Pegylated liposome adriamycin and application thereof in treating advanced and refractory invasive fibroma

Abstract

The invention belongs to the field of drug delivery, and relates to a liposome preparation, in particular to pegylated liposome adriamycin and application thereof in treating advanced and refractory invasive fibroma. The pegylated liposome doxorubicin comprises doxorubicin hydrochloride, polyethylene glycol-lipid, cholesterol and phospholipid. The pegylated liposome doxorubicin liposome disclosed by the invention is small in particle size, high in encapsulation efficiency and high in stability, and has a remarkable curative effect on advanced and refractory invasive fibroma.

Description

Technical Field

[0001] This invention belongs to the field of drug delivery and relates to liposome formulations, specifically a polyethylene glycol-modified liposomal doxorubicin and its use in the treatment of advanced and refractory aggressive fibromas. Background Technology

[0002] PEGylated liposomes are a core representative of "stealth" nanomedicine platforms. They covalently link the hydrophilic polymer polyethylene glycol (PEG) to the liposome surface, significantly prolonging in vivo circulation time, reducing immune clearance, and improving drug accumulation efficiency in lesions (especially tumors). In 1990, Blume and Klibanov et al. used synthetic PEG-DSPE (≈2kDa) to replace traditional ganglioside monoglycerides (GM1) for long-cycle modification, finding that the PEG chains formed a hydration barrier on the surface, preventing plasma protein adsorption and extending the half-life from 0.5h to over 5h. PEG-DSPE modification is simple to synthesize and cost-effective, and later became the underlying technology for PEGylated liposomes. Currently, PEGylated liposomes are commonly used in anticancer chemotherapy, nucleic acid and vaccine delivery, and imaging diagnostics and treatment.

[0003] Doxorubicin, an anthracycline cytotoxic antibiotic, is effective against solid tumors such as breast cancer, ovarian cancer, and soft tissue sarcoma due to its dual mechanism of action involving topoisomerase II inhibition and DNA insertion. It remains a first-line chemotherapy drug. However, conventional doxorubicin hydrochloride injection has a non-selective distribution in the body, resulting in high exposure to normal tissues such as the heart, bone marrow, and gastrointestinal tract. This leads to side effects such as dose-dependent cardiomyopathy, severe bone marrow suppression, and hair loss, significantly limiting its clinical use and therapeutic window. To improve the therapeutic index of doxorubicin, researchers began encapsulating it in liposomes in the 1980s. While traditional liposomes can partially reduce cardiac uptake, they are rapidly cleared by the reticuloendothelial system (RES) in the bloodstream, with a half-life of only about 0.5 hours, resulting in tumor accumulation of less than 5% and limited clinical benefit. In the 1990s, Allen et al. proposed modifying the surface of liposomes with methoxy polyethylene glycol-distearate phosphatidylethanolamine (MPEG-DSPE) to form a hydration barrier, which significantly reduced plasma protein adsorption and macrophage phagocytosis, extending the plasma half-life of liposomes to >70 hours. This technology laid the foundation for polyethylene glycol-modified liposomal doxorubicin.

[0004] PLD (Pegylated Liposomal Doxorubicin), also known as polyethylene glycol-modified liposomal doxorubicin, is a doxorubicin formulation packaged in a polyethylene glycol (PEG)-modified liposomal carrier. In 1995, the US FDA approved Doxil for the treatment of AIDS-related Kaposi's sarcoma, later expanding its use to ovarian cancer and multiple myeloma. Although PLDs have been used clinically for nearly thirty years, there is still room for improvement in their encapsulation efficiency, drug loading, target specificity, heat / enzyme-responsive release, and consistency in scale-up production. Developing next-generation PLD formulations is of great significance for improving the efficacy of tumor treatment, reducing toxic side effects, and meeting the needs of personalized treatment. Summary of the Invention

[0005] The inventors had previously recruited patients with advanced or refractory desmoidoma who had received either pegylated liposomal doxorubicin or placebo treatment, and registered the recruitment at ClinicalTrials.gov (NCT05561036). Results showed that patients treated with pegylated liposomal doxorubicin had significantly prolonged progression-free survival, induced durable remission, and demonstrated good safety profiles compared to the placebo group. There is still significant room for improvement in the development of next-generation pegylated liposomal doxorubicin formulations, particularly in terms of dosage form improvement and enhanced tumor efficacy. Therefore, this invention provides a pegylated liposomal doxorubicin and its use in the treatment of advanced and refractory aggressive desmoidoma.

[0006] The technical solution of the present invention is achieved through the following: In a first aspect, the present invention provides a polyethylene glycol-modified liposomal doxorubicin, characterized in that it comprises doxorubicin hydrochloride, polyethylene glycol-lipid, cholesterol, and phospholipids.

[0007] Preferably, the polyethylene glycol-lipid is selected from one or more of DSPE-PEG2000, DSPE-PEG5000, DSPE-mPEG2000, DSPE-mPEG3400, DSPE-mPEG5000, mPEG2000-PCL2000, mPEG2000-PCL5000, mPEG2000-PCL10000, mPEG5000-PCL2000, mPEG5000-PCL10000, and mPEG5000-PCL15000. More preferably, the polyethylene glycol-lipid is selected from one or more of DSPE-PEG2000, DSPE-PEG5000, DSPE-mPEG2000, DSPE-mPEG3400, and DSPE-mPEG5000. Most preferably, the polyethylene glycol-lipid is selected from one or more of DSPE-PEG2000, DSPE-mPEG2000, and DSPE-mPEG5000.

[0008] Preferably, the phospholipid is selected from one or more of hydrogenated soybean phospholipid (HSPC), hydrogenated lecithin, soybean lecithin, distearate phosphatidylcholine (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioctanoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate choline (PLPC), and 1-palmitoyl-2-arachidonicoyl-sn-glycerol-3-phosphate choline (PAPC). More preferably, the phospholipid is selected from one or more of hydrogenated soybean phospholipid (HSPC), hydrogenated lecithin, soybean lecithin, distearate phosphatidylcholine (DSPC), and 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC). Most preferably, the phospholipid is selected from one or more of hydrogenated soybean phospholipid (HSPC), hydrogenated lecithin, and 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC).

[0009] Preferably, the mass ratio of polyethylene glycol to lipids, cholesterol, and phospholipids is 0.1~7:0.1~7:1~20. More preferably, the mass ratio of polyethylene glycol to lipids, cholesterol, and phospholipids is 1~3:1~3:2~5. Most preferably, the mass ratio of polyethylene glycol to lipids, cholesterol, and phospholipids is 1~2:1~2:3~4.

[0010] Preferably, the concentration of doxorubicin hydrochloride in the polyethylene glycol-modified liposomal doxorubicin is (0.1~10) mg / mL. More preferably, the concentration of doxorubicin hydrochloride in the polyethylene glycol-modified liposomal doxorubicin is (0.5~5) mg / mL. Most preferably, the concentration of doxorubicin hydrochloride in the polyethylene glycol-modified liposomal doxorubicin is (1~4) mg / mL.

[0011] In a second aspect, the present invention provides a method for preparing polyethylene glycol-modified liposome doxorubicin, characterized by comprising the following steps: 1) Preparation of blank polyethylene glycol-modified liposomes: 11) Accurately weigh polyethylene glycol-lipids, cholesterol and phospholipids, add organic solvent, and dissolve at 30~80℃ to obtain an alcoholic solution of mixed lipids; 12) Prepare an ammonium sulfate solution with a concentration of 200~400mM; 13) Under conditions of 40~90℃, the alcoholic solution of the mixed lipids in step 11) is added to the ammonium sulfate solution in step 12) to obtain a blank liposome suspension. 14) After the blank liposome suspension is squeezed through a polycarbonate membrane with gradually decreasing pore size, liposomes containing ammonium sulfate in the inner aqueous phase are obtained. The ammonium sulfate in the outer aqueous phase of the liposome is removed by ultrafiltration, and the liposome is obtained.

[0012] 2) Drug loading of polyethylene glycol liposomes: Take 1-20 mL of the blank polyethylene glycol liposomes prepared in step 1) and add doxorubicin hydrochloride solution dropwise at a temperature of 40-70℃ to obtain polyethylene glycol liposome doxorubicin.

[0013] Preferably, the organic solvent in step 11) is selected from one or more of methanol, anhydrous ethanol, diethyl ether, acetone, and chloroform.

[0014] Preferably, the total mass ratio of polyethylene glycol-lipids, cholesterol, and phospholipids to the volume ratio of the organic solvent in step 11) is (30~300) mg:1 mL. More preferably, the total mass ratio of polyethylene glycol-lipids, cholesterol, and phospholipids to the volume ratio of the organic solvent in step 11) is (50~200) mg:1 mL.

[0015] Preferably, the volume of the ammonium sulfate solution in step 13) is 3 to 20 times that of the organic solvent. More preferably, the volume of the ammonium sulfate solution in step 13) is 5 to 18 times that of the organic solvent. Most preferably, the volume of the ammonium sulfate solution in step 13) is 8 to 13 times that of the organic solvent.

[0016] Preferably, in step 14), the blank liposome suspension is extruded sequentially through polycarbonate membranes with pore sizes of 0.8~0.2μm and 0.4~0.1μm.

[0017] Preferably, the concentration of doxorubicin hydrochloride in the polyethylene glycol-modified liposomal doxorubicin is (0.1~10) mg / mL. More preferably, the concentration of doxorubicin hydrochloride in the polyethylene glycol-modified liposomal doxorubicin is (0.5~5) mg / mL. Most preferably, the concentration of doxorubicin hydrochloride in the polyethylene glycol-modified liposomal doxorubicin is (1~4) mg / mL.

[0018] Thirdly, the present invention provides the use of polyethylene glycol-modified liposomal doxorubicin in the treatment of advanced and refractory aggressive fibromas.

[0019] Preferably, the present invention provides the use of pegylated liposomal doxorubicin in the preparation of a drug for treating advanced and refractory aggressive fibromas, characterized in that the drug comprises pegylated liposomal doxorubicin and pharmaceutically acceptable excipients.

[0020] The beneficial effects of this invention are as follows: The polyethylene glycol-modified liposome doxorubicin of this invention uses a combination of hydrogenated lecithin and DOPC phospholipids, and is compatible with polyethylene glycol-lipids DSPE-PEG2000, DSPE-mPEG2000, and DSPE-mPEG5000 to improve liposome particle size, increase encapsulation efficiency and liposome stability, thereby achieving a more significant therapeutic effect on advanced and refractory invasive fibromas. Attached Figure Description

[0021] Figure 1 In vitro release rate of PEGylated liposomal doxorubicin. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to these embodiments. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0023] Cholesterol was purchased from Beijing Solarbio Science & Technology Co., Ltd. Hydrogenated lecithin (CAS No. 92128-87-5), DSPE-PEG2000, DSPE-mPEG2000, and DSPE-mPEG5000 were purchased from Aivito (Shanghai) Pharmaceutical Technology Co., Ltd.

[0024] Example 1: Preparation of polyethylene glycol-modified liposome doxorubicin A method for preparing pegylated liposome doxorubicin includes the following steps: 1) Preparation of blank polyethylene glycol-modified liposomes: 11) Accurately weigh polyethylene glycol-lipids, cholesterol and phospholipids according to Table 1, add 4 mL of anhydrous ethanol, and dissolve at 50 °C to obtain an alcoholic solution of mixed lipids. 12) Prepare an ammonium sulfate solution with a concentration of 280 mM; 13) At a temperature of 65°C, the alcoholic solution of the mixed lipids from step 11) is added to the ammonium sulfate solution from step 12) to obtain a blank liposome suspension; the volume of the ammonium sulfate solution is 50 mL. 14) After the blank liposome suspension was extruded through polycarbonate membranes with pore sizes of 0.4 μm and 0.2 μm, liposomes containing ammonium sulfate in the inner aqueous phase were obtained. The ammonium sulfate in the outer aqueous phase of the liposomes was removed by ultrafiltration, and the liposomes were obtained. 2) Drug loading of polyethylene glycol-modified liposomes: Take 8 mL of the blank polyethylene glycol-modified liposomes prepared in step 1), and add doxorubicin hydrochloride solution dropwise at a temperature of 60°C to obtain polyethylene glycol-modified liposome doxorubicin; the concentration of doxorubicin hydrochloride in the polyethylene glycol-modified liposome doxorubicin is 2 mg / mL.

[0025] Table 1. Polyethylene glycol – lipids, cholesterol and phospholipids dosage (unit: mg) Example 2: Determination of particle size of polyethylene glycol-modified liposome doxorubicin The polyethylene glycol-modified liposome doxorubicin prepared in Example 1 was diluted with water and then placed in a particle size analyzer to determine its particle size and distribution. Each group was tested in triplicate. The results are shown in Table 2.

[0026] Table 2. Results of average particle size and particle size distribution (n=3) Polyethylene glycol (PEG)-lipids alter the hydrophilic / hydrophobic balance and phase transition behavior of liposome membranes, thereby regulating the compactness and elasticity of the bilayer. The effects of PEG modification with different degrees of polymerization on surface potential and electrostatic repulsion further influence particle aggregation. Appropriate selection of PEG-lipids allows for the control of liposome particle size. The type of phospholipid also significantly affects the particle size and size distribution of liposomes. Selecting suitable phospholipids and combining them with appropriate PEG-lipids allows for more precise control of liposome size to meet drug delivery requirements. In this invention, the combination of hydrogenated lecithin and DOPC in the PEGylated liposome doxorubicin significantly reduces liposome particle size and maintains a stable particle size distribution. Compatibility with DSPE-mPEG2000 further reduces liposome size.

[0027] Example 3: Determination of encapsulation efficiency and storage stability of polyethylene glycol-modified liposomal doxorubicin Encapsulation efficiency: 2 mL of each of the polyethylene glycol-modified liposomes and doxorubicin prepared in Example 1 was added to a centrifuge tube, followed by 10 mL of methanol. The mixture was ultrasonically disrupted and then centrifuged at 10,000 rpm for 15 min. The supernatant was collected, and the content of doxorubicin hydrochloride in the supernatant was determined by chromatography, denoted as "total drug content". 2 mL of each of the polyethylene glycol-modified liposomes and doxorubicin prepared in Example 1 was placed in an ultrafiltration centrifuge tube and centrifuged at 8,000 rpm for 10 min. The content of doxorubicin hydrochloride in the filtrate was determined by chromatography, denoted as "free drug content". The results are shown in Table 3.

[0028] Encapsulation efficiency (%) = ×100% Storage stability: The polyethylene glycol-modified liposome doxorubicin prepared in Example 1 was stored in a refrigerator at 4°C for 1 month. The encapsulation efficiency was measured again after 1 month. The results are shown in Table 3.

[0029] Table 3 Results of encapsulation efficiency and storage stability measurements (n=3) Different combinations of phospholipids with polyethylene glycol-lipids exhibit differences in membrane fluidity, phase transition temperature, and surface charge, leading to variations in encapsulation efficiency. This invention utilizes a combination of hydrogenated lecithin and DOPC phospholipids in polyethylene glycol-modified liposomes, significantly improving liposome encapsulation efficiency and reducing leakage during storage. Compared to DSPE-PEG2000 or DSPE-mPEG2000, the combination with DSPE-mPEG5000 achieves significantly higher encapsulation efficiency and stability.

[0030] Example 4: Determination of in vitro release rate of pegylated liposomal doxorubicin Polyethylene glycol-modified liposomal doxorubicin (groups 4, 5, and 6) prepared in Example 1 were used. The in vitro release rate was determined at 40°C using a mixed solution containing 2M ammonium chloride and 0.2M histidine as the release medium. Commercially available polyethylene glycol-modified liposomal doxorubicin was used as a control. The results showed that the in vitro release rate of the polyethylene glycol-modified liposomal doxorubicin of this invention was essentially consistent with that of the commercially available formulation.

[0031] The inventors had previously recruited patients with advanced or refractory desmoidoma who had received either polyethylene glycol liposomal doxorubicin or placebo, and registered the recruitment at ClinicalTrials.gov (NCT05561036). Results showed that patients treated with polyethylene glycol liposomal doxorubicin had significantly prolonged progression-free survival, induced durable remission, and demonstrated good safety profiles compared to the placebo group. This embodiment confirms that the in vitro release rate of the PEGylated liposomal doxorubicin of this invention is substantially consistent with that of commercially available formulations, indicating the feasibility of using the PEGylated liposomal doxorubicin of this invention for the treatment of advanced and refractory aggressive desmoidoma.

[0032] The basic principles, main features, and advantages of this invention have been described above. Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made without departing from the spirit and scope of the invention, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection claimed by this invention is defined by the appended claims and their equivalents.

Claims

1. A pegylated liposomal doxorubicin characterized in that, doxorubicin hydrochloride, polyethylene glycol-lipid, cholesterol, phospholipid.

2. The pegylated liposomal doxorubicin of claim 1, wherein, The polyethylene glycol-lipid is selected from one or more of DSPE-PEG2000, DSPE-PEG5000, DSPE-mPEG2000, DSPE-mPEG3400, DSPE-mPEG5000, mPEG2000-PCL2000, mPEG2000-PCL5000, mPEG2000-PCL10000, mPEG5000-PCL2000, mPEG5000-PCL10000, mPEG5000-PCL15000.

3. The pegylated liposomal doxorubicin of claim 1, wherein, The phospholipid is selected from one or more of hydrogenated soybean phospholipid (HSPC), hydrogenated lecithin, soybean lecithin, distearoylphosphatidylcholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioctanoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC), 1-palmitoyl-2-arachidoyl-sn-glycero-3-phosphocholine (PAPC).

4. The pegylated liposomal doxorubicin of claim 2 or 3, wherein: The mass ratio of the polyethylene glycol-lipid, cholesterol, and phospholipid is 0.1-7:0.1-7:1-20.

5. The pegylated liposomal doxorubicin of claim 1, wherein: ###0002### The concentration of doxorubicin hydrochloride in the polyethylene glycol liposome doxorubicin is (0.1-10) mg / mL.

6. A method for preparing pegylated liposomal doxorubicin characterized in that, The method comprises the following steps: 1) preparing a blank polyethylene glycol liposome: 11) accurately weighing the polyethylene glycol-lipid, cholesterol, and phospholipid, adding an organic solvent, dissolving at 30-80°C to obtain an alcohol solution of the mixed lipids; 12) preparing an ammonium sulfate solution with a concentration of 200-400 mM; 13) under the condition of a temperature of 40-90°C, adding the alcohol solution of the mixed lipids in step 11) to the ammonium sulfate solution in step 12) to obtain a blank liposome suspension; 14) after the blank liposome suspension is successively extruded through polycarbonate membranes with gradually decreasing pore sizes, a liposome containing ammonium sulfate in the inner water phase is obtained, and the ammonium sulfate in the outer water phase of the liposome is removed by ultrafiltration, thereby obtaining the polyethylene glycol liposome doxorubicin. 2) drug loading of the polyethylene glycol liposome: taking 1-20 mL of the blank polyethylene glycol liposome prepared in step 1), under the condition of a temperature of 40-70°C, dropwise adding a doxorubicin hydrochloride solution to obtain the polyethylene glycol liposome doxorubicin.

7. The production method according to claim 6, wherein In step 11), the total mass of the polyethylene glycol-lipid, cholesterol, and phospholipid to the volume of the organic solvent is (30-300) mg:1 mL.

8. The production method according to claim 6, wherein In step 13), the volume of the ammonium sulfate solution is 3-20 times the volume of the organic solvent.

9. The production method according to claim 6, wherein The concentration of doxorubicin hydrochloride in the polyethylene glycol liposome doxorubicin is (0.1-10) mg / mL. The method comprises the following steps:

10. Use of pegylated liposomal doxorubicin for the preparation of a medicament for the treatment of advanced and refractory aggressive fibromatosis, characterized in that, The medicament comprises the pegylated liposomal doxorubicin of claim 1, and a pharmaceutically acceptable adjuvant.

Images