A long-acting fulvestrant liposome for injection and its preparation method

Abstract

The present invention belongs to the technical field of anti-tumor drug preparations and relates to a long-acting fulvestrant liposome for injection and a method for preparing the same. The long-acting fulvestrant liposome for injection comprises a fulvestrant inclusion complex, a liposome membrane material, a lyoprotectant, and an aqueous medium. The long-acting fulvestrant liposome for injection exhibits good stability, high encapsulation efficiency, long-lasting sustained release, and high bioavailability.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor drug preparations, and particularly relates to a long-acting fulvestrant liposome for injection and a preparation method thereof. Background Art

[0002] Breast cancer is a disease in which abnormal breast cells grow uncontrollably and form tumors. If left unchecked, the tumors can spread throughout the body and be fatal. According to the World Health Organization, approximately 2.3 million women worldwide will be diagnosed with breast cancer in 2022, and 670,000 will die from it. Women in every country can develop breast cancer at any age after puberty, but the incidence increases in later life, making it the most deadly cancer in women.

[0003] Fulvestrant is a novel estrogen receptor downregulator, primarily used for postmenopausal (including natural and artificial menopause) estrogen receptor-positive locally advanced or metastatic breast cancer that relapses after or during adjuvant anti-estrogen therapy, or progresses during anti-estrogen therapy. Currently, the commercially available fulvestrant injection is injected into the buttocks muscle and takes a long time, causing significant pain and inconvenience to patients. The excipient benzyl benzoate in the injection formulation is also prone to allergic reactions. Fulvestrant also has a first-pass effect in the liver, resulting in very low oral bioavailability and rapid pharmacokinetics, making it difficult to maintain blood concentrations for long periods of time. Therefore, the development of a clinical formulation of fulvestrant that is less painful, long-acting, and highly bioavailable is urgent.

[0004] In recent years, liposome technology has developed rapidly, with the emergence of novel liposome technologies such as lipid nanoparticles (LNPs), immunoliposomes, long-circulating liposomes, magnetic liposomes, membrane-fusogenic liposomes, and flexible liposomes. Currently marketed liposome injectable products in China include doxorubicin hydrochloride liposome injection, amphotericin B liposome injection, paclitaxel liposome injection, mitoxantrone hydrochloride liposome injection, and irinotecan hydrochloride liposome injection, primarily for various tumor indications. Among these, long-circulating liposome technology is theoretically more suitable for the development of clinical formulations for fulvestrant. Compared to traditional liposomes, long-circulating liposomes offer the following advantages: First, traditional liposomes are easily recognized by opsonins in the blood (such as the complement system and apolipoproteins) and rapidly captured and cleared by the mononuclear phagocytic system (MPS, such as the liver and spleen), resulting in a short half-life. Long-circulating liposomes are modified with hydrophilic substances (such as polyethylene glycol, PEG) to form a steric barrier that hinders plasma protein adsorption and immune system recognition, thereby significantly extending circulation time. For example, the half-life of doxorubicin long-circulating liposomes (Doxil) has been extended from 0.2 hours for the free drug to 41-70 hours, and the area under the drug concentration-time curve (AUC) has increased by over 200-fold. Second, long-circulating liposomes leverage the high permeability and retention of tumor tissue to accumulate at diseased sites (such as tumors and areas of inflammation). Compared to traditional liposomes, drug concentration in tumor tissue can be increased by 4-16 times, and through a sustained-release mechanism, the drug is continuously delivered to the target site, minimizing damage to normal tissues. Third, traditional liposomes are rapidly taken up by the MPS and tend to accumulate in the liver and spleen, leading to organ toxicity. However, long-circulating liposomes reduce MPS affinity, limiting drug release at non-target sites, thereby mitigating toxicity to organs such as the heart and kidneys. Finally, and most importantly, their nanoparticle size (typically 20-200 nm) and surface modifications (such as PEG brush layers) reduce liposome aggregation and drug leakage, resulting in improved drug stability and drug encapsulation efficiency compared to traditional liposomes.

[0005] Chinese Patent Publication No. CN 107362142A discloses a fulvestrant liposome injection and its preparation process. The liposome injection is primarily composed of fulvestrant, soybean lecithin, distearoylphosphatidylcholine, cholesterol, and a surfactant. Chinese Patent Publication No. CN 103221052 A discloses an inclusion complex comprising fulvestrant and cyclodextrin.

[0006] However, traditional liposome technology or inclusion compound technology is difficult to solve the sudden release phenomenon of fulvestrant. Summary of the Invention

[0007] To develop a more clinically applicable Fulvestrant preparation. The present invention is a major upgrade based on the technology of long-acting liposomes, developing a cyclodextrin-included long-acting liposome technology, which is particularly suitable for drugs such as Fulvestrant that are poorly soluble in water, easily oxidized, have low bioavailability, and have a short half-life. Compared with traditional liposomes, this effective delivery carrier can improve the drug-lipid ratio, enhance the stability of the liposomes, inhibit the dissociation of the drug from the inclusion complex, and thus maintain the integrity of the lipid bilayer, better control the release of drugs in the body, prolong the time effect, and improve the efficacy. The schematic diagram of traditional liposomes and cyclodextrin-included long-acting liposomes is shown in Figure 2. Figure 1 shown.

[0008] To achieve the above object, the long-acting fulvestrant liposomes for injection of the present invention contain a fulvestrant inclusion compound, a liposome membrane material, a lyoprotectant and an aqueous medium, and the weight ratio of the four is 1:2-20:5-30:100-800.

[0009] The weight ratio of fulvestrant to cyclodextrin in the fulvestrant inclusion compound is 1:2-10.

[0010] Furthermore, the cyclodextrin is selected from one or more of hydroxypropyl beta-cyclodextrin (HP-β-CD) and sulfobutyl beta-cyclodextrin (SBECD). The liposome membrane material is selected from hydrogenated soybean lecithin (HSPC), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-MPEG2000), and cholesterol. The lyoprotectant is selected from one or more of mannitol, trehalose, sucrose, lactose, maltose, and polyvinylpyrrolidone (PVP). The aqueous medium is selected from one or more of citrate buffer, phosphate buffer, and carbonate buffer solution, and the aqueous medium includes buffers of different pH values.

[0011] The present invention also provides a method for preparing long-acting fulvestrant liposomes for injection:

[0012] (1) Weigh fulvestrant and cyclodextrin according to the prescribed amount, dissolve them in ethanol and room temperature water for injection respectively, control the temperature below 20°C, and slowly add them to the fulvestrant ethanol solution while stirring. After the inclusion is completed, filter and sterilize, and then spray dry at low temperature to obtain the fulvestrant inclusion complex;

[0013] (2) Weigh the prescribed amount of HSPC, cholesterol, and DSPE-MPEG2000 and dissolve them in the solvent. Rotary evaporate at 30°C to 40°C until a lipid film forms on the inner wall of the round-bottom flask. Quickly freeze at -5°C ± 2°C for 0.5 to 1.5 h.

[0014] (3) dissolving the prescribed amount of fulvestrant inclusion complex in a phosphate buffer solution at pH 7.4, and adding the solution to the round-bottom flask in (2) to hydrate the lipid film to form crude liposomes;

[0015] (4) The crude liposomes are then homogenized 2 to 5 times in a microfluidizer at a homogenization pressure of 200 to 1500 bar to obtain a long-acting liposome solution;

[0016] (5) Add lyophilization protective agent, filter through 0.45 μm and 0.22 μm filter membranes in sequence, divide into packages, and freeze-dry to obtain long-acting fulvestrant liposomes for injection.

[0017] The organic solvent used in the preparation process is selected from one or more of ethanol, chloroform, dichloromethane and acetone.

[0018] The long-acting fulvestrant liposomes for injection provided by the present invention, including their cyclodextrin inclusion complex, liposome membrane material, and aqueous medium, improve the surface tension of the liposome bilayer, reduce the fluidity of the liposome membrane, reduce the risk of allergies caused by the solvent, improve the quality controllability and stability of the liposomes, and improve the use compliance of clinical patients. At the same time, cyclodextrin inhibits the dissociation of the drug from the inclusion complex, thereby maintaining the integrity of the lipid bilayer and better controlling the release of the drug in the body. At the same time, the long-circulating liposomes reduce liposome aggregation and drug leakage through nanoscale particle size and surface modification, improve the drug burst release phenomenon, prolong the circulation time in the blood, and improve the drug efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Schematic diagram of long-acting liposomes containing traditional liposomes and cyclodextrin.

[0020] Figure 2 : Cell viability of samples after incubation with human breast cancer MCF-7 cells for 48 h (n=3). DETAILED DESCRIPTION

[0021] In order to make the purpose and technical solution of the present invention more clear, the present invention is further described below in conjunction with the embodiments, but the scope of protection of the present invention is not limited to these embodiments, and the embodiments are only used to illustrate the present invention. It should be understood by those skilled in the art that any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the scope of protection of the present invention.

[0022] Example 1

[0023] Table 1. Example 1 prescription:

[0024]

[0025] Preparation method: Fulvestrant and HP-β-CD were weighed according to the prescription amount, dissolved in ethanol and room temperature water for injection (concentration 10% W / V), respectively, and slowly added to the fulvestrant ethanol solution while stirring at 10°C. After the inclusion is completed, it was sterilized by filtration and spray-dried at low temperature (10°C) to obtain the fulvestrant inclusion complex; HSPC, cholesterol, and DSPE-MPEG2000 were weighed in the prescription amount and dissolved in 500ml of chloroform. The mixture was rotary evaporated at 30°C to 40°C until a lipid film was formed on the inner wall of the round-bottom flask, and then quickly frozen at -5°C for 1h. The prescription amount of fulvestrant inclusion complex was dissolved in 500ml The solution was added to a phosphate buffer solution at pH 7.4 into a round-bottom flask to hydrate the lipid film to form crude liposomes; the crude liposomes were then homogenized three times using a microfluidizer at a homogenization pressure of 1000 bar; an aqueous solution of a lyoprotectant containing 15 g of sucrose was added, and the mixture was filtered through 0.45 μm and 0.22 μm filter membranes in sequence, packaged, and freeze-dried to obtain long-acting fulvestrant liposomes for injection.

[0026] Example 2

[0027] Table 2. Example 2 prescription:

[0028]

[0029] Preparation method: Fulvestrant and HP-β-CD were weighed according to the prescription amount, dissolved in ethanol and room temperature water for injection (concentration of 10% W / V), respectively, and slowly added to the fulvestrant ethanol solution while stirring at 5°C. After the inclusion was completed, the mixture was filtered, sterilized, and spray-dried at low temperature (10°C) to obtain the fulvestrant inclusion complex; HSPC, cholesterol, and DSPE-MPEG2000 were weighed and dissolved in 500 ml of chloroform, and rotary evaporated at 30°C-40°C until a lipid film was formed on the inner wall of the round-bottom flask, and then quickly frozen at -7°C for 0.5 h. The prescription amount of fulvestrant inclusion complex was dissolved in 100 ml of pH 6.5 phosphate buffer, and the solution was added to the round-bottom flask to hydrate the lipid film to form crude liposomes; the crude liposomes were then homogenized four times by a microfluidizer with a homogenization pressure of 1000 bar; an aqueous solution of lyoprotectant containing 15 g of trehalose was added, and the mixture was sequentially homogenized by 0.45 μm, 0 Filter through a 22 μm filter membrane, divide into portions, and freeze-dry to obtain long-acting fulvestrant liposomes for injection.

[0030] Example 3

[0031] Table 3. Example 3 prescription:

[0032]

[0033] Preparation method: Fulvestrant and HP-β-CD were weighed according to the prescription amount, dissolved in ethanol and room temperature water for injection (concentration 10% W / V), respectively, and slowly added to the Fulvestrant solution while stirring at 15°C. After the inclusion was completed, the mixture was sterilized by filtration and spray-dried at low temperature (10°C) to obtain the Fulvestrant inclusion complex; HSPC, cholesterol, and DSPE-MPEG2000 were weighed and dissolved in 500 ml of ethanol, and rotary evaporated at 30°C-40°C until a lipid film was formed on the inner wall of a round-bottom flask, and then quickly frozen at -3°C for 1.5 h. The prescription amount of Fulvestrant inclusion complex was dissolved in 800 ml of pH 7.0 phosphate buffer, and the solution was added to the round-bottom flask to hydrate the lipid film to form crude liposomes; the crude liposomes were then homogenized three times by a microfluidizer with a homogenization pressure of 1000 bar; an aqueous solution containing 55 g of lactose lyoprotectant was added, and the mixture was sequentially homogenized by 0.45 μm, 0 Filter through a 22 μm filter membrane, divide into portions, and freeze-dry to obtain long-acting fulvestrant liposomes for injection.

[0034] Example 4

[0035] Table 4. Example 4 prescription:

[0036]

[0037] Preparation method: Fulvestrant and SBECD were weighed according to the prescription amount, dissolved in ethanol and room temperature water for injection (concentration 10% W / V), respectively, and slowly added to the fulvestrant solution while stirring at 20°C. After the inclusion is completed, the mixture is sterilized by filtration and spray-dried at low temperature (15°C) to obtain the fulvestrant inclusion complex; HSPC, cholesterol, and DSPE-MPEG2000 were weighed in the prescription amount and dissolved in 500ml of dichloromethane, and rotary evaporated at 30°C to 40°C until a lipid film was formed on the inner wall of the round-bottom flask, and then quickly frozen at -5°C for 0.5h. The prescription amount of fulvestrant inclusion complex was dissolved in 500ml The solution was added to a citrate buffer solution at pH 7.4 into a round-bottom flask to hydrate the lipid film to form crude liposomes; the crude liposomes were then homogenized three times using a microfluidizer with a homogenization pressure of 1000 bar; an aqueous solution of a lyoprotectant containing 100 g of mannitol was added, and the mixture was filtered through 0.45 μm and 0.22 μm filter membranes in sequence, packaged, and freeze-dried to obtain long-acting fulvestrant liposomes for injection.

[0038] Example 5

[0039] Table 5. Example 5 prescription:

[0040]

[0041] Preparation method: Fulvestrant and SBECD were weighed according to the prescription amount, dissolved in ethanol and room temperature water for injection (concentration of 10% W / V), respectively, and slowly added to the Fulvestrant solution while stirring at 10°C. After the inclusion was completed, the mixture was sterilized by filtration and spray-dried at low temperature (2°C) to obtain the Fulvestrant inclusion complex; HSPC, cholesterol, and DSPE-MPEG2000 were weighed and dissolved in 500 ml of acetone, and rotary evaporated at 30°C-40°C until a lipid film was formed on the inner wall of the round-bottom flask, and then quickly frozen at -5°C for 1.5 h. The prescription amount of Fulvestrant inclusion complex was dissolved in 200 ml of pH 7.4 phosphate buffer, and the solution was added to the round-bottom flask to hydrate the lipid film to form crude liposomes; the crude liposomes were then homogenized three times by a microfluidizer with a homogenization pressure of 1000 bar; an aqueous solution of lyoprotectant containing 100 g of maltose was added, and the mixture was sequentially homogenized by 0.45 μm, 0 Filter through a 22 μm filter membrane, divide into portions, and freeze-dry to obtain long-acting fulvestrant liposomes for injection.

[0042] Example 6

[0043] Table 6. Example 6 prescription:

[0044]

[0045] Preparation method: Fulvestrant and SBECD were weighed according to the prescription amount, dissolved in ethanol and room temperature water for injection (concentration 10% W / V), respectively, and slowly added to the fulvestrant solution while stirring at 10°C. After the inclusion is completed, it was sterilized by filtration and spray-dried at low temperature (5°C) to obtain the fulvestrant inclusion complex; HSPC, cholesterol, and DSPE-MPEG2000 were weighed in the prescription amount and dissolved in 500ml of acetone. The mixture was rotary evaporated at 30°C to 40°C until a lipid film was formed on the inner wall of the round-bottom flask, and then quickly frozen at -7°C for 1.5h. The prescription amount of fulvestrant inclusion complex was dissolved in 400ml The solution was added to a carbonate buffer solution at pH 8.0 into a round-bottom flask to hydrate the lipid film to form crude liposomes; the crude liposomes were then homogenized three times using a microfluidizer at a homogenization pressure of 700 bar; an aqueous solution of a lyoprotectant containing 30 g of polyvinyl pyrrolidone was added, and the mixture was filtered through 0.45 μm and 0.22 μm filter membranes in sequence, packaged, and freeze-dried to obtain long-acting fulvestrant liposomes for injection.

[0046] Example 7

[0047] Table 7. Example 7 prescription:

[0048]

[0049] Preparation method: Fulvestrant and HP-β-CD were weighed according to the prescription amount, dissolved in ethanol and room temperature water for injection (concentration 10% W / V), respectively, and slowly added to the Fulvestrant solution while stirring at 5°C. After the inclusion was completed, the mixture was sterilized by filtration and spray-dried at low temperature (10°C) to obtain the Fulvestrant inclusion complex; HSPC, cholesterol, and DSPE-MPEG2000 were weighed and dissolved in 500 ml of acetone, and rotary evaporated at 30°C-40°C until a lipid film was formed on the inner wall of a round-bottom flask, and then quickly frozen at -3°C for 0.5 h. The prescription amount of Fulvestrant inclusion complex was dissolved in 100 ml of pH 7.4 phosphate buffer, and the solution was added to the round-bottom flask to hydrate the lipid film to form crude liposomes; the crude liposomes were then homogenized 5 times by a microfluidizer with a homogenization pressure of 800 bar; an aqueous solution of lyoprotectant containing 60 g of mannitol was added, and the mixture was sequentially homogenized by 0.45 μm, 0 Filter through a 22 μm filter membrane, divide into portions, and freeze-dry to obtain long-acting fulvestrant liposomes for injection.

[0050] Example 8

[0051] Table 8. Example 8 prescription:

[0052]

[0053] Preparation method: Fulvestrant and HP-β-CD were weighed according to the prescription amount, dissolved in ethanol and room temperature water for injection (concentration 10% W / V), respectively, and slowly added to the Fulvestrant solution while stirring at 15°C. After the inclusion was completed, the mixture was filtered, sterilized, and spray-dried at low temperature (10°C) to obtain the Fulvestrant inclusion complex; HSPC, cholesterol, and DSPE-MPEG2000 were weighed and dissolved in 1000 ml of chloroform, and rotary evaporated at 30°C-40°C until a lipid film was formed on the inner wall of a round-bottom flask, and then quickly frozen at -5°C for 1 hour. The prescription amount of Fulvestrant inclusion complex was dissolved in 200 ml of carbonate buffer solution at pH 7.4, and the solution was added to the round-bottom flask to hydrate the lipid film to form crude liposomes; the crude liposomes were then homogenized 5 times by a microfluidizer with a homogenization pressure of 1300 bar; an aqueous solution of lyoprotectant containing 66 g of sucrose was added, and the mixture was sequentially homogenized by 0.45 μm, 0 Filter through a 22 μm filter membrane, divide into portions, and freeze-dry to obtain long-acting fulvestrant liposomes for injection.

[0054] Example 9

[0055] Table 9. Example 9 prescription:

[0056]

[0057] Preparation method: Fulvestrant and SBECD were weighed according to the prescription amount, dissolved in ethanol and room temperature water for injection (concentration of 10% W / V) respectively, and slowly added to the Fulvestrant solution while stirring at 10°C. After the inclusion was completed, the mixture was filtered, sterilized, and spray-dried at low temperature (10°C) to obtain the Fulvestrant inclusion complex; HSPC, cholesterol, and DSPE-MPEG2000 were weighed and dissolved in 1000 ml of ethanol, and rotary evaporated at 30°C-40°C until a lipid film was formed on the inner wall of the round-bottom flask, and then quickly frozen at -5°C for 1 hour. The prescription amount of Fulvestrant inclusion complex was dissolved in 200 ml of pH 7.4 phosphate buffer, and the solution was added to the round-bottom flask to hydrate the lipid film to form crude liposomes; the crude liposomes were then homogenized 8 times by a microfluidizer with a homogenization pressure of 1300 bar; added to an aqueous solution of lyophilized protective agent containing 32 g of sucrose, and sequentially homogenized by 0.45 μm, 0 Filter through a 22 μm filter membrane, divide into portions, and freeze-dry to obtain long-acting fulvestrant liposomes for injection.

[0058] Comparative Example 1

[0059] Table 10. Comparative Example 1 Prescription:

[0060]

[0061] Preparation method: Weigh the prescribed amount of HSPC, cholesterol, and fulvestrant and dissolve them in 1000 ml of chloroform. Rotary evaporate at 30°C-40°C until a lipid film is formed on the inner wall of a round-bottom flask. Quickly freeze at -5°C for 1 hour. Add 200 ml of pH 7.4 phosphate buffer to the round-bottom flask to hydrate the lipid film to form crude liposomes. The crude liposomes are then homogenized 5 times using a microfluidizer with a homogenization pressure of 1300 bar. Add a lyoprotectant aqueous solution containing 32 g of sucrose, filter through 0.45 μm and 0.22 μm filter membranes in sequence, package, and freeze-dry to obtain fulvestrant liposomes for injection.

[0062] Comparative Example 2

[0063] Table 11. Comparative Example 2 Prescription:

[0064]

[0065] Preparation method: Fulvestrant and SBECD were weighed according to the prescription amount, dissolved in ethanol and room temperature water for injection (concentration 10% W / V) respectively, and slowly added to the Fulvestrant solution while stirring at 10°C. After the inclusion was completed, the mixture was sterilized by filtration and spray-dried at low temperature (10°C) to obtain the Fulvestrant inclusion complex; HSPC and cholesterol were weighed in the prescription amount and dissolved in 1000 ml of chloroform, and rotary evaporated at 30°C-40°C until a lipid film was formed on the inner wall of the round-bottom flask, and then quickly frozen at -5°C for 1 hour. The prescription amount of Fulvestrant inclusion complex was dissolved in 200 ml of pH 7.4 phosphate buffer, and the solution was added to the round-bottom flask to hydrate the lipid film to form crude liposomes; the crude liposomes were then homogenized 8 times by a microfluidizer with a homogenization pressure of 1300 bar; added to an aqueous solution of lyophilized protective agent containing 32 g of sucrose, and sequentially passed through 0.45 μm, 0 Filter through a 22 μm filter membrane, divide into portions, and freeze-dry to obtain long-acting fulvestrant liposomes for injection.

[0066] Comparative Example 3

[0067] Table 12. Comparative Example 3 Prescription:

[0068]

[0069] Preparation method: Fulvestrant and SBECD were weighed according to the prescription amount, dissolved in ethanol and room temperature water for injection (concentration 10% W / V) respectively, and slowly added to the Fulvestrant solution while stirring at 25°C. After the inclusion was completed, the mixture was filtered, sterilized, and spray-dried (25°C) to obtain the Fulvestrant inclusion complex; HSPC, cholesterol, and DSPE-MPEG2000 were weighed and dissolved in 1000 ml of chloroform, and rotary evaporated at 30°C-40°C until a lipid film was formed on the inner wall of the round-bottom flask, and then quickly frozen at -5°C for 1 hour. The prescription amount of Fulvestrant inclusion complex was dissolved in 200 ml of pH 7.4 phosphate buffer, and the solution was added to the round-bottom flask to hydrate the lipid film to form crude liposomes; the crude liposomes were then homogenized 8 times by a microfluidizer with a homogenization pressure of 1300 bar; added to an aqueous solution of lyophilized protective agent containing 32 g of sucrose, and sequentially filtered through 0.45 μm, 0 Filter through a 22 μm filter membrane, divide into portions, and freeze-dry to obtain long-acting fulvestrant liposomes for injection.

[0070] Comparative Example 4

[0071] Table 13. Comparative Example 4 Prescription:

[0072]

[0073] Preparation method: Fulvestrant and SBECD were weighed according to the prescription amount, dissolved in ethanol and room temperature water for injection (concentration 10% W / V) respectively, and slowly added to the Fulvestrant solution while stirring at 10°C. After the inclusion was completed, the mixture was sterilized by filtration and spray-dried at low temperature (10°C) to obtain the Fulvestrant inclusion complex; HSPC, cholesterol, and DSPE-MPEG2000 were weighed and dissolved in 1000 ml of ethanol, and rotary evaporated at 30°C-40°C until a lipid film was formed on the inner wall of the round-bottom flask; the prescription amount of Fulvestrant inclusion complex was dissolved in 200 ml of pH 7.4 phosphate buffer, and the solution was added to the round-bottom flask to hydrate the lipid film to form crude liposomes; the crude liposomes were then homogenized 8 times by a microfluidizer with a homogenization pressure of 1300 bar; added to an aqueous solution of lyophilized protective agent containing 32 g of sucrose, and sequentially passed through 0.45 μm, 0 Filter through a 22 μm filter membrane, divide into portions, and freeze-dry to obtain long-acting fulvestrant liposomes for injection.

[0074] Comparative Example 5

[0075] Table 14. Comparative Example 5 Prescription:

[0076]

[0077] Preparation method: Fulvestrant and SBECD were weighed according to the prescription amount, dissolved in ethanol and room temperature water for injection (concentration 10% W / V) respectively, and slowly added to the Fulvestrant solution while stirring at 10°C. After the inclusion was completed, the mixture was filtered, sterilized, and spray-dried at low temperature (10°C) to obtain the Fulvestrant inclusion complex; HSPC, cholesterol, and DSPE-MPEG2000 were weighed and dissolved in 1000 ml of ethanol, and rotary evaporated at 30°C-40°C until a lipid film was formed on the inner wall of the round-bottom flask, and refrigerated at 4°C for 1.5 h. The prescription amount of Fulvestrant inclusion complex was dissolved in 200 ml of pH 7.4 phosphate buffer, and the solution was added to the round-bottom flask to hydrate the lipid film to form crude liposomes; the crude liposomes were then homogenized 8 times by a microfluidizer with a homogenization pressure of 1300 bar; added to an aqueous solution of lyophilized protective agent containing 32 g of sucrose, and sequentially homogenized by 0.45 μm, 0 Filter through a 22 μm filter membrane, divide into portions, and freeze-dry to obtain long-acting fulvestrant liposomes for injection.

[0078] Trial 1: Critical Quality Control of Liposome Formulation

[0079] (1) Properties: The examples and comparative examples are all white loose masses.

[0080] (2) Particle size and zeta potential

[0081] Sample preparation: An appropriate amount of liposome sample prepared in the Examples and Comparative Examples was added to phosphate buffer and diluted to 5 ml. The sample was filtered through a 0.22 μm PES filter membrane. An appropriate amount of the solution was added to a sample cell. The particle size and potential were measured using a zetasize dynamic light scattering particle size analyzer (model: Nanozs, manufacturer: Malvern Panalytical). The results are shown in the table below.

[0082] Table 15 Average particle size and Zeta potential test results of samples

[0083]

[0084] The experiments showed that compared with Comparative Examples 1-5, Examples 1-9 had smaller average particle sizes and were more stable, indicating good process reproducibility. Furthermore, smaller particle sizes resulted in stronger targeting and better membrane stability. The charge of the liposomes reduced aggregation and fusion, increasing stability. When the absolute value of the Zeta potential (mV) was between -30 and -50 mV, the liposomes were relatively stable, indicating that the Examples were significantly better than the Comparative Examples. It should be noted that, through single-factor experiments, the quick freezing step of the liposome film significantly affected the quality of the drug-loaded liposomes. Drug-loaded liposomes prepared without quick freezing had loose lipid arrangement, low drug loading, and poor sustained-release effects. The compactness of the lipid film after refrigeration at 4°C was also unsatisfactory. The quick freezing treatment of the lipid film in the present invention resulted in highly ordered hydrophobic tail chains in the film, a tighter hydrogen bond network between polar head groups, and a gel phase, resulting in a densified membrane structure and improved drug distribution and encapsulation capabilities.

[0085] (3) Encapsulation efficiency and in vitro release

[0086] The liposome encapsulation efficiency was determined by dialysis. An appropriate amount of liposomes was accurately weighed and placed in a dialysis bag, which was then sealed and magnetically stirred. The dialysate was collected at 0.5, 1, 2, 4, 6, 8, 12, and 24 hours, and the same amount of hydration medium was added at the same time. The samples were filtered through a 0.22 μm PES membrane and determined by HPLC. The encapsulation efficiency was calculated based on the results.

[0087] The in vitro release of liposomes was determined using dialysis. An appropriate amount of liposomes was precisely weighed and placed in a dialysis bag. The bag was then placed in 5% calf serum (PBS) buffer (pH 7.4) at 37°C. The bag was sealed and magnetically stirred. The dialysate was collected at 0.5, 1, 2, 4, 6, 8, 12, and 24 hours, and the same amount of hydration medium was added. The samples were filtered through a 0.22 μm PES membrane and assayed by HPLC. The in vitro release was calculated based on the results. The encapsulation efficiency and in vitro release results are shown in the table below.

[0088] Table 16 Sample encapsulation efficiency and in vitro release test results

[0089]

[0090] The test showed that fulvestrant is poorly soluble in water and easily oxidized. Compared with Comparative Example 1 without cyclodextrin inclusion, Comparative Example 3 without low-temperature controlled encapsulation, and Comparative Example 4 without quick freezing, the liposomes prepared by Examples 1 to 9 after cyclodextrin inclusion had significantly improved encapsulation efficiency, reduced drug leakage, and increased drug loading. Compared with Comparative Examples 1 to 5, the liposomes prepared by Examples 1 to 9 after cyclodextrin inclusion had a significant sustained-release effect on drug release in the liposomes without burst release.

[0091] Experiment 2: Liposome Compatibility and Long-term Stability

[0092] An appropriate amount of the long-acting fulvestrant liposomes for injection prepared in the examples was taken and diluted with 5% glucose injection to contain 2.5 mg / ml of fulvestrant. The compatibility stability after 8 hours was examined. The finished liposomes prepared in some examples (Examples 2, 4, 6, and 8) were directly taken and their stability was examined under long-term conditions of 2 to 8°C and accelerated conditions of 25°C for 6 months. The results are shown in the table below.

[0093] Table 17 Liposome compatibility stability results

[0094]

[0095] Note: “☆” represents a colorless to milky white clear liquid with no precipitation; the content at 0h is 100%.

[0096] Table 18 Long-term and accelerated stability results of liposomes

[0097]

[0098] Note: “★” represents white loose lumps.

[0099] The experiments showed that after liposomes prepared with different formulations were mixed with 5% glucose injection, they were stable at a concentration of 2.5 mg / ml for 8 hours, indicating good compatibility stability. The liposomes prepared with different formulations showed no significant changes in properties, encapsulation efficiency, and other related aspects after being stored at 2-8° C. for 6 months and at 25° C. for 6 months. The fulvestrant liposomes for injection prepared according to the present invention had good stability and were suitable for clinical use.

[0100] Test 3: In vitro pharmacodynamics test

[0101] The finished products prepared in Comparative Example 1, Comparative Example 2 and Example 9 were selected and their toxic effects on human breast cancer cells MCF-7 were investigated by CCK-8 method. MCF-7 cells with good growth in the logarithmic growth phase were digested with 0.25% trypsin for 3 minutes until the cells became round. An appropriate amount of MEM complete culture medium containing 10% fetal bovine serum was added to terminate the digestion. The cells were collected into a centrifuge tube and centrifuged at 1200 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in 5 ml of MEM complete culture medium containing 10% fetal bovine serum. After counting, the cells were plated in a 96-well plate, with 100 μl per well, containing about 3000 cells, and placed at 37°C and 5% The cells were cultured overnight in a CO2 cell culture incubator. The liposome samples were diluted with culture medium and 100 μl was added to each well to make the final drug concentrations of 0.01, 0.05, 0.1, 0.5, 1.0, and 10.0 μg / ml. The cells were placed in a cell culture incubator at 37°C and 5% carbon dioxide and cultured. At 48 h, the drug solution in the 96-well plate was discarded and 200 μl of complete culture medium containing 10% CCK-8 was added. After further culture for 2 h, the absorbance value was measured at a wavelength of 450 nm using a microplate reader, and the cell viability was calculated. The cell viability results are shown in Table 1. Figure 2 .

[0102] The results showed that with the increase of fulvestrant drug concentration, the survival rate of human breast cancer MCF-7 cells continued to decrease, showing a certain dose-dependency; the half inhibitory concentration (IC 50 ) value: Example 9 < Comparative Example 2 < Comparative Example 1. This indicates that cyclodextrin-encapsulated liposomes significantly inhibited cell proliferation in human breast cancer MCF-7 cells compared to liposomes without drug inclusion. Further formulation into long-acting liposomes further effectively increased the toxicity of fulvestrant against MCF-7 cells. This mechanism may be that glycoproteins present on the cell membrane of MCF-7 cells excrete the hydrophobic drug, reducing the intracellular drug concentration and, consequently, the drug's efficacy. Formulating the drug into long-acting inclusion liposomes can effectively enhance the drug's efficacy.

Claims

1. A long-acting fulvestrant liposome for injection, characterized in that: The long-acting fulvestrant liposome for injection is composed of 1 part of fulvestrant inclusion compound, 2-20 parts of liposome membrane material, 5-30 parts of lyoprotectant, and 100-800 parts of aqueous medium by weight; the weight ratio of fulvestrant to cyclodextrin in the fulvestrant inclusion compound is 1:2-10; The preparation method of the long-acting fulvestrant liposome for injection comprises the following steps: (1) Weigh fulvestrant and cyclodextrin, dissolve them in ethanol and room temperature water for injection at 5°C to 20°C, respectively, mix slowly, stir, filter and sterilize after inclusion, and spray dry at 2°C to 15°C to obtain fulvestrant inclusion complex; (2) Weigh hydrogenated soybean lecithin, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol and dissolve them in an organic solvent. Rotary evaporate at 30°C to 40°C until a lipid film is formed. Freeze at -5°C ± 2°C for 0.5 to 1.5 h. (3) dissolving the fulvestrant inclusion complex in a buffer solution, adding it to the lipid film, and hydrating it to form crude liposomes; (4) The crude liposomes are homogenized 2 to 5 times in a microfluidizer at a homogenization pressure of 200 to 1500 bar to obtain a long-acting liposome solution; (5) Add lyophilization protective agent, microfilter, subpackage, and freeze-dry to obtain long-acting fulvestrant liposomes for injection.

2. The long-acting fulvestrant liposome for injection according to claim 1, characterized in that: The cyclodextrin is hydroxypropyl beta-cyclodextrin and / or sulfobutyl beta-cyclodextrin.

3. The long-acting fulvestrant liposome for injection according to claim 1, characterized in that: The freeze-drying protective agent is selected from at least one of mannitol, trehalose, sucrose, lactose, maltose and polyvinyl pyrrolidone.

4. The long-acting fulvestrant liposome for injection according to claim 1, characterized in that: The aqueous medium is selected from at least one of citrate buffer, phosphate buffer and carbonate buffer.

5. The preparation method according to claim 1, characterized in that The organic solvent is selected from one or more of ethanol, chloroform, dichloromethane and acetone.

6. The long-acting fulvestrant liposome for injection according to claim 1, characterized in that: The pH of the buffer solution is 6.5-8.

0.

7. Use of the long-acting fulvestrant liposome for injection according to claim 1 in the preparation of an anti-tumor drug.

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