drug liposome is prepared by passive drug delivery of carbappa pre-race drug; wherein the carbappa pre-race drug is the carbappa pre-race drug as described above, or the carbappa pre-race drug obtained by the above synthesis method.

[0016] This invention also discloses a method for preparing the above-described carbamazepine predrug liposome, comprising the following steps:

[0017] (1) Dissolve the carbacco pre-race drug and lipid in an organic solvent and mix well to obtain carbacco pre-race drug mother liquor and lipid mother liquor respectively. Mix the carbacco pre-race drug mother liquor and the lipid mother liquor, and remove the organic solvent by rotary evaporation under reduced pressure until a film is formed.

[0018] (2) Add an aqueous medium to the film and incubate at a constant temperature to obtain crude cabazitaxel liposomes;

[0019] (3) Homogenize the crude carbamate pre-race drug liposome to obtain the carbamate pre-race drug liposome.

[0020] The present invention also discloses the application of the above-mentioned carbamazepine pre-race drug or the above-mentioned carbamazepine pre-race drug liposome in a drug delivery system.

[0021] The present invention also discloses the use of the above-mentioned carbamazepine predrug or the above-mentioned carbamazepine predrug liposome in the preparation of antitumor drugs.

[0022] The present invention also discloses the application of the above-mentioned carbamazepine prodrug or the above-mentioned carbamazepine prodrug liposome in an injection, oral or topical administration system.

[0023] Implementing the embodiments of the present invention will have the following beneficial effects:

[0024] (1) This invention is the first to design and synthesize a cabazitaxel prodrug with a structure similar to phospholipids. The synthesis method is simple and highly reproducible. By linking cabazitaxel with a branched fatty alcohol with a structure similar to phospholipids, the affinity between the drug and phospholipids can be improved, the encapsulation efficiency and stability of the drug can be improved, the exposure of the drug in normal tissues can be effectively reduced, and the delivery efficiency is high. At the same time, based on the special high redox microenvironment naturally formed in the tumor site, we further introduced tumor microenvironment sensitive bridging-disulfide bonds, which enable the designed prodrug to intelligently respond to the high redox microenvironment naturally formed in the tumor site for drug release, further improving the safety of the drug and providing a new strategy for low-toxicity and high-efficiency chemotherapy drug delivery.

[0025] (2) In this invention, cabazitaxel is loaded onto the membrane of liposomes by passive drug delivery. The resulting cabazitaxel prodrug liposomes have small particle size, uniform distribution, high encapsulation efficiency, and high stability. At the same time, the preparation method is simple and highly feasible.

[0026] (3) This invention investigated the formulation properties and in vitro and in vivo antitumor activity of cabazitaxel prodrug liposomes. The results showed that cabazitaxel prodrug liposomes exhibited good storage stability and good plasma chemical stability, good safety profile, and intelligent response drug release, demonstrating good in vitro and in vivo antitumor activity. In terms of efficacy and safety, cabazitaxel prodrug liposomes have certain advantages.

[0027] In summary, the intelligent responsive prodrug liposome strategy designed in this invention can improve the delivery efficiency of chemotherapeutic drugs, potentially increase the tolerable dose of chemotherapeutic drugs, and enhance the antitumor effect of chemotherapeutic drugs. It provides a new direction and new ideas for the delivery of chemotherapeutic drugs, meets the urgent clinical need for highly effective and low-toxicity chemotherapeutic drugs, promotes the application of prodrug liposome technology in tumor treatment, and provides new ideas and new choices for developing highly effective and low-toxicity chemotherapeutic drug delivery strategies. Attached Figure Description

[0028] Figure 1 This is the mass spectrum of the carbamate prodrug in Example 1 of the present invention.

[0029] Figure 2 The carbazine pre-race drug in Example 1 of this invention 1 H-NMR spectrum.

[0030] Figure 3 This is a purity diagram of the carbamate prodrug liposomes in Example 1 of the present invention.

[0031] Figure 4 This is a transmission electron microscope image of the carbamate prodrug liposome in Example 2 of the present invention.

[0032] Figure 5 This is a graph showing the particle size variation of the carbamate prodrug liposomes in Examples 2, 3, and 4 of the present invention, indicating their stability at room temperature and 4°C.

[0033] Figure 6 The images show the hemolysis test results of the carbamate prodrug liposomes and carbamate solution in Examples 2, 3, and 4 of this invention.

[0034] Figure 7 The diagram shows the in vitro release test of the carbamate prodrug liposome in Examples 2, 3, and 4 of this invention.

[0035] Figure 8 The image shows the rat plasma chemical stability of the carbamate prodrug liposomes in Examples 2, 3, and 4 of this invention.

[0036] Figure 9 The images show the cytotoxicity of the carbazide prodrug liposomes and carbazide solution in Examples 2, 3, and 4 of this invention.

[0037] Figure 10 The graphs show the changes in tumor volume during in vivo antitumor experiments of the carbazide prodrug liposomes and carbazide solution in Examples 2, 3, and 4 of this invention (two-sided t-test: *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001).

[0038] Figure 11 This is a comparison of tumor burden rates in in vivo antitumor experiments of the carbamate prodrug liposomes and carbamate solution in Examples 2, 3, and 4 of the present invention (two-sided t-test: *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001).

[0039] Figure 12 The graphs show the changes in body weight during in vivo antitumor experiments of the liposomes and solution of the carbazide prodrug in Examples 2, 3, and 4 of this invention (two-sided t-test: *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001). Detailed Implementation

[0040] The present invention will be further described below with reference to specific embodiments, but this does not limit the present invention in any way.

[0041] This invention discloses a carbacino pre-race drug, the structural formula of which is as follows:

[0042]

[0043] Specifically, this invention addresses the drawbacks of cabazitaxel's poor affinity for phospholipids in liposomes, leading to low encapsulation efficiency and poor stability. To address these issues, a cabazitaxel prodrug with a structure similar to phospholipids was designed and synthesized. By linking a branched fatty alcohol with a similar structure to the phospholipid tail chain, the affinity between the drug and lipids is improved, enhancing drug loading and stability, resulting in favorable formulation properties and in vitro / in vivo antitumor effects. Furthermore, leveraging the unique high redox microenvironment naturally occurring at tumor sites, a tumor microenvironment-sensitive bridging mechanism—disulfide bonds—is introduced. This enables the designed prodrug to intelligently respond to the high redox microenvironment naturally occurring at tumor sites, further improving drug safety and representing a novel strategy for low-toxicity and highly efficient chemotherapy drug delivery.

[0044] Furthermore, the present invention also discloses a method for synthesizing a carbamazepine pre-race drug as described in any embodiment of the present invention, comprising the following steps:

[0045] (1) The dicarboxylic acid is dehydrated by acetic anhydride to obtain a dicarboxylic acid anhydride; the dicarboxylic acid is 2,2'-dithiodiacetic acid.

[0046] In one specific embodiment, step (1) specifically includes: dissolving the dicarboxylic acid in acetic anhydride, stirring at room temperature for 1 h to 4 h under nitrogen protection to react the dicarboxylic acid into dicarboxylic anhydride, and after the reaction is complete, adding toluene, and removing toluene and acetic anhydride by rotary evaporation under reduced pressure in a water bath at 30 °C to 32 °C.

[0047] In one specific embodiment, the molar ratio of the dicarboxylic acid to acetic anhydride is 1:(1-50).

[0048] In one specific embodiment, toluene is added in batches, wherein the total volume ratio of acetic anhydride to toluene is 1:(1-10).

[0049] (2) Fatty alcohols undergo ring-opening esterification with dicarboxylic acid anhydrides under the action of a catalyst to obtain intermediate products; the fatty alcohol is octyl lauryl alcohol.

[0050] In one specific embodiment, step (2) specifically includes: taking a fatty alcohol and a catalyst, dissolving them together with a dicarboxylic acid anhydride in dichloromethane, and carrying out a ring-opening esterification reaction at room temperature for 8 to 12 hours, and obtaining the intermediate product octyldodecyl dithiodiacetic acid monoester by separation by a chromatography column.

[0051] In one specific embodiment, the catalyst is 4-dimethylaminopyridine (DMAP).

[0052] In one specific embodiment, the molar ratio of catalyst, fatty alcohol and diacid anhydride is 1:(1-10):(1-15).

[0053] In one specific embodiment, the reaction in step (2) is carried out under nitrogen protection conditions.

[0054] In one specific embodiment, the eluent includes cyclohexane, ethyl acetate, and glacial acetic acid.

[0055] (3) The intermediate product undergoes an esterification reaction with cabazitaxel under the action of a catalyst to obtain the cabazitaxel prodrug.

[0056] In one specific embodiment, step (3) specifically includes: taking the catalyst and dissolving it together with the intermediate product in anhydrous dichloromethane, activating it under nitrogen protection and ice bath conditions for 0.5h to 4h, adding the cabazitaxel dissolved in dichloromethane and reacting it at room temperature for 24h to 48h, and then purifying it by liquid phase separation to obtain the cabazitaxel prodrug.

[0057] In one specific embodiment, the catalyst comprises one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt), 4-dimethylaminopyridine (DMAP), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), N,N-diisopropylethylamine (DIPEA), and p-toluenesulfonic acid.

[0058] In one specific embodiment, the molar ratio of catalyst, cabazitaxel, and intermediate product is 1:(0.1-5):(0.1-5).

[0059] In one specific embodiment, a mobile phase consisting of acetonitrile, methanol, and water was used for the separation and purification of the product.

[0060] The reaction equation is as follows:

[0061]

[0062] Furthermore, this invention addresses the issue of cabazitaxel's poor water solubility and the lack of ionizable groups near physiological pH, making active drug loading difficult. Therefore, this invention designs a passive drug loading method to encapsulate cabazitaxel in a lipid bilayer. Specifically, this invention discloses a cabazitaxel liposome, which is prepared by passively loading cabazitaxel. The cabazitaxel is the cabazitaxel as described in any embodiment of this invention, or the cabazitaxel obtained by the synthesis method described in any embodiment of this invention.

[0063] Furthermore, the present invention also discloses a method for preparing carbamazepine pre-race drug liposomes as described in any embodiment of the present invention, comprising the following steps:

[0064] (1) Dissolve the carbamazepine and lipid in an organic solvent and mix well to obtain the carbamazepine mother liquor and lipid mother liquor respectively. Mix the carbamazepine mother liquor and lipid mother liquor and remove the organic solvent by rotary evaporation under reduced pressure until a film is formed.

[0065] In one specific embodiment, the lipids are phospholipids, cholesterol, and PEG-modified lipids. Specifically, by adding PEG-modified lipids, the present invention can reduce complement adsorption, decrease phagocytosis by mononuclear macrophages, prolong the in vivo circulation time of liposomes, and further improve drug delivery efficiency.

[0066] In one specific embodiment, the phospholipid includes one or more of the following: lecithin, hydrogenated soybean lecithin (HSPC), distearylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), dioleoyllecithin (DOPC), disqualoylphosphatidylcholine (DEPC), distearylphosphatidylethanolamine (DSPE), and dioleoylphosphatidylethanolamine (DOPE).

[0067] In one specific embodiment, the PEG modifier is an amphiphilic polymer.

[0068] In one specific embodiment, the PEG modifier includes one or more of the following: distearate phosphatidylethanolamine-methoxy polyethylene glycol (DSPE-mPEG), distearate phosphatidylethanolamine-polyethylene glycol-folic acid (DSPE-PEG-FA), vitamin E polyethylene glycol succinate (TPGS), and cholesterol-PEG (CH-PEG).

[0069] In one specific embodiment, the molecular weight of the PEG modifier is 1000-5000.

[0070] In one specific embodiment, the molar ratio of cabazitaxel, phospholipids, cholesterol, and PEG modifier is 1:(1-100):(0-40):(0-20).

[0071] In one specific embodiment, the organic solvent includes one or more of dichloromethane, chloroform, methanol, and ethanol.

[0072] (2) Add an aqueous medium to the membrane and incubate at a constant temperature to obtain crude carbamate liposomes.

[0073] In one specific embodiment, the water-soluble medium includes purified water, a 0.9% NaCl solution, a PBS solution, and a 5% glucose solution.

[0074] In one specific embodiment, the constant temperature incubation temperature is 5°C to 20°C higher than the phase transition temperature.

[0075] In one specific embodiment, the incubation time at a constant temperature is 10 min to 60 min.

[0076] (3) Homogenize the crude carbamate pre-race drug liposomes to obtain carbamate pre-race drug liposomes.

[0077] In one specific embodiment, homogenization includes one or more of ultrasonication, high-pressure homogenization, and extrusion.

[0078] In one specific embodiment, the ultrasonic power is 10W to 500W; the ultrasonic time is 1min to 30min.

[0079] In one specific embodiment, the particle size of the carbamate prodrug liposomes is 50 nm to 200 nm.

[0080] In one specific embodiment, the particle size of the carbamazepine liposomes includes, but is not limited to, 50 nm, 80 nm, 100 nm, 120 nm, 150 nm, and 200 nm.

[0081] Specifically, this invention uses a passive drug delivery method to encapsulate cabazitaxel onto the membrane of liposomes. The resulting cabazitaxel prodrug liposomes have small particle size, uniform distribution, high encapsulation efficiency, and high stability. At the same time, the preparation method is simple and highly feasible.

[0082] In one specific embodiment, the preparation can be carried out using solvent injection, reverse evaporation, microfluidic injection, secondary emulsification, cross-flow injection, organic solvent freeze-drying, spray drying, fluidized bed coating, single-phase solution freeze-drying, or supercritical fluid method.

[0083] Furthermore, the present invention also discloses the application of a carbamazepine pre-race drug as described in any embodiment of the present invention or a carbamazepine pre-race drug liposome as described in any embodiment of the present invention in a drug delivery system.

[0084] Furthermore, the present invention also discloses the use of a carbamazepine pre-race drug as described in any embodiment of the present invention or a carbamazepine pre-race drug liposome as described in any embodiment of the present invention in the preparation of antitumor drugs.

[0085] Furthermore, the present invention also discloses the use of a carbamazepine pre-race drug as described in any embodiment of the present invention or a carbamazepine pre-race drug liposome as described in any embodiment of the present invention in an injection, oral, or topical drug delivery system.

[0086] The following are specific embodiments.

[0087] Example 1: Synthesis of Cabazitaxel as a pre-race drug

[0088] 1.45 g (8 mmol) of 2,2'-dithiodiacetic acid was weighed and added to 10 mL of acetic anhydride. After complete dissolution, the mixture was stirred at room temperature for 2 h. After the reaction was complete, toluene, an azeotrope, was added in small amounts several times. Toluene and high-boiling acetic anhydride were removed by vacuum distillation using a rotary evaporator to obtain dithiodiacetic anhydride. The dithiodiacetic anhydride formed was dissolved in an appropriate amount of anhydrous dichloromethane, and then 1.19 g (4 mmol) of octyl lauryl alcohol solution dissolved in anhydrous dichloromethane and 4-dimethylaminopyridine (DMAP) were added slowly with stirring. The reaction was carried out at room temperature for 12 h, and the reaction progress was monitored by thin-layer chromatography. After the reaction was complete, crude octyl lauryl alcohol-dithiodiacetic acid monoester was obtained. It was purified by column chromatography using a cyclohexane-ethyl acetate elution system with 0.1% glacial acetic acid. 138.6 mg (0.3 mmol) of octyl lauryl dithiodiacetic acid monoester was weighed and dissolved in dichloromethane. 115 mg (0.6 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 63.6 mg (0.3 mmol) of 1-hydroxybenzotriazole, and 14.7 mg (0.12 mmol) of 4-dimethylaminopyridine dissolved in dichloromethane were added dropwise, and the mixture was activated in an ice bath for 2 h. Then, 208.7 mg (0.25 mmol) of cabazitaxel dissolved in dichloromethane was added, and the reaction was carried out at room temperature for 48 h. After the reaction was complete, the target product was separated and purified using the preparative solution to obtain the cabazitaxel prodrug.

[0089] The structure of the prodrug in Example 1 was determined using mass spectrometry and proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figures 1-2 As shown. The results of the 1H NMR spectrum analysis are as follows:

[0090] 1H-NMR (400MHz, DMSO-d6): δ7.97(d,J=7.2Hz,2H,Ar-H),7.85(d,J=9.2Hz,1H,-NH-),7.74(t,J=7.3Hz,1H,Ar-H) ,7.66(t,J=7.5Hz,2H,Ar-H),7.42(t,J=7.6Hz,2H,Ar-H),7.37(d,J=7.4Hz,2H,Ar-H),7.17(t,J=7.1Hz,1H,Ar-H ),5.81(t,J=8.8Hz,1H,40-H),5.36(d,J=7.1Hz,1H),5.13(d,J=8.0Hz,1H),5.06(t,J=8.5Hz,1H,-OH),4.94(d, J=10.0Hz,1H,10-H),4.68(s,1H,19-H),4.48(s,1H,19-H),4.07–3.94(m,4H,COOCH2SSCH2COO),3.86(s,2H,BA20 side chain OCH2),3.77–3.74(m,1H,11-H),3.73(s,2H,CH2COOCH2SS),3.58(d,J=7.0Hz,1H),3.32(s,3H,14-OCH3),,3.27(s,3H, 10-OCH3),3.20(s,3H),2.24(s,3H,OOCCH3),1.79(s,3H,CH3),1.50(s,2H),1.38(s,9H,C(CH3)3),1.31–1.17(m,32H,H atoms on the BA20 side chain),0.97(d,J=6.2Hz,6H,4-C(CH3)2),0.85(t,J=6.7Hz,6H,BA20 side chain CH3).

[0091] After high-performance liquid chromatography analysis, the purity of the cabazitaxel prodrug was 99.59%. Figure 3 As shown.

[0092] Examples 2-5

[0093] Considering that different drug-to-lipid ratios will further affect drug encapsulation and formulation stability, this invention synthesizes carbazide pre-race drug liposomes with different drug-to-lipid ratios based on Example 1, namely Examples 2-5, as detailed below:

[0094] Example 2

[0095] The carbazitaxel predrug, HSPC, and DSPE-mPEG from Example 1 were added. 2KDissolved in chloroform, stock solutions were prepared at concentrations of 1.33 mg / mL, 4.58 mg / mL, and 1.439 mg / mL. 2.66 mg of the prodrug, 22.883 mg of HSPC, and DSPE-mPEG were taken. 2K 4.317 mg was mixed in a round-bottom flask at a drug-to-liposome ratio of 1:10. The chloroform was removed under reduced pressure using a rotary evaporator to obtain a uniform thin film. Preheated purified water to 60°C was added to the round-bottom flask and hydrated at 60°C for 30 min to obtain crude cabazitaxel prodrug liposomes. After treatment with an ultrasonic cell disruptor (150W, 5 min), cabazitaxel prodrug liposomes were obtained with a particle size of 106.9±1.210 nm, a PDI of 0.199±0.045, and a potential of -35.0±2.66 mV. The encapsulation efficiency was determined to be 99±0.045% by low-speed centrifugation. The particle size and morphology of the prepared prodrug liposomes were determined by transmission electron microscopy, and the results are as follows: Figure 4 Transmission electron microscopy (TEM) images showed that the prepared prodrug liposomes were uniformly spherical. The stability of the cabazitaxel prodrug liposomes was investigated at room temperature and 4°C, respectively. Figure 5 As shown, the particle size did not change significantly after 14 days at room temperature and 30 days at 4℃.

[0096] Example 3

[0097] The carbazitaxel predrug, HSPC, and DSPE-mPEG from Example 1 were added. 2K Dissolved in chloroform, stock solutions were prepared at concentrations of 1.33 mg / mL, 8.91 mg / mL, and 2.88 mg / mL, respectively. 2.66 mg of the prodrug, 44.565 mg of HSPC, and DSPE-mPEG were taken. 2K 8.635 mg was mixed in a round-bottom flask at a drug-to-liposome ratio of 1:20. The chloroform was removed under reduced pressure using a rotary evaporator to obtain a uniform thin film. Preheated purified water to 60°C was added to the round-bottom flask and hydrated at 60°C for 30 min to obtain crude cabazitaxel liposomes. After treatment with an ultrasonic cell disruptor (150W, 5 min), cabazitaxel liposomes were obtained with a particle size of 115.2±1.000 nm, a PDI of 0.237±0.026, and a potential of -35.0±2.66 mV. The encapsulation efficiency was determined to be 98.10% by low-speed centrifugation. The stability of the cabazitaxel liposomes was investigated at room temperature and 4°C. Figure 5 As shown, the particle size did not change significantly after 14 days at room temperature and 30 days at 4℃.

[0098] Example 4

[0099] The carbazitaxel predrug, HSPC, and DSPE-mPEG from Example 1 were added. 2KDissolved in chloroform, stock solutions were prepared at concentrations of 1.33 mg / mL, 13.37 mg / mL, and 4.32 mg / mL, respectively. 2.66 mg of the prodrug, 66.848 mg of HSPC, and DSPE-mPEG were taken. 2K 12.952 mg was mixed in a round-bottom flask at a drug-to-liposome ratio of 1:30. The chloroform was removed under reduced pressure using a rotary evaporator to obtain a uniform thin film. Preheated purified water to 60°C was added to the round-bottom flask and hydrated at 60°C for 30 min to obtain crude cabazitaxel liposomes. After treatment with an ultrasonic cell disruptor (150W, 5 min), cabazitaxel liposomes were obtained with a particle size of 122.1±2.967 nm, a PDI of 0.261±0.022, and a potential of -35.0±2.66 mV. The encapsulation efficiency was determined to be 97.82% by low-speed centrifugation. The stability of the cabazitaxel liposomes was investigated at room temperature and 4°C. Figure 5 As shown, the particle size did not change significantly after 14 days at room temperature and 30 days at 4℃.

[0100] Example 5

[0101] Cabazitaxel, HSPC, cholesterol, and DSPE-mPEG were added. 2K Dissolved in chloroform, stock solutions were prepared at concentrations of 1.33 mg / mL, 5.21 mg / mL, 1.24 mg / mL, and 1.50 mg / mL. 2.66 mg of the prodrug, 66.848 mg of HSPC, 1.244 mg of cholesterol, and DSPE-mPEG were taken. 2K 12.952 mg was mixed in a round-bottom flask at a drug-to-liposome ratio of 1:10. The chloroform was removed under reduced pressure using a rotary evaporator to obtain a uniform thin film. Preheated purified water to 60°C was added to the round-bottom flask and hydrated at 60°C for 30 min to obtain crude cabazitaxel liposomes. After treatment with an ultrasonic cell disruptor (150W, 5 min), cabazitaxel liposomes were obtained with a particle size of 126.0±2.879 nm, a PDI of 0.194±0.026, and a potential of -51.8±0.608 mV. The encapsulation efficiency was determined to be 96.23% by low-speed centrifugation.

[0102] The results of Examples 2-5 show that when phospholipids, cholesterol, and DSPE-mPEG are regulated... 2KWhen the molar ratio and drug-lipid ratio are within a suitable range, cabazitaxel prodrug exhibits strong affinity for phospholipids. The encapsulation efficiency of the prepared cabazitaxel prodrug liposomes can reach over 95%, with particle sizes all less than 150 nm. This facilitates targeted accumulation of tumors through the high permeability and long retention effect of solid tumors. Simultaneously, the potential of the prepared cabazitaxel prodrug liposomes, measured by a particle size analyzer, is between -30 mV and -60 mV, which helps prevent nanoparticle aggregation through charge repulsion. Furthermore, its stability at room temperature and under cold storage is excellent.

[0103] Example 6: Verification of the stability of lyophilized and reconstituted carbapoxetine predrug liposomes

[0104] One mL of the cabazitaxel octyl lauryl alcohol prodrug liposomes prepared in Examples 2, 3, and 4 was placed in a vial. 5% sucrose and 10% sucrose were used as freeze-drying protectants, respectively, and then the samples were freeze-dried in a freeze dryer for 24 hours to obtain white cakes. The resulting freeze-dried powders were reconstituted with physiological saline and isotonic glucose solution, respectively, and the particle size distribution was measured. The results are shown in Table 1.

[0105] Table 1. Particle size and particle size distribution of lyophilized and reconstituted liposomes of cabazitaxel.

[0106]

[0107] According to the results in Table 1, the lyophilized and reconstituted liposomes of the carbamate prodrug in Examples 2-4 have high stability. Among them, when 10% sucrose is used as the lyophilization protectant and 5% Glu is used as the solvent, the particle size and particle size distribution are better after lyophilization and reconstitution.

[0108] Example 7: In vitro hemolysis experiment of carbamazepine prodrug liposomes

[0109] Because the cabazitaxel injection used clinically contains polysorbate 80, it is prone to causing hemolysis. Liposome formulations, which primarily use phospholipids as membrane materials, have good biocompatibility and can effectively avoid hemolysis. Fresh red blood cell suspensions were mixed with cabazitaxel solution and the cabazitaxel prodrug prepared in Examples 2, 3, and 4 at a ratio of 1:19, and positive control groups (20% Triton X-100) and negative control groups (PBS solution) were set up, respectively. The mixtures were incubated in a shaker at 37°C for 1 hour. Afterwards, the red blood cells were centrifuged at low speed (3000 rpm, 10 min), and the results are as follows. Figure 6As shown, after incubation of the cabazitaxel solution with the red blood cell suspension, the red blood cells ruptured, and the supernatant was distinctly blood-red. However, after incubation of the cabazitaxel prodrug liposomes from Examples 2-4 with the red blood cell suspension, the supernatant was clear, and no blood color was observed to the naked eye. Furthermore, the supernatant was added to a 96-well plate, and the absorbance was measured at 540 nm to calculate the hemolysis rate ((absorbance A preparation - A negative) / (A positive - A negative) * 100%). The results are shown in Table 2.

[0110] Table 2. Hemolysis rate test results for cabazitaxel solution and Examples 2-4.

[0111] Cabazitaxel solution 32.76±3.27％ Drug to lipid ratio 1 : 10 1.44±0.28％ Drug to lipid ratio 1 : 20 2.16±0.15％ Drug to lipid ratio 1 : 30 1.68±0.15％

[0112] The results showed that the cabazitaxel solution caused significant hemolysis, with a hemolysis fraction of 32%, while the cabazitaxel prodrug liposomes in Examples 2-4 significantly improved the hemolysis, with a hemolysis fraction of less than 5%, demonstrating good biocompatibility and safety.

[0113] Example 8: In vitro release study of carbamazepine prodrug liposomes

[0114] The cabazitaxel octyl lauryl alcohol prodrug liposomes prepared in Examples 2, 3, and 4 were diluted to 0.2 mg / mL, and 500 μL was added to 30 mL of PBS (pH 7.4) containing 30% ethanol (v / v). Blank, 50 mM H2O2, and 1 mM glutathione (GSH) experimental groups were set up. Samples were taken at 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h, and the CTX concentration was determined using high-performance liquid chromatography (HPLC). The results are as follows: Figure 7 As shown, in the blank medium, the three carbamazepine prodrug liposomes of Examples 2-4 released slowly, with the release rate of the parent drug being less than 5% within 24 hours. This indicates that the prodrug liposomes can avoid premature leakage of the drug during systemic circulation, thereby reducing the systemic toxicity of carbamazepine. The redox response drug release of the carbamazepine prodrug liposomes was investigated using H2O2 and GSH as triggers. Under conditions of 50 mM H2O2 and 1 mM GSH, the carbamazepine prodrug liposomes of Examples 2-4 could release the drug relatively rapidly, with release rates of approximately 40% and 80%, respectively. This indicates that the carbamazepine prodrug liposomes of Examples 2-4 can intelligently respond to release at high redox sites in tumors and have good tumor selectivity.

[0115] Example 9: Plasma chemical stability study of carbapoxetine prodrug liposomes

[0116] The carbapazioxicam liposomes prepared in Examples 2, 3, and 4 were mixed with blank rat plasma (1:9, v / v) and incubated in a 37°C constant-temperature shaker (100 rpm). At 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h, 50 μL of sample was taken, and 150 μL of acetonitrile was added. After vortexing for 3 min and centrifugation at 13000 rpm for 10 min, the supernatant was collected to determine the plasma drug concentration. The results are as follows: Figure 8 As shown, the drug degraded slowly with prolonged incubation time, and the carbamazepine pre-race drug liposomes of Examples 2-4 exhibited good plasma chemical stability. After 24 hours of incubation with plasma, 81.08%, 70.43%, and 35.79% of the drug, respectively, remained undegraded, releasing 9.10%, 25.48%, and 42.27% of the parent drug, respectively. The drug exhibited the best plasma chemical stability at a drug-to-lipid ratio of 1:10, followed by ratios of 1:20 and 1:30. Overall, the carbamazepine pre-race drug liposomes of Examples 2-4 all demonstrated good plasma chemical stability, ensuring the pharmacokinetic behavior of the drug in vivo.

[0117] Example 10: Cytotoxicity analysis of liposomes of cabazitaxel prodrug

[0118] The MTT assay was used to investigate the toxicity of cabazitaxel prodrug liposomes to mouse prostate cancer (RM-1) cells. First, morphologically sound cells were digested, diluted to an appropriate concentration with fresh culture medium, and then homogenized. 2000 cells were added to each well of a 96-well plate and incubated for 12 hours to allow cell attachment. After cell attachment, cabazitaxel solution, and the cabazitaxel prodrug liposome solutions prepared in Examples 2, 3, and 4 were added, respectively. Serial dilutions were performed using cell culture medium, with 200 μL of the test solution added to each well, and three parallel wells were used for each concentration. Forty-eight hours after drug administration, the 96-well plate was removed, the old culture medium was discarded, and 185 μL of MTT solution diluted with fresh culture medium was added to each well (1500 μL fresh culture medium + 35 μL 5 mg / mL MTT solution). The plate was incubated for 4 hours, then the culture medium was discarded. The 96-well plate was inverted onto filter paper to thoroughly absorb any remaining liquid. Then, 200 μL of DMSO was added to each well, and the plate was shaken for 10 minutes to dissolve the blue-purple crystals. The absorbance was measured at 490 nm using a microplate reader. The results are as follows: Figure 9 As shown, since the prodrugs require a smart response activation process to exert their effects in cells, the cytotoxicity of the cabazitaxel prodrug liposomes in Examples 2-4 is weaker than that of the cabazitaxel solution. Due to the high redox sensitivity of the introduced disulfide bonds, they can all smartly respond to the abnormally high redox levels of tumor cells, exhibiting good anti-tumor cell proliferation effects. Among them, the cytotoxicity is strongest at a drug-liposome ratio of 1:30.

[0119] Example 11: In vivo antitumor effect study of liposomes containing carbazide prodrug

[0120] Take 100 μL of cell suspension containing 3 million mouse prostate cancer cells (RM-1, 3 x 10⁻⁶). 7 A mouse model of ectopic prostate cancer was established by subcutaneous inoculation of cells / mL into the right posterior back of male C57 mice. The tumor volume on the back of the tumor-bearing mice reached 100 mm². 3 At approximately 10:00 AM, mice were randomly divided into groups of 5. A negative control group (saline), a carbazide solution group, and the carbazide prodrug liposome groups from Examples 2, 3, and 4 were included. The mice were administered the drug every two days for a total of 5 doses at a dose of 6 mg / kg. Tumor volume (major axis * minor axis * minor axis / 2) and body weight were measured and recorded daily, and curves showing changes in tumor volume and body weight over time were plotted. At the end of the experiment, the mice were euthanized, tumor tissue was dissected, and the tumor bearing rate (tumor weight / mouse body weight * 100%) was calculated.

[0121] The results are as follows Figures 10-12 As shown. In the saline group, which served as a negative control, tumors progressed rapidly, with tumor volume exceeding 2000 mm² only on day 10. 3 In mice with a tumor burden exceeding 10%, the negative control group was sacrificed prematurely for animal ethics reasons. In contrast, the cabazitaxel prodrug liposome groups in Examples 2, 3, and 4 (10LIPO-6, 20LIPO-6, and 30LIPO-6) all demonstrated good antitumor effects. Among them, in terms of tumor volume and tumor burden, the cabazitaxel prodrug liposome group with a drug-to-lipid ratio of 1:10 in Example 2 showed the best antitumor effect, with significant differences compared to the cabazitaxel solution group (CTX-6) and the cabazitaxel prodrug liposome group with a drug-to-lipid ratio of 1:30 (30LIPO-6). Furthermore, mice treated with cabazitaxel solution (CTX-6) experienced a significant decrease in body weight, while the body weight of mice treated with cabazitaxel prodrug liposomes (10LIPO-6, 20LIPO-6, and 30LIPO-6) did not change significantly, indicating that the designed intelligent responsive prodrug liposomes have good safety. In summary, the carbazide predrug liposomes of this embodiment have significant anti-tumor effects, with few toxic side effects and high safety.

[0122] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing carbamazepine pre-race drug liposomes, characterized in that, The structural formula of the carbazitaxel pre-race drug is as follows: ； The method for preparing the carbamate prodrug liposomes includes the following steps: (1) 2.66 mg of cabazitaxel pre-race drug, 22.883 mg of HSPC and DSPE-mPEG were administered. 2K 4.317 mg was dissolved in chloroform and mixed thoroughly to prepare stock solutions of 1.33 mg / mL cabazitaxel, 4.58 mg / mL HSPC, and 1.439 mg / mL DSPE-mPEG. 2K After the stock solution is prepared, the cabazitaxel predrug stock solution, the HSPC stock solution, and the DSPE-mPEG are added. 2K The stock solution was mixed and the chloroform was removed by rotary evaporation under reduced pressure to obtain a uniform thin film. (2) After preheating the purified water to 60°C, add it to the membrane and hydrate it at 60°C for 30 min to obtain crude cabazitaxel pre-race drug liposomes; (3) The crude carbapoxetine liposomes were subjected to ultrasonic treatment to obtain the carbapoxetine liposomes; the ultrasonic power was 150W; and the ultrasonic time was 5min.

2. The method for preparing carbazide pre-race drug liposomes according to claim 1, characterized in that, The method for synthesizing the aforementioned carbamazepine pre-race drug includes the following steps: (1) A dicarboxylic acid is dehydrated by acetic anhydride to obtain a dicarboxylic acid anhydride; the dicarboxylic acid is 2,2'-dithiodiacetic acid; (2) The fatty alcohol undergoes a ring-opening esterification reaction with the diacid anhydride under the action of a catalyst to obtain an intermediate product; the fatty alcohol is octyl lauryl alcohol; (3) The intermediate product undergoes an esterification reaction with cabazitaxel under the action of a catalyst to obtain the cabazitaxel prodrug; the reaction equation is as follows: 。 3. The method for preparing carbazide pre-race drug liposomes according to claim 2, characterized in that, In step (1), the dicarboxylic acid is dissolved in acetic anhydride and stirred at room temperature for 1-4 hours under nitrogen protection to react the dicarboxylic acid into dicarboxylic anhydride. After the reaction is complete, toluene is added and the toluene and acetic anhydride are removed by rotary evaporation under reduced pressure in a water bath at 30-32°C. The molar ratio of the dicarboxylic acid to the acetic anhydride is 1:(1~50).

4. The method for preparing carbazide pre-race drug liposomes according to claim 2, characterized in that, In step (2), fatty alcohol and catalyst are dissolved together with the diacid anhydride in dichloromethane and a ring-opening esterification reaction is carried out at room temperature for 8-12 hours. The intermediate product octyldodecyl dithiodiacetic acid monoester is obtained by separation by chromatography column. The catalyst is 4-dimethylaminopyridine; The molar ratio of the catalyst, the fatty alcohol, and the diacid anhydride is 1:(1~10):(1~15); In step (3), the catalyst and the intermediate product are dissolved in anhydrous dichloromethane. After activation in an ice bath under nitrogen protection for 0.5h to 4h, the carbatazone dissolved in dichloromethane is added and esterification reaction is carried out at room temperature for 24h to 48h. The carbatazone prodrug is then obtained by preparative liquid phase separation and purification. The catalyst comprises one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 4-dimethylaminopyridine, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, and p-toluenesulfonic acid. The molar ratio of the catalyst, the cabazitaxel, and the intermediate is 1:(0.1~5):(0.1~5).

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