A highly stable amphotericin B liposome and its preparation method

Abstract

This invention discloses a highly stable amphotericin B liposome and its preparation method, belonging to the field of pharmaceutical formulation technology. This invention utilizes a methanol-water-chloroform ternary mixed solvent system to achieve complete molecular-level dissolution of the raw materials, eliminating the conventional drying process and directly hydrating and homogenizing. It also provides optimal formulation ratios and process parameters. The preparation method includes steps such as oil phase preparation, aqueous phase preparation, homogenization, incubation, cooling, filtration, volumetric filling, and lyophilization. This invention creatively proposes optimal formulation composition and process conditions, resulting in a product with high encapsulation efficiency, uniform particle size, and strong stability. Furthermore, the preparation process is simple and easily industrialized, solving the problems of complex preparation processes and poor stability of existing injectable amphotericin B liposomes, and possesses good clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, specifically relating to a highly stable amphotericin B liposome and its preparation method. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Amphotericin B is a broad-spectrum antifungal drug with significant efficacy against infections caused by various deep fungi, and its clinical application is widespread. However, its poor water solubility and high toxicity limit its clinical use. Liposomes, as drug carriers, have good biocompatibility. The slow release of amphotericin B encapsulated in liposomes maintains a low blood concentration of free amphotericin B in patients, reduces renal filtration, and improves nephrotoxicity, thus becoming an important route for improving the administration characteristics of amphotericin B.

[0004] AmBisome, the world's first approved nanoliposome drug (in Europe in 1990), was developed by NeXstar (USA). The liposome's bilayer structure consists of a specific ratio of amphoteric molecules phosphatidylcholine (HSPC), diesterol glycerol phosphate (DSPG), and cholesterol. AmBisome binds to phospholipids and cholesterol in a non-covalent manner through hydrogen bonding and intermolecular forces.

[0005] The existing method for preparing amphotericin B liposomes for injection includes the following steps: Step 1. Oil phase preparation: The drug and phospholipid raw material are dissolved in an organic solvent to ensure uniform mixing at the molecular level to form an oil phase solution. In the dissolved state, drug molecules and the polar heads of phospholipids form ion-pair complexes through charge interaction. Step 2. Spray drying to form lipid powder: The solution containing the complex is rapidly dried using a spray drying process to remove the organic solvent, resulting in a dry, free-flowing lipid powder. Step 3. Hydration to form MLVs (multilayer liposomes): The lipid powder is added to an aqueous medium for hydration. Phospholipid molecules spontaneously arrange themselves into a bilayer structure in the aqueous phase, encapsulating the aqueous phase to form multilayer vesicles (MLVs). The drug is embedded in the lipid bilayer in the form of a complex, achieving initial encapsulation. Step 4. Homogenization to form SUVs (small single-compartment liposomes): The particle size of the MLVs is reduced and homogenized using high-pressure homogenization or extrusion to form small single-compartment liposomes (SUVs) with particle sizes in the nanometer range. This step improves the uniformity and stability of the liposomes and further enhances the encapsulation effect of the drug. Step 5. Filter for sterilization, fill, and freeze-dry to form solid liposome freeze-dried cakes. Rehydration before use will restore the liposome suspension.

[0006] Currently available methods for preparing amphotericin B liposomes all require removing organic solvents from the liposome complex dissolved in the oil phase through spray drying or rotary evaporation. On the one hand, spray drying equipment occupies a large area, and on the other hand, the spray drying process has a long cycle, resulting in low production efficiency, complex processes, and high difficulty in industrialization. Even under complex process conditions, the encapsulation efficiency still needs to be improved, and some formulations have problems such as uneven particle size distribution, poor stability, and reduced encapsulation efficiency during storage. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the purpose of this invention is to provide a highly stable amphotericin B liposome and its preparation method.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a method for preparing highly stable amphotericin B liposomes, comprising the following steps: Step 1, oil phase preparation: Add methanol and water to amphotericin B and liposome membrane material, stir continuously, add chloroform, heat, add hydrochloric acid solution to adjust the pH to 1-3, stir until clear, adjust the pH to neutral and cool for later use; The liposome membrane material contains phospholipid compounds and sterol compounds; Step 2, Aqueous phase preparation: Dissolve the freeze-drying protectant and pH adjuster in water and stir to dissolve; Step 3, Homogenization: The oil phase obtained in Step 1 and the aqueous phase obtained in Step 2 are mixed and homogenized in a homogenizer at a pressure of 3000~3500psi to a particle size of 30~100nm. Step 4, incubation, cooling and filtration: The homogenized liquid from step 3 is incubated, cooled to ≤25℃, and then subjected to pre-filtration and sterilization filtration in sequence; Step 5, volume adjustment, filling and freeze drying: adjust the volume to 3.5~4.5mg / g according to the content test results, fill at a controlled temperature of 2~8℃, and cap after freeze drying.

[0009] In step 1, the volume ratio of methanol to water added to amphotericin B and the liposome membrane material is 1:1.2~1.5; the mass ratio of methanol to amphotericin B is 1.8×10⁻⁶. 3 ~2.4×10 3 The stirring speed is 18~22 rpm.

[0010] In step 1, the amount of chloroform added is 1.25 to 2.6 times the volume of methanol.

[0011] In step 1, control the reaction temperature at 45~55℃, add hydrochloric acid solution and stir until completely dissolved; increase the rotation speed to 30~34 rpm, add sodium hydroxide solution, stir until clear, and cool down to 15~25℃.

[0012] Furthermore, in step 1, the antioxidant is dissolved in chloroform.

[0013] Further, in step 1, add hydrochloric acid solution and stir for 6-10 minutes until completely dissolved; increase the speed to 30-34 rpm, add sodium hydroxide solution, stir for 6-8 minutes until clear, cool to 15-25℃, and reduce the speed to 5-7 rpm for later use (let stand for no more than 24 hours).

[0014] In step 1, the present invention selects 45~55℃ as the core temperature control point for oil phase dissolution. This is an optimal design based on multiple dimensions, including raw material dissolution characteristics, compatibility of mixed solvent systems, drug stability, and subsequent process connections. The core reasons are as follows: 1. Compatible with methanol-water-chloroform ternary mixed solvent systems to achieve complete molecular-level dissolution of raw materials: Amphotericin B is a lipid-soluble amphoteric molecule with poor solubility in both water and pure organic phases. The hydrophobic fatty acid tails of the liposome membrane material (HSPC / DSPG-Na / cholesterol) tend to aggregate at low temperatures. The comparative study in this invention found that 45~55℃ is the optimal dissolution temperature for this ternary mixed solvent. At this temperature, the solvent molecule motion rate is moderate, which can break the hydrogen bond association between amphotericin B molecules and disperse the hydrophobic aggregates of phospholipids / cholesterol, so that the drug and membrane material are uniformly dispersed at the molecular level, forming a clear, particle-free oil phase solution, which lays a uniform foundation for the subsequent self-assembly of liposomes.

[0015] 2. The temperature is moderate, avoiding raw material degradation and balancing dissolution efficiency and stability: The polyene double bonds of amphotericin B are easily oxidized at ≥60℃, and the unsaturated fatty acid chains of phospholipids undergo ester bond hydrolysis at high temperatures. At <40℃, the dissolution rate is too slow, requiring extended stirring time (>15min), which increases the risk of oxidation from contact with oxygen. 45~55℃ is the equilibrium temperature between dissolution efficiency and degradation risk, and complete dissolution can be achieved in just 8min, significantly shortening the residence time of the raw material in the dissolution stage and reducing oxidation or hydrolytic degradation.

[0016] 3. Seamlessly connects with subsequent homogenization temperatures (50~60℃), reducing energy consumption for process temperature control: The optimal temperature for the subsequent homogenization step in this invention is 50~60℃, and the oil phase dissolution temperature is controlled at 45~55℃. This allows the oil phase to be directly mixed with the water phase and enter the homogenization process without additional heating or cooling, reducing temperature fluctuations in the process. This reduces the energy consumption of temperature control equipment in industrial production, avoids the aggregation of raw material molecules caused by sudden temperature changes, and ensures the homogeneity of the oil-water phase mixture.

[0017] 4. Promotes the dispersion of the antioxidant α-tocopherol, enhancing its antioxidant effect: α-Tocopherol is a lipid-soluble antioxidant that tends to accumulate locally in the chloroform phase at low temperatures and cannot be uniformly dispersed in the oil phase system. At 45~55℃, α-Tocopherol can be completely dispersed in a ternary mixed solvent, forming a uniform antioxidant film on the surface of the drug / phospholipid molecules, effectively inhibiting the oxidation reaction during the dissolution stage.

[0018] In step 2, weigh the freeze-drying protectant and pH adjuster, add water, stir to dissolve, and obtain an aqueous solution of freeze-drying protectant with pH 5.5~6.0.

[0019] In step 3, the homogenization temperature is set to 50~60℃, and the liquid obtained in step 1 is homogenized repeatedly through a homogenizer (Y-type homogenization chamber, 75 micrometer pore size) until the particle size D50 reaches 40~100nm. Further, it is stopped when the particle size D50 reaches 45~95nm.

[0020] In step 4, the homogenized solution is transferred to an incubation tank and incubated for 2-4 hours.

[0021] Further, in step 4, the cooling and filtration steps are as follows: the liquid is cooled to 22°C by a heat exchanger, and then sequentially conveyed to a sterile storage tank through a pre-filter cartridge (Cobetter, SMDS / SAFS material, 40 inches, 2 in parallel) and a sterile filter cartridge (Cobetter, SMDS / SAFS material, 40 inches, 1 for each stage); 20 kg of sucrose hydrate is added to the incubation tank, and after being processed through the same process, it is added to the sterile storage tank, stirred for 15 min, and then cooled to 5°C and kept warm.

[0022] As optional materials, the phospholipid compounds include one or more of the following: lecithin and its derivatives, soybean lecithin and its derivatives, cephalin and its derivatives, cardiolipin and its derivatives, phosphatidylinositol and its derivatives, phosphatidylserine and its derivatives, phosphatidylglycerol and its derivatives, phosphatidic acid and its derivatives, phosphatidylethanolamine and its derivatives, phosphatidylcholine and its derivatives, phosphatidylinositol and its derivatives, sphingomyelin and its derivatives, and hydrogenated products of the above substances.

[0023] Phospholipid compounds are mixtures of phosphatidylcholine or its derivatives and diesterol glycerol phosphate or its derivatives.

[0024] Furthermore, the phospholipid compound is a mixture of distearate phosphatidylglycerol and hydrogenated soybean phospholipids.

[0025] In step 1, the liposome membrane material contains hydrogenated soybean phosphatidylcholine (HSPC), sodium distearate phosphatidylglycerol (DSPG-Na), and cholesterol, with a mass ratio of 2~3:1.2~1.8:1.

[0026] In step 1, the antioxidant is α-tocopherol; in step 2, the freeze-drying protectant is sucrose; and the pH adjuster is disodium succinate hexahydrate, which is used in combination with hydrochloric acid or sodium hydroxide to adjust the pH.

[0027] The mass ratio of materials in steps 1 and 2 is: Amphotericin B: Liposome membrane material: Antioxidant: Lyophilization protectant: pH adjuster = 40~60: 200~300mg: 0.5~2mg: 800~1000: 30~40.

[0028] This invention has found that the ratio optimization of liposome membrane materials in the prior art is insufficient, resulting in poor structural stability of liposomes and affecting drug storage and clinical efficacy.

[0029] This invention selects hydrogenated soybean phosphatidylcholine (HSPC), sodium distearate phosphatidylglycerol (DSPG-Na), and cholesterol as liposome membrane materials. The three are compounded in a specific weight ratio to form a stable bilayer structure. α-Tocopherol is added as an antioxidant to effectively prevent lipid oxidation. Sucrose is used as a freeze-drying protectant to improve the stability of the formulation after freeze-drying. Disodium succinate hexahydrate is used as a pH adjuster to maintain the pH stability of the formulation.

[0030] This invention employs a process of preparing oil and aqueous phases separately and then mixing and homogenizing them. By controlling the homogenization pressure and temperature, the uniformity of liposome particle size is ensured. The invention develops steps for oil phase preparation, homogenization incubation, and cooling filtration. The optimal homogenization temperature range is 50-60℃, and the optimal temperature range after incubation is 20-25℃. This temperature range is conducive to the formation of a stable liposome membrane, reducing drug leakage and improving encapsulation efficiency. Strict control of filling and lyophilization conditions further enhances the stability of the formulation.

[0031] Under the preparation process conditions provided by this invention, and based on the above optimized formulation, highly stable amphotericin B liposomes are provided.

[0032] Therefore, this invention provides an injectable amphotericin B liposome with a reasonable formulation, simple process, and high stability, which has important practical significance.

[0033] In a second aspect, the present invention provides highly stable amphotericin B liposomes prepared by the preparation method described in the first aspect.

[0034] The encapsulation efficiency of the highly stable amphotericin B liposomes is higher than 94%; after storage at 25°C for 24 months, the increase in related substances is no more than 5%.

[0035] Thirdly, the present invention provides an amphotericin B liposome injection solution, which is prepared by dissolving the highly stable amphotericin B liposomes described in the second aspect in a solvent.

[0036] This invention employs a methanol-water-chloroform ternary mixed solvent system to achieve complete molecular-level dissolution of raw materials, eliminating the conventional drying process and directly hydrating and homogenizing. It also provides optimal formulation ratios and process parameters. In conventional processes, under high vacuum conditions, as the low-boiling-point organic solvent rapidly evaporates, the lipid components are concentrated and hydrated in the remaining aqueous phase. Through initial self-assembly, they gradually form drug-containing phospholipid clusters and large vesicles of varying morphologies. The liposome structures are heterogeneous, resulting in poor encapsulation and instability. The process of this invention involves direct hydration and homogenization. Under high pressure, after multiple crushing and self-assembly processes, small single-chamber liposome structures are formed, each containing a certain volume of aqueous phase encapsulated by a drug-containing phospholipid bilayer. Therefore, the resulting drug-containing phospholipid bilayer has uniform particle size, excellent encapsulation, and product stability. Furthermore, the process is simple and easy to industrialize.

[0037] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows: 1. High encapsulation rate: Under the formulation ratio and process parameters provided by this invention, the prepared drug can be efficiently encapsulated in the liposome bilayer or the internal aqueous phase, with an encapsulation rate of 94% or more, reducing the toxicity risk of free drugs.

[0038] 2. High stability: The liposome membrane material ratio and formulation composition of the present invention result in a long storage period at 25°C, small particle size changes, and stable encapsulation efficiency, effectively solving the problem of poor stability of existing formulations and avoiding the increase in toxicity caused by the decrease in encapsulation efficiency during drug storage.

[0039] 3. Simple process and easy to industrialize: The preparation process does not require complex equipment, key process parameters are easy to control, suitable for large-scale production, and reduces production costs. Attached Figure Description

[0040] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0041] Figure 1 These are microscopic structural diagrams of liposomes; where a is a microscopic structural diagram of the product of Example 1 of the present invention, and b is a microscopic structural diagram of a commercially available product. Detailed Implementation

[0042] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0043] The present invention will be further described below with reference to the embodiments.

[0044] Example 1: Preparation of Amphotericin B Liposomes for Injection (Formula 1) 1. Formula composition (1000 vials): Amphotericin B 50mg / vial, HSPC 125mg / vial, DSPG-Na 73mg / vial, Cholesterol 51mg / vial, α-Tocopherol 1mg / vial, Sucrose 900mg / vial, Disodium succinate hexahydrate 35mg / vial, Methanol (100kg), Chloroform (250kg), Water for injection (120kg in the oil phase + appropriate amount in the aqueous phase).

[0045] 2. Preparation steps: (1) Preparation of oil phase: Add amphotericin B, DSPG-Na, HSPC and cholesterol to the oil phase tank, add methanol and room temperature water for injection, and start stirring (50% speed, 20 rpm); take 1 kg of chloroform to dissolve α-tocopherol and pour it into the oil phase tank, rinse the container with chloroform 3 times (1 kg each time), add chloroform to make up to 250 kg; control the temperature at 50℃, add 200 mL of 1 mol / L hydrochloric acid solution to adjust the pH value to 2.0, stir for 8 min until completely dissolved; increase the speed to 80%, slowly add 180 mL of 1 mol / L sodium hydroxide solution to adjust the pH value to 6.5, stir for 7 min until clear, cool down to 19℃, reduce the speed to 15% for later use (store for no more than 24 h).

[0046] (2) Preparation of aqueous phase: Weigh sucrose and disodium succinate hexahydrate into a hydration tank, add room temperature water for injection to a final volume of 50 kg, and stir to dissolve.

[0047] (3) Homogenization: Set the homogenization temperature to 55°C, and pass the oil phase obtained in step 1 and the aqueous phase liquid obtained in step 2 through a homogenizer (Y-type homogenization chamber, 75 micrometers of pores) under a pressure of 3500 psi for repeated homogenization until the particle size D50 reaches 65 nm.

[0048] (4) Incubation and rinsing: Transfer the homogenized liquid to the incubation tank, rinse the homogenization system twice with 10 kg of sucrose hydrated liquid prepared in step 2 after filtration with pH 5.0, add the rinsing liquid into the incubation tank, add sucrose hydrated liquid to make up to 8 kg, and incubate for 3 hours.

[0049] (5) Cooling and filtration: The liquid is cooled to 22°C by a heat exchanger and then conveyed to the sterile storage tank through a pre-filter cartridge (Cobetter, SMDS / SAFS material, 40 inches, 2 in parallel) and a sterile filter cartridge (Cobetter, SMDS / SAFS material, 40 inches, 1 for each stage); 20 kg of sucrose hydrate is added to the incubation tank, and after being processed in the same way, it is added to the sterile storage tank, stirred for 15 min, and cooled to 5°C and kept warm.

[0050] (6) Volume adjustment and freeze drying: After sterilization and filtration, sample and measure the content. Adjust the volume to 4.0 mg / g with sterilized sucrose hydrate solution, fill at 5℃, freeze dry after half-stopping, and then cap to obtain the finished product.

[0051] Example 2: Preparation of Amphotericin B Liposomes for Injection (Formula 2) 1. Formula composition (1000 vials): Amphotericin B 50mg / vial, HSPC 125mg / vial, DSPG-Na 73mg / vial, Cholesterol 51mg / vial, α-Tocopherol 1mg / vial, Sucrose 900mg / vial, Disodium succinate hexahydrate 35mg / vial, Methanol (total 90kg), Chloroform (total 220kg), Water for injection (oil phase 110kg + appropriate amount of aqueous phase).

[0052] 2. Preparation steps: (1) Preparation of oil phase: Add the prescribed amount of amphotericin B and liposome membrane material to the oil phase tank, add methanol and water for injection, and stir (45% speed); dissolve α-tocopherol in 0.8 kg of chloroform and add it, rinse the container 3 times (0.8 kg each time), add chloroform to make up to 220 kg; control the temperature at 48℃, add hydrochloric acid solution and stir for 6 min to dissolve, increase the speed to 75%, add sodium hydroxide solution and stir for 6 min to clarify, cool down to 18℃, and adjust the speed to 15% for later use.

[0053] (2) Preparation of aqueous phase: Weigh sucrose and disodium succinate hexahydrate, add water for injection to make up to 45 kg, and stir to dissolve.

[0054] (3) Homogenization: Homogenization temperature 52℃, homogenization pressure 3100psi, reciprocating homogenization until particle size D50 reaches 58nm.

[0055] (4) Incubation and rinsing: Transfer the material solution to the incubation tank, rinse the homogenization system twice with 10kg of pH5.0 sucrose hydrate, add sucrose hydrate to make up the volume, and incubate for 3 hours.

[0056] (5) Cooling and filtration: Cool down to 20°C, filter through a designated filter element and then transport to a sterile storage tank. Stir for 12 minutes and cool down to 4°C for heat preservation.

[0057] (6) Volume adjustment and freeze drying: Volume adjustment to 3.8 mg / g, temperature control at 4℃ for filling, freeze drying and capping to obtain the finished product.

[0058] Example 3: Preparation of Amphotericin B Liposomes for Injection (Formula 3) 1. Formula composition (1000 vials): Amphotericin B 50mg / vial, HSPC 125mg / vial, DSPG-Na 73mg / vial, Cholesterol 51mg / vial, α-Tocopherol 1mg / vial, Sucrose 900mg / vial, Disodium succinate hexahydrate 35mg / vial, Methanol (total volume 110kg), Chloroform (total volume 280kg), Water for injection (oil phase 130kg + appropriate amount of aqueous phase).

[0059] 2. Preparation steps: (1) Oil phase preparation: Add each raw material to the oil phase tank, add methanol and water for injection, and stir (55% speed); dissolve α-tocopherol in 1.2 kg of chloroform and add it, rinse 3 times (1.2 kg each time), add chloroform to make up to 280 kg; control the temperature at 52℃, add hydrochloric acid solution and stir for 7 min to dissolve, increase the speed to 85%, add sodium hydroxide solution and stir for 8 min to clarify, cool down to 20℃, and adjust the speed to 15% for later use.

[0060] (2) Preparation of aqueous phase: Weigh sucrose and disodium succinate hexahydrate, add water for injection to make up to 55 kg, and stir to dissolve.

[0061] (3) Homogenization: Homogenization temperature 58℃, homogenization pressure 3300psi, homogenization until particle size D50 reaches 72nm.

[0062] (4) Incubation and rinsing: Transfer the liquid to the incubation tank, rinse the homogenization system twice, add sucrose hydrate to make up the volume, and incubate for 3 hours.

[0063] (5) Cooling and filtration: Cool down to 23°C, filter through the filter element and then transport to a sterile storage tank, stir for 18 minutes, cool down to 6°C and keep warm.

[0064] (6) Volume adjustment and freeze drying: Volume adjusted to 4.2 mg / g, temperature controlled at 6℃ for filling, freeze-drying and then capping to obtain the finished product.

[0065] Example 4: Preparation of Amphotericin B Liposomes for Injection (Formula 4) 1. Formula composition (1000 vials): Amphotericin B 50mg / vial, HSPC 125mg / vial, DSPG-Na 73mg / vial, Cholesterol 51mg / vial, α-Tocopherol 1mg / vial, Sucrose 900mg / vial, Disodium succinate hexahydrate 35mg / vial, Methanol (total volume 80kg), Chloroform (total volume 200kg), Water for injection (oil phase 100kg + appropriate amount of aqueous phase).

[0066] 2. Preparation steps: (1) Oil phase preparation: Add the raw materials to the oil phase tank, add methanol and water for injection, and stir (40% speed); dissolve α-tocopherol in 0.6 kg of chloroform and add it, rinse 3 times (0.6 kg each time), add chloroform to make up to 200 kg; control the temperature at 45℃, add hydrochloric acid solution and stir for 5 min to dissolve, increase the speed to 70%, add sodium hydroxide solution and stir for 5 min to clarify, cool down to 17℃, and adjust the speed to 15% for later use.

[0067] (2) Preparation of aqueous phase: Weigh sucrose and disodium succinate hexahydrate, add water for injection to make up to 40 kg, and stir to dissolve.

[0068] (3) Homogenization: Homogenization temperature 50℃, homogenization pressure 3000psi, homogenization until particle size D50 reaches 45nm.

[0069] (4) Incubation and rinsing: Transfer the liquid to the incubation tank, rinse the homogenization system twice, add sucrose hydrate to make up the volume, and incubate for 3 hours.

[0070] (5) Cooling and filtration: Cool down to 21°C, filter through the filter element and then transport to a sterile storage tank. Stir for 10 minutes and cool down to 3°C for heat preservation.

[0071] (6) Volume adjustment and freeze drying: Volume adjustment to 3.5 mg / g, temperature control at 3℃ for filling, freeze drying and capping to obtain the finished product.

[0072] Example 5: Preparation of Amphotericin B Liposomes for Injection (Formula 5) 1. Formula composition (1000 vials): Amphotericin B 50mg / vial, HSPC 125mg / vial, DSPG-Na 73mg / vial, Cholesterol 51mg / vial, α-Tocopherol 1mg / vial, Sucrose 900mg / vial, Disodium succinate hexahydrate 35mg / vial, Methanol (total volume 120kg), Chloroform (total volume 300kg), Water for injection (oil phase 150kg + appropriate amount of aqueous phase).

[0073] 2. Preparation steps: (1) Oil phase preparation: Add the raw materials to the oil phase tank, add methanol and water for injection, and stir (60% speed); dissolve α-tocopherol in 2kg of chloroform and add it, rinse 3 times (2kg each time), add chloroform to make up to 300kg; control the temperature at 55℃, add hydrochloric acid solution and stir for 10min to dissolve, increase the speed to 90%, add sodium hydroxide solution and stir for 10min to clarify, cool down to 21℃, and adjust the speed to 15% for later use.

[0074] (2) Preparation of aqueous phase: Weigh sucrose and disodium succinate hexahydrate, add water for injection to make up to 60 kg, and stir to dissolve.

[0075] (3) Homogenization: Homogenization temperature 60℃, homogenization pressure 3500psi, homogenization until particle size D50 reaches 95nm.

[0076] (4) Incubation and rinsing: Transfer the liquid to the incubation tank, rinse the homogenization system twice, add sucrose hydrate to make up the volume, and incubate for 3 hours.

[0077] (5) Cooling and filtration: Cool down to 25°C, filter through the filter element and then transport to a sterile storage tank, stir for 20 minutes, cool down to 8°C and keep warm.

[0078] (6) Volume adjustment and freeze drying: Volume adjustment to 4.5 mg / g, temperature control at 8℃ for filling, freeze drying and capping to obtain the finished product.

[0079] Comparative Example 1: 1. Formula composition (1000 vials): Amphotericin B 50mg / vial, HSPC 213mg / vial, DSPG-Na 100mg / vial, Cholesterol 50mg / vial, α-Tocopherol 2mg / vial, Sucrose 1200mg / vial, Disodium succinate hexahydrate 60mg / vial, Methanol (total volume 120kg), Chloroform (total volume 300kg), Water for injection (oil phase 150kg + appropriate amount of aqueous phase).

[0080] 2. Preparation steps: (1) Oil phase preparation: Add raw materials to the oil phase tank, add methanol and water for injection, and stir (60% speed); dissolve α-tocopherol in 2kg of chloroform and add it, rinse 3 times (2kg each time), add chloroform to make up to 300kg; control the temperature at 55℃, add hydrochloric acid solution and stir for 10min to dissolve, increase the speed to 90%, add sodium hydroxide solution and stir for 10min to clarify, cool down to 21℃, and adjust the speed to 15% for later use.

[0081] (2) Preparation of aqueous phase: Weigh sucrose and disodium succinate hexahydrate, add water for injection to make up to 60 kg, and stir to dissolve.

[0082] (3) Homogenization: Homogenization temperature 60℃, homogenization pressure 3500psi, homogenization until particle size D50 reaches 95nm.

[0083] (4) Incubation and rinsing: Transfer the liquid to the incubation tank, rinse the homogenization system twice, and add sucrose hydrate to make up the volume.

[0084] (5) Cooling and filtration: Cool down to 25°C, filter through the filter element and then transport to a sterile storage tank, stir for 20 minutes, cool down to 8°C and keep warm.

[0085] (6) Volume adjustment and freeze drying: Volume adjustment to 4.5 mg / g, temperature control at 8℃ for filling, freeze drying and capping to obtain the finished product.

[0086] Comparative Example 2: (1) Weigh 840 mg of distearate phosphatidylglycerol (DSPG), 2130 mg of hydrogenated soybean phosphatidylcholine (HSPC), 520 mg of cholesterol (CHOL), and 500 mg of amphotericin B into a certain amount of a mixed solution of dichloromethane and methanol acidified with 1 mol / L hydrochloric acid, and stir to dissolve at 30-40 °C to obtain a mixed solution. The volume ratio of dichloromethane to methanol is 2:1.

[0087] (2) The above mixed solution is dried to obtain a dry powder.

[0088] (3) Add the above dried powder to a buffer solution with pH=5.5 (in the buffer solution: the mass percentage of sucrose is 10%, the mass percentage of disodium succinate is 3%, and the pH of the buffer solution is adjusted to 5.5 with hydrochloric acid), and hydrate at 60°C for 1 hour; (4) The hydrated solution was homogenized 7 times at a pressure of 300 bar. (5) After homogenization, the sample is filtered with a filter membrane pore size of 0.22 μm and a filtration pressure of 2 bar. The sample after sterile filtration is then freeze-dried and capped to obtain the finished product.

[0089] Comparative Example 3 (1) Prescription composition: Same as in Example 5.

[0090] (2) Preparation steps: After the oil phase preparation and before the aqueous phase mixing, a vacuum spray drying step is added: The oil phase solution is placed in a spray dryer with an inlet air temperature of 60°C, an outlet air temperature of 35°C, and a vacuum degree of -0.08 to -0.09 MPa, and dried until lipid powder is obtained; the powder is then mixed with the aqueous phase and homogenized. The process parameters (temperature, pressure, filtration, freeze drying, etc.) for homogenization and subsequent steps are the same as in Example 5.

[0091] Comparative Example 4 (1) Prescription composition: Same as in Example 5.

[0092] (2) Preparation steps: Only the homogenization temperature was changed to 70°C, and all other process parameters (homogenization pressure 3500psi, pore size 75μm, cooling to 25°C, filling temperature 8°C, etc.) were the same as in Example 5.

[0093] Comparative Example 5 (1) Prescription composition: Same as in Example 5.

[0094] (2) Preparation steps: Only the cooling temperature of the heat exchanger after incubation was changed to 35°C. All other process parameters (homogenization temperature 60°C, pressure 3500psi, filter element, filling temperature 8°C, etc.) were the same as in Example 5.

[0095] Experimental example, stability test The amphotericin B liposomes for injection prepared in the examples and comparative examples were stored at 25°C for 24 months, and the particle size, encapsulation efficiency and related substances were tested periodically.

[0096] 1. Detection methods for related substances (high performance liquid chromatography, HPLC) (1) Chromatographic conditions Chromatographic column: C18 column (250mm × 4.6mm, 5μm); Mobile phase: methanol-0.05 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.0 with phosphoric acid) = 85:15 (v / v); Flow rate: 1.0 mL / min; Detection wavelength: 303 nm; Column temperature: 30℃; Injection volume: 20 μL; Run time: twice the retention time of the main component peak, record the chromatogram.

[0097] (2) Determination method Take an appropriate amount of this product, dissolve and dilute it with methanol-water (1:1) to prepare a test solution containing 0.5 mg of amphotericin B per mL; accurately measure 1 mL, place it in a 100 mL volumetric flask, dilute to the mark with the above solvent, and shake well to prepare a control solution. Inject 20 μL of the control solution into the liquid chromatograph, adjust the detection sensitivity so that the peak height of the main component is 20%~30% of full scale; then accurately measure 20 μL each of the test solution and the control solution, inject them separately into the liquid chromatograph, and record the chromatograms. Calculate the content of related substances according to the self-comparison method: the sum of the peak areas of all impurities in the test solution should not exceed 1.3 times the peak area of ​​the main peak in the control solution (13%, which meets the standard limit).

[0098] 2. Content determination method (high performance liquid chromatography, HPLC, compatible with related substance chromatographic conditions) (1) Chromatographic conditions Consistent with related substance detection (no need to readjust the chromatographic column).

[0099] (2) Determination method Take 10 vials of this product, mix the contents thoroughly, accurately weigh an appropriate amount (approximately equivalent to 25 mg of amphotericin B), place it in a 50 mL volumetric flask, dissolve and dilute to the mark with methanol-water (1:1), and shake well to prepare the test solution; separately take an appropriate amount of amphotericin B reference standard, and prepare a reference solution containing 0.5 mg per mL using the same method. Accurately measure 20 μL of each of the above two solutions, inject them separately into the liquid chromatograph, and record the chromatograms. Calculate the content based on peak area using the external standard method. The amphotericin B content in this product should be 90%~115% of the labeled amount (meeting the standard limit).

[0100] The test results are shown in Tables 1 and 2.

[0101] Table 1. Product stability results of the examples

[0102] Table 2. Stability results of comparative and commercially available products.

[0103] The results showed that the liposomes prepared in each embodiment had a stable and uniform particle size, with no significant changes during storage. The encapsulation efficiency remained above 94%, and the growth of related substances was slow, meeting the formulation quality standards. This indicates that the amphotericin B liposomes for injection prepared by the present invention have good stability.

[0104] The stability results demonstrate that the product prepared in this invention has a different molecular structure from the comparative example and commercially available products. The reason for the poor stability of the prior art is that the liposome bilayer structure is heterogeneous and easily damaged, as well as the amphotericin B drug molecule is oxidized and hydrolyzed, and the two promote each other: structural damage leads to drug leakage, and the free drug is more easily oxidized / hydrolyzed, producing a large number of related substances; at the same time, the degradation products further damage the lipid membrane structure, ultimately leading to a significant decrease in encapsulation efficiency and content, and abnormal particle size distribution.

[0105] Compared with Example 5, Comparative Example 1 showed that HSPC, DSPG-Na, sucrose, and disodium succinate hexahydrate (pH adjuster) were all added in excess. The core problem caused by this was the imbalance of the liposome membrane material ratio and the abnormal concentration of excipients, which led to the disorder of the physicochemical environment of the system. This disrupted the balance of intermolecular forces in the liposome bilayer and the acid-base and osmotic pressure stability of the formulation, ultimately resulting in the loss of liposome structural stability and accelerated drug degradation.

[0106] Comparative Example 2 illustrates the traditional process of "dissolving in a pure organic phase → drying into powder → subsequent hydration and homogenization," while Example 5 illustrates the innovative process of this invention: "dissolving in a methanol-water mixed phase → direct hydration and homogenization → precise temperature control and cooling." The difference in results between the two is essentially due to the difference in the self-assembly state and structural uniformity of liposomes caused by the process routes. Furthermore, the combined effects of solvent and temperature control mean that the existing "drying into powder followed by hydration" process fundamentally fails to achieve the ordered self-assembly of liposomes, resulting in a "pseudo-liposome structure" (phospholipid clusters / large vesicles) rather than the "true liposome structure" (dense small single-chamber liposomes) of this invention. This, coupled with the disadvantages of a pure organic phase, high temperature, and high interfacial tension, ultimately leads to a complete loss of formulation stability. In contrast, the innovative process of this invention, through "dissolving in a mixed solvent + direct hydration and homogenization + precise temperature control throughout," fundamentally ensures the ordered self-assembly and structural density of liposomes, which is the core foundation for high formulation stability and high encapsulation efficiency.

[0107] In addition, under high vacuum conditions, conventional processes cause the low-boiling-point organic solvents to evaporate rapidly, and the lipid components are concentrated and hydrated in the remaining aqueous phase. Through preliminary self-assembly, they gradually form drug-containing phospholipid clusters, large vesicles, and other structures with different morphologies. The liposome structures are heterogeneous, resulting in poor encapsulation and instability.

[0108] according to Figure 1 Microscopic observation reveals that the liposomes in this invention have more uniform particle size and better encapsulation effect, which can effectively improve product stability and safety.

[0109] The process of this invention involves direct hydration and homogenization, followed by multiple crushing and self-assembly processes under high pressure to form small single-chamber liposome structures in which a certain volume of aqueous phase is encapsulated by a drug-containing phospholipid bilayer. Therefore, the resulting drug-containing phospholipid bilayer exhibits uniform particle size, excellent encapsulation effect, and product stability. Furthermore, it requires less organic solvent, has simple process steps, and is easy to industrialize and promote.

[0110] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing highly stable amphotericin B liposomes, characterized in that, Includes the following steps: Step 1, oil phase preparation: Add methanol and water to amphotericin B and liposome membrane material, stir continuously, add chloroform, heat, add hydrochloric acid solution to adjust the pH to 1-3, stir until clear, adjust the pH to neutral and cool for later use; the liposome membrane material contains phospholipid compounds and sterol compounds; Step 2, Aqueous phase preparation: Dissolve the freeze-drying protectant and pH adjuster in water and stir to dissolve; Step 3, Homogenization: The oil phase obtained in Step 1 and the aqueous phase obtained in Step 2 are mixed and homogenized in a homogenizer at a pressure of 3000~3500psi to a particle size of 30~100nm. Step 4, incubation, cooling and filtration: The homogenized liquid from step 3 is incubated, cooled to ≤25℃, and then subjected to pre-filtration and sterilization filtration in sequence; Step 5: Adjust volume, fill and freeze dry.

2. The method for preparing highly stable amphotericin B liposomes according to claim 1, characterized in that, In step 1, the volume ratio of methanol to water added to amphotericin B and the liposome membrane material is 1:1.2~1.5; the mass ratio of methanol to amphotericin B is 1.8×10⁻⁶. 3 ~2.4×10 3 ; Preferably, the amount of chloroform added is 1.25 to 2.6 times the volume of methanol; Preferably, the antioxidant is dissolved in chloroform.

3. The method for preparing highly stable amphotericin B liposomes according to claim 1, characterized in that, In step 1, the reaction temperature is controlled at 45~55℃.

4. The method for preparing highly stable amphotericin B liposomes according to claim 1, characterized in that, In step 1, add hydrochloric acid solution and stir until completely dissolved; Increase the rotation speed, add sodium hydroxide solution, stir until clear, and cool to 15~25℃.

5. The method for preparing highly stable amphotericin B liposomes according to claim 1, characterized in that, In step 3, the homogenization temperature is set to 50~60℃, and the liquid obtained in step 1 is homogenized repeatedly through a homogenizer until the particle size D50 reaches 40~100nm; further, it is stopped when the particle size D50 reaches 45~95nm.

6. The method for preparing highly stable amphotericin B liposomes according to claim 1, characterized in that, In step 1, the phospholipid compounds include one or more of the following: lecithin and its derivatives, soybean phospholipids and their derivatives, cephalin and its derivatives, cardiolipin and its derivatives, phosphatidylinositol and its derivatives, phosphatidylserine and its derivatives, phosphatidylglycerol and its derivatives, phosphatidic acid and its derivatives, phosphatidylethanolamine and its derivatives, phosphatidylcholine and its derivatives, phosphatidylinositol and its derivatives, sphingomyelin and its derivatives, phospholipids and their derivatives, and hydrogenated products.

7. The method for preparing highly stable amphotericin B liposomes according to claim 1, characterized in that, In step 1, the liposome membrane material contains hydrogenated soybean phosphatidylcholine, sodium distearate phosphatidylglycerol, and cholesterol in a weight ratio of 2~3:1.2~1.8:

1.

8. The method for preparing highly stable amphotericin B liposomes according to claim 1, characterized in that, The mass ratio of materials in steps 1 and 2 is: Amphotericin B: Liposome membrane material: Antioxidant: Lyophilization protectant: pH adjuster = 40~60: 200~300mg: 0.5~2mg: 800~1000: 30~40.

9. The highly stable amphotericin B liposomes prepared by the preparation method according to any one of claims 1 to 8 are characterized in that, The encapsulation efficiency of the highly stable amphotericin B liposomes is higher than 90%; after storage at 25°C for 24 months, the increase in related substances is no more than 5%.

10. A liposome injection of amphotericin B, characterized in that, It is prepared from the highly stable amphotericin B liposomes as described in claim 9.

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