Fulvestrant long-acting liposome for injection and preparation method thereof

Abstract

The invention belongs to the technical field of antitumor drug preparations, and relates to a fulvestrant long-acting liposome for injection and a preparation method thereof. The fulvestrant long-acting liposome for injection comprises a fulvestrant clathrate compound, a liposome membrane material, a freeze-drying protective agent and an aqueous medium. The fulvestrant long-acting liposome for injection has the clinical effects of good stability, high encapsulation efficiency, long-acting slow release and high bioavailability.

Description

Technical Field

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

[0003] Breast cancer is a disease in which abnormal breast cells grow out of control and form tumors. If not controlled, the tumors can spread throughout the body and be fatal. According to the World Health Organization, in 2022, approximately 2.3 million women worldwide will be diagnosed with breast cancer and 670,000 will die from it. Women in every country in the world can develop breast cancer at any age after puberty, but the incidence increases in later life, making it the most deadly cancer in women.

[0004] Fulvestrant is a new type of estrogen receptor down-regulator, mainly used for postmenopausal (including natural menopause and artificial menopause) estrogen receptor-positive locally advanced or metastatic breast cancer that relapses after or during anti-estrogen adjuvant therapy, or progresses during anti-estrogen therapy. At present, the commercially available Fulvestrant injection is injected into the buttocks muscle and the injection time is long, which brings great pain and inconvenience to patients. The excipient benzyl benzoate in the injection prescription has a large number of drug allergic reactions. At the same time, Fulvestrant has a first-pass effect in the liver, resulting in a very low oral bioavailability, and it is eliminated quickly in pharmacokinetics, making it difficult to maintain blood drug concentrations for a long time. Therefore, it is urgent to develop a clinical preparation of Fulvestrant that is less painful, long-acting, and highly bioavailable.

[0005] In recent years, liposome technology has developed rapidly, and new liposome technologies such as lipid nanoparticles (LNP), immunoliposomes, long-circulating liposomes, magnetic liposomes, membrane fusion liposomes and flexible liposomes have emerged. The liposome injection products currently on the market in China include doxorubicin hydrochloride liposome injection, amphotericin B liposome injection, paclitaxel liposome injection, mitoxantrone hydrochloride liposome injection and irinotecan hydrochloride liposome injection, etc., and the main indications are various tumors. Among them, long-circulating liposome technology is theoretically more suitable for the development of clinical preparations of fulvestrant. Compared with traditional liposomes, long-circulating liposomes have the following advantages: First, traditional liposomes are easily recognized by opsonins (such as complement system and apolipoprotein) in the blood, and are quickly captured and cleared by the mononuclear phagocytic system (MPS, such as liver and spleen), resulting in a short half-life. Long-circulating liposomes are modified with hydrophilic substances (such as polyethylene glycol PEG) on the surface to form a steric hindrance layer, which hinders the adsorption of plasma proteins and recognition by the immune system, thereby significantly prolonging the circulation time. For example, the half-life of doxorubicin long-circulating liposomes (Doxil) was extended from 0.2 hours of free drugs to 41-70 hours, and the area under the drug concentration-time curve (AUC) increased by more than 200 times. Second, long-circulating liposomes utilize the high permeability and retention effect of tumor tissues to enrich at lesion sites (such as tumors and inflammatory areas). Compared with traditional liposomes, the concentration of drugs in tumor tissues can be increased by 4-16 times, and they continue to act on the target through a sustained release mechanism, reducing damage to normal tissues. Third, traditional liposomes are easily accumulated in the liver and spleen due to rapid uptake by MPS, leading to organ toxicity, while long-circulating liposomes reduce MPS affinity and reduce drug release at non-target sites, thereby reducing toxicity to organs such as the heart and kidneys. The last and most important point is that long-circulating liposomes reduce liposome aggregation and drug leakage through nanoscale particle size (usually 20-200 nm) and surface modification (such as PEG brush layer), which improves drug stability and drug encapsulation rate compared to traditional liposomes.

[0006] The Chinese patent with publication number CN 107362142A discloses a fulvestrant liposome injection and its preparation process, wherein the liposome injection is mainly composed of fulvestrant, soybean lecithin and distearoylphosphatidylcholine, cholesterol and a surfactant. The Chinese patent with publication number CN 103221052 A discloses an inclusion complex containing fulvestrant and cyclodextrin.

[0007] However, traditional liposome technology or inclusion compound technology is difficult to solve the problem of the burst release of fulvestrant. Summary of the invention

[0008] To develop a more clinically suitable fulvestrant preparation. The present invention is a greater upgrade based on the technology of long-acting liposomes, and has developed 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 liposomes, inhibit the dissociation of drugs from inclusion complexes, 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 the figure. Figure 1 shown.

[0009] To achieve the above object, the long-acting fulvestrant liposome for injection of the present invention contains fulvestrant inclusion compound, liposome membrane material, lyophilization protective agent and aqueous medium, and the weight ratio of the four is 1:2-20:5-30:100-800.

[0010] Wherein, the weight ratio of fulvestrant to cyclodextrin contained in the fulvestrant inclusion compound is 1:2-10.

[0011] 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 lyophilization protectant is selected from one or more of mannitol, trehalose, sucrose, lactose, maltose, and polyvinyl pyrrolidone (PVP). The aqueous medium is selected from one or more of citrate buffer, phosphate buffer, and carbonate buffer solution, and the aqueous medium contains buffers of different pH values.

[0012] The present invention also provides a method for preparing long-acting fulvestrant liposomes for injection: (1) Weigh fulvestrant and cyclodextrin according to the prescription 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 compound; (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 is formed on the inner wall of the round-bottom flask. Freeze at -5°C ± 2°C for 0.5 to 1.5 h. (3) dissolving the prescribed amount of Fulvestrant inclusion complex in a phosphate buffer at pH 7.4, and adding the solution to the round-bottom flask in (2) to hydrate the lipid film to form crude liposomes; (4) The crude liposomes are then homogenized 2 to 5 times by a microfluidizer at a homogenization pressure of 200 to 1500 bar to obtain a long-acting liposome solution; (5) Add a 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.

[0013] The organic solvent used in the preparation process is selected from one or more of ethanol, chloroform, dichloromethane and acetone.

[0014] The long-acting fulvestrant liposome for injection provided by the present invention, its cyclodextrin inclusion compound, 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 liposome, and also improve the use compliance of clinical patients. At the same time, cyclodextrin inhibits the dissociation of the drug from the inclusion compound, 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 liposome reduces liposome aggregation and drug leakage through nanometer-scale particle size and surface modification, improves the drug burst release phenomenon, prolongs the circulation time in the blood, and improves the drug efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Schematic diagram of long-acting liposomes containing traditional liposomes and cyclodextrin.

[0016] Figure 2 : Cell viability of samples after incubation with human breast cancer MCF-7 cells for 48 h (n=3). DETAILED DESCRIPTION

[0017] In order to make the purpose and technical solution of the present invention clearer, the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited to these embodiments, and the embodiments are only used to explain 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 protection scope of the present invention.

[0018] Example 1 Table 1. Example 1 prescription:

[0019] Preparation method: Weigh fulvestrant and HP-β-CD according to the prescription amount, dissolve them in ethanol and room temperature water for injection (concentration 10% W / V), respectively, and slowly add them to the fulvestrant ethanol solution while stirring at 10°C. After the inclusion is completed, filter and sterilize, and then spray dry at low temperature (10°C) to obtain the fulvestrant inclusion complex; weigh the prescription amount of HSPC, cholesterol, and DSPE-MPEG2000, dissolve them in 500ml of chloroform, and evaporate them at 30°C~40°C until a lipid film is formed on the inner wall of the round-bottom flask, and freeze them at -5°C for 1h. Dissolve the prescription amount of fulvestrant inclusion complex in 500ml The solution was added into a round-bottom flask in a phosphate buffer solution at pH 7.4 to hydrate the lipid film to form crude liposomes; the crude liposomes were homogenized 3 times by a microfluidizer with a homogenization pressure of 1000 bar; an aqueous solution of a lyophilization protective agent containing 15 g of sucrose was added, and the mixture was filtered through 0.45 μm and 0.22 μm filter membranes in turn, packaged, and freeze-dried to obtain long-acting fulvestrant liposomes for injection.

[0020] Example 2 Table 2. Example 2 prescription:

[0021] 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 at 5°C while stirring. After the inclusion was completed, the mixture was filtered and sterilized and spray-dried at low temperature (10°C) to obtain the fulvestrant inclusion complex; the prescription amount of 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 frozen at -7°C for 0.5 h, and 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 4 times by a microfluidizer with a homogenization pressure of 1000 bar; an aqueous solution of a lyophilized protective agent containing 15 g of trehalose was added, and the mixture was successively 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.

[0022] Example 3 Table 3. Example 3 prescription:

[0023] 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; the prescription amount of 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 the round-bottom flask, and then frozen at -3°C for 1.5 h, and 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 3 times by a microfluidizer with a homogenization pressure of 1000 bar; an aqueous solution containing 55 g of lactose lyophilization protective agent was added, and the mixture was successively 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.

[0024] Example 4 Table 4. Example 4 prescription:

[0025] Preparation method: Weigh fulvestrant and SBECD according to the prescription amount, dissolve them in ethanol and room temperature water for injection (concentration 10% W / V) respectively, slowly add them to the fulvestrant solution while stirring at 20°C, filter and sterilize after inclusion, and spray dry at low temperature (15°C) to obtain fulvestrant inclusion complex; weigh the prescription amount of HSPC, cholesterol, and DSPE-MPEG2000, dissolve them in 500ml of dichloromethane, evaporate at 30°C~40°C until a lipid film is formed on the inner wall of the round-bottom flask, freeze at -5°C for 0.5h, and dissolve the prescription amount of fulvestrant inclusion complex in 500ml The solution was added into a round-bottom flask in a citrate buffer solution at pH 7.4 to hydrate the lipid film to form crude liposomes; the crude liposomes were homogenized three times by a microfluidizer at a homogenization pressure of 1000 bar; an aqueous solution of a lyophilization protective agent containing 100 g of mannitol was added, and the mixture was filtered through 0.45 μm and 0.22 μm filter membranes in turn, packaged, and freeze-dried to obtain long-acting fulvestrant liposomes for injection.

[0026] Example 5 Table 5. Example 5 prescription:

[0027] 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 (2°C) to obtain the Fulvestrant inclusion complex; the prescription amount of 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, and 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 3 times by a microfluidizer with a homogenization pressure of 1000 bar; an aqueous solution of a lyophilization protective agent containing 100 g of maltose was added, and the mixture was successively 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.

[0028] Example 6 Table 6. Example 6 prescription:

[0029] Preparation method: Weigh fulvestrant and SBECD according to the prescription amount, dissolve them in ethanol and room temperature water for injection (concentration 10% W / V) respectively, slowly add them to the fulvestrant solution while stirring at 10°C, filter and sterilize after inclusion, and spray dry at low temperature (5°C) to obtain fulvestrant inclusion complex; weigh the prescription amount of HSPC, cholesterol, and DSPE-MPEG2000, dissolve them in 500ml of acetone, evaporate at 30°C~40°C until a lipid film is formed on the inner wall of the round-bottom flask, freeze at -7°C for 1.5h, and dissolve the prescription amount of fulvestrant inclusion complex in 400ml The solution was added into a round-bottom flask in a carbonate buffer solution at pH 8.0 to hydrate the lipid film to form crude liposomes; the crude liposomes were homogenized three times by a microfluidizer at a homogenization pressure of 700 bar; an aqueous solution of a lyophilization protective agent containing 30 g of polyvinyl pyrrolidone was added, and the mixture was filtered through 0.45 μm and 0.22 μm filter membranes in turn, packaged, and freeze-dried to obtain long-acting fulvestrant liposomes for injection.

[0030] Example 7 Table 7. Example 7 prescription:

[0031] 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; the prescription amount of 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 frozen at -3°C for 0.5 h, and 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 a lyophilization protectant containing 60 g of mannitol was added, and the mixture was successively 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.

[0032] Example 8 Table 8. Example 8 prescription:

[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 at 15°C while stirring. 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; the prescription amount of 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 frozen at -5°C for 1 hour, and the prescription amount of Fulvestrant inclusion complex was dissolved in 200 ml of carbonate buffer solution with a pH of 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 a lyophilization protective agent containing 66 g of sucrose was added, and the mixture was successively 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 9 Table 9. Example 9 prescription:

[0035] 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; the prescription amount of 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 frozen at -5°C for 1 hour, and 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 a lyophilization protective agent containing 32 g of sucrose, and successively 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.

[0036] Comparative Example 1 Table 10. Comparative Example 1 Prescription:

[0037] Preparation method: Weigh the prescribed amount of HSPC, cholesterol, and fulvestrant and dissolve them in 1000 ml of chloroform, perform rotary evaporation at 30°C-40°C until a lipid film is formed on the inner wall of a round-bottom flask, and freeze at -5°C for 1 hour. Add 200 ml of pH 7.4 phosphate buffer into the round-bottom flask to hydrate the lipid film to form crude liposomes. The crude liposomes are then homogenized 5 times by a microfluidizer with a homogenization pressure of 1300 bar. Add a lyophilization protective agent aqueous solution containing 32 g of sucrose, filter through 0.45 μm and 0.22 μm filter membranes in turn, package, and freeze-dry to obtain fulvestrant liposomes for injection.

[0038] Comparative Example 2 Table 11. Comparative Example 2 Prescription:

[0039] 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; the prescription amount of HSPC and cholesterol 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 frozen at -5°C for 1 hour, and 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 a lyophilization protective agent containing 32 g of sucrose, and successively 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.

[0040] Comparative Example 3 Table 12. Comparative Example 3 Prescription:

[0041] 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 sterilized by filtration and spray dried (25°C) to obtain the Fulvestrant inclusion complex; the prescription amount of 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 frozen at -5°C for 1 hour, and 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 a lyophilization protective agent containing 32 g of sucrose, and successively 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] Comparative Example 4 Table 13. Comparative Example 4 Prescription:

[0043] 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; the prescription amount of 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 a lyophilized protective agent containing 32 g of sucrose, and successively 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.

[0044] Comparative Example 5 Table 14. Comparative Example 5 Prescription:

[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 was completed, the mixture was sterilized by filtration and spray dried at low temperature (10°C) to obtain the Fulvestrant inclusion complex; the prescription amount of 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, and 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 a lyophilized protective agent containing 32 g of sucrose, and successively 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.

[0046] Experiment 1: Critical Quality Control of Liposome Formulation

[0047] (1) Properties: The properties of the examples and comparative examples are all white loose lumps.

[0048] (2) Particle size and zeta potential Sample preparation: Take an appropriate amount of liposome samples prepared in the examples and comparative examples, add them into phosphate buffer and dilute to 5 ml, filter through a 0.22 μm PES filter membrane, take an appropriate amount of the drug solution and add it into the sample pool, and use a zetasize dynamic light scattering particle size analyzer (model: Nanozs, manufacturer: Malvern Panalytical) to detect the particle size and potential. The results are shown in the table below.

[0049] Table 15 Average particle size and Zeta potential test results of samples

[0050] The test shows that compared with comparative examples 1 to 5, embodiments 1 to 9 have a smaller average particle size and are more stable, indicating good process repeatability. At the same time, the smaller the particle size, the stronger the targeting and the better the stability of the membrane. The charge of the liposome can reduce mutual aggregation and fusion, increase stability, and when the absolute value of the Zeta potential (mV) is -30 to -50 mV, the liposome is relatively stable, that is, the embodiment is significantly better than the comparative example. It should be noted that after a single factor experimental investigation, the quick freezing step of the liposome film has a great influence on the quality of the drug-loaded liposome. The drug-loaded liposome prepared without quick freezing treatment has loose lipid arrangement, low drug loading and poor sustained release effect. The compactness of the lipid film after 4°C cold storage treatment is also not ideal. The quick freezing treatment of the lipid film in the present invention can make the hydrophobic tail chains in the film highly ordered, the hydrogen bond network between the polar head groups is tighter, and a gel phase is formed to achieve densification of the membrane structure and improve the drug distribution and encapsulation capacity.

[0051] (3) Encapsulation efficiency and in vitro release 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 placed in a hydration medium and sealed. Magnetic stirring was started, and the dialysis fluid was taken at 0.5, 1, 2, 4, 6, 8, 12, and 24 hours, respectively, and an equal amount of hydration medium was added at the same time. The samples were filtered through a 0.22 μm PES filter membrane and determined by HPLC. The encapsulation efficiency was calculated based on the results.

[0052] The in vitro release of liposomes was determined by dialysis. An appropriate amount of liposomes was accurately weighed and placed in a dialysis bag, which was then placed in a PBS buffer solution (temperature 37°C) containing 5% calf serum at pH 7.4. The bag was sealed and magnetic stirring was started. The dialyzed liquid was taken 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 sample was filtered through a 0.22μm PES filter membrane and determined 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.

[0053] Table 16 Sample encapsulation efficiency and in vitro release test results

[0054] The test shows that Fulvestrant is insoluble 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 treatment, the liposomes prepared by Examples 1 to 9 after cyclodextrin inclusion have significantly improved encapsulation efficiency, reduced drug leakage, and increased drug loading; compared with Comparative Examples 1 to 5, the liposomes prepared by cyclodextrin inclusion in Examples 1 to 9 have obvious sustained-release effect of drug release in liposomes, and no burst release phenomenon.

[0055] Experiment 2: Liposome compatibility and long-term stability An appropriate amount of the long-acting fulvestrant liposomes for injection prepared in the example was taken, diluted to 2.5 mg / ml of fulvestrant by adding 5% glucose injection, and the compatibility stability after being placed for 8 hours was examined; the finished liposomes prepared in some examples (Examples 2, 4, 6, and 8) were directly taken and placed at 2-8°C for a long time and at 25°C for 6 months to examine their stability. The results are shown in the table below.

[0056] Table 17 Liposome compatibility stability results

[0057] Note: “☆” represents a colorless to milky white clear liquid with no precipitation; the content at 0h is 100%.

[0058] Table 18 Long-term and accelerated stability results of liposomes

[0059] Note: “★” represents white loose lumps.

[0060] The experiment showed that after the liposomes prepared with different prescriptions were matched with 5% glucose injection, they were stable at a concentration of 2.5 mg / ml for 8 hours, and the matching stability was good; the liposomes prepared with different prescriptions had no obvious changes in properties, encapsulation rate, and related aspects under the conditions of long-term storage 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 could be used in clinical practice.

[0061] Test 3: In vitro pharmacodynamics test 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 taken, digested with 0.25% trypsin for 3 min until the cells became round, and 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, centrifuged at 1200 rpm for 5 min, 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 continued to be 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 by an enzyme reader, and the cell survival rate was calculated. The cell survival rate results are shown in Figure 2 .

[0062] The experiment showed that with the increase of fulvestrant drug concentration, the survival rate of human breast cancer cell MCF-7 continued to decrease, showing a certain dose-dependency; the half inhibitory concentration (IC 50 ) value: Example 9 < Comparative Example 2 < Comparative Example 1, that is, the liposomes encapsulated by cyclodextrin have a significant cell proliferation inhibitory effect on human breast cancer cells MCF-7 compared with liposomes without encapsulation of drugs, and further preparing long-acting liposomes can effectively increase the toxicity of Fulvestrant drugs to human breast cancer cells MCF-7; its mechanism may be that the glycoproteins present on the cell membrane of human breast cancer cells MCF-7 expel the hydrophobic drugs taken in, thereby reducing the drug concentration in the cells and thus reducing the drug efficacy. The drug can be effectively improved by preparing the drug into inclusion compound long-acting liposomes.

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 lyophilization protective agent, and 100-800 parts of aqueous medium by weight ratio; the weight ratio of fulvestrant to cyclodextrin in the fulvestrant inclusion compound is 1:2-10.

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 liposome membrane material is selected from at least one of hydrogenated soybean lecithin, distearoylphosphatidylethanolamine-polyethylene glycol, and cholesterol.

4. 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.

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

6. A method for preparing the long-acting fulvestrant liposome for injection according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: (1) Weighing fulvestrant and cyclodextrin, dissolving them in ethanol and room temperature water for injection respectively at low temperature, slowly mixing and stirring, filtering and sterilizing after the inclusion is completed, and spray drying at low temperature to obtain fulvestrant inclusion complex; (2) Weigh hydrogenated soybean lecithin, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol and dissolve them in an organic solvent, evaporate them at 30°C to 40°C until a lipid film is formed, and freeze them at -5°C ± 2°C for 0.5 to 1.5 h; (3) dissolving the fulvestrant inclusion complex in a buffer solution, adding the solution to the lipid film, and hydrating the solution to form crude liposomes; (4) The crude liposomes are homogenized 2 to 5 times by a microfluidizer at a homogenization pressure of 200 to 1500 bar to obtain a long-acting liposome solution; (5) Add a lyophilization protective agent, perform microfiltration, subpackage, and freeze-dry to obtain long-acting fulvestrant liposomes for injection.

7. The preparation method according to claim 6, characterized in that: The organic solvent is selected from one or more of ethanol, chloroform, dichloromethane and acetone.

8. The preparation method according to claim 6, characterized in that: The pH of the buffer solution is 6.5-8.

0.

9. The preparation method according to claim 6, characterized in that: The step (1) comprises: weighing fulvestrant and cyclodextrin, dissolving them in ethanol and room temperature injection water respectively at 5°C to 20°C, slowly mixing and stirring, filtering and sterilizing after the inclusion, and spray drying at 2°C to 15°C to obtain the fulvestrant inclusion complex.

10. Use of the long-acting fulvestrant liposome for injection according to claim 1 in the preparation of anti-tumor drugs.

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