Amphotericin B liposome and a preparation method thereof

Abstract

The application relates to the field of biological medicines, and specifically discloses amphotericin B liposomes and a preparation method thereof. The preparation method comprises the following steps: S1, mixing amphotericin B, liposome raw materials and an organic solvent to obtain a mixed solution; S2, shearing the mixed solution and a freeze-drying protection solution to form a lipid vesicle, then performing pre-freezing treatment, and then performing low-pressure freeze-drying to obtain a raw material powder; and S3, dissolving, hydrating, homogenously extruding, filtering and freeze-drying the raw material powder in the step S2 to obtain the amphotericin B liposomes. The application also discloses the amphotericin B liposomes prepared by the above method. The application has the characteristics that the removal of the organic solvent does not affect the stability of the medicine, and the obtained amphotericin B liposomes have a higher encapsulation rate.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biopharmaceuticals, more particularly, it relates to a liposome of amphotericin B and a preparation method thereof. BACKGROUND

[0002] Amphotericin B belongs to the polyene antibiotic drugs, which can effectively treat deep fungal infection, and is one of the preferred treatment drugs for systemic fungal infection. However, the polyene antibiotic is easy to combine with the sterol components of the host cells, resulting in cell membrane damage, cell permeability and lysis, so that amphotericin B can cause acute hemolysis in the treatment of patients. In addition, due to the low solubility of amphotericin B, it is difficult to reach an effective treatment concentration under low dose, and high dose will cause serious adverse reactions, which limits its application in clinic.

[0003] It is found that the encapsulation of amphotericin B in liposomes can reduce its systemic toxicity, reduce the adverse reactions of the preparation, and improve the stability of the drug. After the administration of amphotericin B liposomes, the concentration of amphotericin B in the plasma is relatively low, and the combination rate with cholesterol on the renal tubular cell membrane is reduced, thereby significantly reducing the nephrotoxicity, improving the effective drug dose in clinic, achieving better therapeutic effect, reducing the side effects of amphotericin B and prolonging the survival period of patients.

[0004] Amphotericin B liposome is a double-layer liposome containing amphotericin B, and the effective component is polyene antibiotic amphotericin B, and the liposome is a closed layer with double-layer structure composed of phospholipid and cholesterols. When preparing amphotericin B liposome at present, the phospholipid raw material, sterol raw material and amphotericin B are mixed in an organic solvent, and then the organic solvent is removed to obtain amphotericin B liposome by freeze-drying. At present, the main means for removing organic solvent is spray drying, but the temperature required by spray drying is high, which has a certain influence on the stability of amphotericin B raw drug.

[0005] It is necessary to provide a new production method which is more suitable for industrial production and does not affect the stability of the drug, and the encapsulation efficiency of amphotericin B liposome is higher. SUMMARY

[0006] In order to obtain amphotericin B liposome with higher encapsulation efficiency, and without affecting the stability of the drug in the process of removing organic solvent, the present application provides a kind of amphotericin B liposome and a preparation method thereof.

[0007] In the first aspect, the present application provides a preparation method of amphotericin B liposome, which adopts the following technical scheme: a preparation method of amphotericin B liposome, comprising the following steps:

[0008] S1, mixing amphotericin B with liposome raw materials and organic solvents to obtain a mixed solution;

[0009] S2, shearing the mixed solution with a freeze-drying protective solution to form a lipid vesicle, then pre-freezing and freeze-drying to obtain a raw material powder;

[0010] S3, dissolving, hydrating, homogenizing, filtering and freeze-drying the raw material powder in step S2 to obtain amphotericin B liposomes.

[0011] By adopting the technical scheme, the low-pressure freeze-drying method is used to remove the organic solvent in step S2 in the application, and the boiling point of the organic solvent is reduced at low pressure. The organic solvent can be removed at a lower temperature at low pressure, which can avoid the influence of high temperature on the stability of the raw material drug. In addition, the freeze-drying process is used to remove the organic solvent in the application, which can realize aseptic operation in the preparation process. Compared with the spray drying method, the cytotoxic raw material drug in the form of dust is scattered in the environment, which has a certain influence on the environment. The freeze-drying method is used to remove the organic solvent in the application to realize aseptic operation in the preparation process. The raw material drug will not be scattered in the air, and the material collection is also more simple. The process realizes aseptic operation, which is more suitable for the industrial production process of sterile injections.

[0012] Moreover, the mixed solution and the freeze-drying protective solution are sheared and mixed to form a lipid vesicle in the application, and then the subsequent freeze-drying operation is performed. The drug vesicle is formed in advance, the encapsulation efficiency of the liposome is improved, the liposome vesicle with smaller particle size and more uniform particle size distribution is obtained, and the encapsulation efficiency and the particle size are higher than those of the traditional spray drying. Finally, the amphotericin B liposomes with a particle size of <100 nm, a PDI of <0.3, a qualified particle size and a uniform particle size are obtained. The finished product is a light yellow freeze-dried powder, and the product is a yellow transparent solution after reconstituted with water for injection.

[0013] Optionally, the pre-freezing treatment parameters in step S2 are as follows:

[0014] First, the pre-freezing treatment is performed at 1.05-1.3 atm under normal pressure or positive pressure, 0±10℃ for 1-5h, and then at normal pressure, -45±10℃ for 1-10h;

[0015] The low-pressure freeze-drying treatment is operated in the following five stages in sequence, and the specific parameters are as follows:

[0016] The first stage involves treatment at -45±20℃ and a vacuum of 1-20Pa for 10-60 minutes; the second stage involves treatment at -20±10℃ and a vacuum of 1-20Pa for 12-48 hours; the third stage involves treatment at 10±5℃ and a vacuum of 1-20Pa for 2-24 hours; the fourth stage involves treatment at 20±10℃ and a vacuum of 1-20Pa for 5-24 hours; and the fifth stage involves treatment at 40±20℃ and a vacuum of 1-20Pa for 5-24 hours.

[0017] By adopting the above technical solution, this application first performs a two-stage pre-freezing treatment. First, treatment is carried out at around 0°C to initially freeze water molecules and some organic solvents in the solution. Then, treatment is carried out at around -45°C for an extended period to completely freeze the remaining water and organic solvents into a solid state. This pre-freezing treatment helps stabilize the lipid vesicle structure. During the freezing process, lipid molecules rearrange into a more stable structure, which helps maintain the integrity and encapsulation efficiency of the liposomes during subsequent freeze-drying. Then, the subsequent low-pressure freeze-drying operation is performed, maintaining low-pressure conditions. Treatment is first carried out at -45°C, followed by gradual temperature increases. Through five stages of gradual temperature increases and maintenance for a certain period, the liposomes are... The frozen water and organic solvent gradually sublimate to form gas, ensuring gradual and thorough removal of the solvent. More specifically, dichloromethane in the organic solvent is removed when the temperature is raised to around -20°C, and methanol in the organic solvent is removed when the temperature is raised to around 10°C. This process ensures more thorough removal of the organic solvent, leaving no residue. Furthermore, the gradual heating in this application slows down the rate of solvent sublimation, reducing internal pressure changes in the liposomes and helping to maintain the integrity of the liposomes and the drug encapsulation rate. Finally, the organic solvent is removed by hydration with the solution followed by shearing, pre-freezing, and lyophilization, achieving pre-formation of drug vesicles and improving the encapsulation rate of the liposomes.

[0018] Optionally, the pre-freezing parameters in step S2 are:

[0019] The pre-freezing parameters in step S2 are:

[0020] First, it is treated at normal pressure and 0℃ for 2 hours, and then at normal pressure and -45℃ for 5 hours for pre-freezing treatment;

[0021] The low-pressure freeze-drying process involves the following five stages, with specific parameters as follows:

[0022] The first stage involves treatment at -45℃ and a vacuum of 1 Pa for 30 minutes; the second stage involves treatment at -10℃ and a vacuum of 1 Pa for 20 hours; the third stage involves treatment at 10℃ and a vacuum of 1 Pa for 20 hours; the fourth stage involves treatment at 20℃ and a vacuum of 1 Pa for 20 hours; and the fifth stage involves treatment at 40℃ and a vacuum of 1 Pa for 20 hours.

[0023] By adopting the above technical solution and using the above parameters, the encapsulation efficiency of amphotericin B liposomes is ultimately higher.

[0024] Optionally, the freeze-drying protection solution in step S2 is obtained by mixing a freeze-drying protectant and water at a mass ratio of 1:(2-5), wherein the freeze-drying protectant is selected from one or more of sucrose, glucose, mannitol, trehalose, and maltose.

[0025] By adopting the above technical solution, the addition of the freeze-drying protective solution in this application achieves the hydration process of the mixed solution and the pre-formation of drug liposome vesicles, which helps to improve the encapsulation rate of liposomes. On the other hand, the addition of the freeze-drying protective solution helps to protect the pre-formed vesicles from rupture during the subsequent freeze-drying solvent removal process, ultimately resulting in liposomes with a higher encapsulation rate.

[0026] Optionally, after the mixed solution and the lyophilized protective solution are sheared to form lipid vesicles in step S2, they are further subjected to aeration treatment. The gas source for aeration treatment is air or inert gas, the aeration flow rate is 1-100 L / min, and the aeration time is 100-150 min.

[0027] By adopting the above technical solution, in this application, after mixing and shearing the mixed solution with the freeze-drying protective solution to form lipid vesicles, the solution is aerated to introduce gas, which increases turbulence and shear force in the solution. This helps disperse the liposomes in the freeze-drying protective solution, promotes the further formation of liposome vesicles and improves their uniformity. Moreover, the aeration treatment can reduce the particle size of the liposomes, making them more uniform. Furthermore, the aeration treatment helps rearrange phospholipid molecules to form a more stable liposome structure, reducing vesicle rupture during subsequent pre-freeze-freeze-drying, and ultimately obtaining a liposome product with a higher encapsulation rate.

[0028] Optionally, in step S2, the amount of lyophilization protectant added is 12-15 times the mass of amphotericin B.

[0029] Optionally, the organic solvent in step S1 may be a mixture of dichloromethane and methanol.

[0030] Optionally, the liposome raw material in step S1 includes at least phospholipid compounds and also includes excipients, wherein the phospholipid compounds include a mixture of distearate phosphatidylglycerol and hydrogenated soybean phospholipids.

[0031] Optionally, the excipients include cholesterol.

[0032] Optionally, the excipients may also include antioxidants.

[0033] Optionally, the antioxidant is selected from DL-α-tocopherol.

[0034] By adopting the above technical solution, the addition of antioxidants to the liposome raw material includes the stability of the liposome raw material and the formed vesicles during the preparation process.

[0035] Optionally, the specific operation of step S1 is as follows: add distearate phosphatidylglycerol, amphotericin B and DL-α-tocopherol to a mixed organic solvent of dichloromethane and methanol, stir until the mixture is clear, adjust the pH to 2-2.5, then add alkali, and after turbidity, add acid again to make the system repeatedly clear three times. Then add hydrogenated soybean lecithin and cholesterol to the system and continue stirring to obtain a mixed solution.

[0036] By adopting the above technical solution, in this application, the pH of phospholipids and amphotericin B is adjusted to a strongly acidic state after being mixed in an organic solvent. This helps to change the pH value of the system, which in turn helps to change the solubility properties of the drug and phospholipids, making them easier to dissolve and disperse in the solvent. Then, after adding alkali and adjusting the pH value again, the drug and phospholipids can be solubilized through multiple solubilization treatments. This process can also affect the interaction and arrangement between phospholipid molecules, helping to form a stable bilayer structure of phospholipid molecules. This affects the stability and structure of the liposomes formed subsequently, and ultimately, the vesicles formed in advance during the freeze-drying process are more stable and have a better encapsulation rate in the final liposomes.

[0037] Optionally, in step S1, the mass ratio of amphotericin B to distearate phosphatidylglycerol is 1:(1.5-1.7), the amount of DL-α-tocopherol added is 1-1.8 wt% of the amount of amphotericin B added, the amount of hydrogenated soybean lecithin added is 4-5 times the mass of amphotericin B, and the mass ratio of amphotericin B to cholesterol added is 1:(0.9-1.1).

[0038] By adopting the above technical solution and selecting the above-mentioned addition amount, the liposomes prepared have better overall performance.

[0039] Optionally, the specific operation of step S3 is as follows: The raw material powder in step S2 is reconstituted with sucrose solution, the pH is adjusted to between 2.5 and 5.0, hydrated, and then the pH is adjusted to 5-6 by sodium succinate and sodium hydroxide. After high pressure homogenization, it is extruded to the target particle size of 60-120 nm, then kept at 40-70℃ for 0.5-2 h, filtered for sterilization, and then freeze-dried to obtain amphotericin B liposomes.

[0040] Secondly, this application provides an amphotericin B liposome, which adopts the following technical solution:

[0041] A liposome of amphotericin B was prepared by the method described above.

[0042] By adopting the above technical solution, the amphotericin B liposomes prepared by the method provided in this application have a higher encapsulation rate, smaller particle size and lower polymer dispersion coefficient compared with liposomes prepared by traditional processes.

[0043] In summary, this application has the following beneficial effects:

[0044] 1. This application employs a shear hydration followed by pre-freezing and lyophilization process to remove organic solvents, thereby achieving the pre-formation of drug vesicles, improving the encapsulation efficiency of liposomes, and obtaining liposome vesicles with smaller particle size and more uniform particle size distribution; moreover, it avoids the impact of high-temperature solvent removal on the stability of the active pharmaceutical ingredient, and compared with spray drying, it can achieve aseptic operation of the process, making it more suitable for the industrial production process of sterile injectables;

[0045] 2. In this application, a two-stage cooling process is first used for pre-freezing. First, the solution is treated at around 0°C to freeze water molecules and some organic solvents. Then, it is treated at around -45°C for an extended period of time to completely freeze the remaining water and organic solvents into a solid state. This pre-freezing process helps stabilize the lipid vesicle structure. During the freezing process, lipid molecules rearrange into a more stable structure, which helps maintain the integrity and encapsulation efficiency of the liposomes during the subsequent freeze-drying process. Then, the subsequent low-pressure freeze-drying operation is carried out. The low-pressure conditions are maintained. First, the solution is treated at -45°C and then gradually heated. Through five stages of gradual heating and maintenance for a certain period of time, the frozen water and organic solvents gradually sublimate to form gas and are removed. This ensures the gradual and complete removal of solvents, resulting in more thorough removal of organic solvents without residue. Moreover, the gradual heating in this application can slow down the rate of solvent sublimation, reduce the pressure changes inside the liposomes, and help maintain the integrity of the liposomes and the encapsulation efficiency of the drug.

[0046] 3. In this application, after mixing and shearing the mixed solution with the freeze-drying protective solution to form lipid vesicles, the solution is aerated. The introduction of gas increases the turbulence and shear force in the solution, which helps the liposomes disperse in the freeze-drying protective solution, promotes the further formation of liposome vesicles and improves their uniformity. Moreover, the aeration treatment can also reduce the particle size of the liposomes, making them more uniform. Furthermore, the aeration treatment helps to rearrange phospholipid molecules to form a more stable liposome structure, reducing vesicle rupture during the subsequent pre-freeze-freeze-drying process, and ultimately obtaining a liposome product with a higher encapsulation rate. Detailed Implementation

[0047] The following detailed description of this application is provided in conjunction with the embodiments. It should be noted that: unless otherwise specified, the conditions in the following embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following embodiments are all from commercially available sources.

[0048] Example 1

[0049] A method for preparing amphotericin B liposomes includes the following steps:

[0050] S1. Add 1.68g DSPG (distearylphosphatidylglycerol), 1.0g amphotericin B, and 12.8mg DL-α-tocopherol to a mixed organic solvent of dichloromethane and methanol. Stir until the mixture is clear, then add hydrochloric acid to adjust the pH to 2. Then add sodium hydroxide. After the mixture becomes turbid, add hydrochloric acid again. Repeat this process three times to clear the mixture. Then add 4.26g HSPC (hydrogenated soybean phosphatidylcholine) and 1.04g cholesterol to the mixture. Continue stirring to obtain an orange-yellow drug-phospholipid mixed solution.

[0051] The mixture of dichloromethane and methanol in a 1:1 mass ratio is used to obtain the product.

[0052] S2. The mixed solution and the freeze-drying protection solution are sheared at high speed (2000 r / min) for 10 min to form lipid vesicles. Then, the mixture is aerated for 2 h and pre-frozen. Finally, it is freeze-dried under low pressure to obtain raw material powder.

[0053] The freeze-drying protective solution is obtained by mixing freeze-drying protectant and water at a mass ratio of 1:3. Sucrose is selected as the freeze-drying protectant, and the amount of freeze-drying protectant added is 13 times the mass of amphotericin B.

[0054] The gas source for aeration was nitrogen, with an aeration flow rate of 50 L / min and an aeration time of 120 min. The pre-freezing parameters were: first, treatment at atmospheric pressure and 0℃ for 2 h, followed by treatment at atmospheric pressure and -45℃ for 5 h for pre-freezing. The low-pressure freeze-drying process consisted of five stages, with the following parameters: Stage 1: Treatment at -45℃ and 1 Pa vacuum for 30 min; Stage 2: Treatment at -10℃ and 1 Pa vacuum for 20 h; Stage 3: Treatment at 10℃ and 1 Pa vacuum for 20 h; Stage 4: Treatment at 20℃ and 1 Pa vacuum for 20 h to complete the low-pressure freeze-drying process and obtain the raw material powder; Stage 5: Treatment at 40℃ and 1 Pa vacuum for 20 h.

[0055] S3. The raw material powder from step S2 is reconstituted with a 20% sucrose solution. The amount of sucrose solution added is three times the mass of the raw material powder. The pH is adjusted to between 3 and 5. The mixture is hydrated for 1 hour. Then, the pH is adjusted to 5.5 by sodium succinate and sodium hydroxide. After homogenization, the mixture is extruded to the target particle size of 60-120 nm. After incubation at 65°C for 1.5 hours, the mixture is filtered to remove bacteria and then freeze-dried to obtain amphotericin B liposomes.

[0056] Example 2

[0057] A method for preparing amphotericin B liposomes includes the following steps:

[0058] S1. Add 1.5g DSPG (distearylphosphatidylglycerol), 1.0g amphotericin B, and 18mg DL-α-tocopherol to a mixed organic solvent of dichloromethane and methanol. Stir until the mixture is clear, then add hydrochloric acid to adjust the pH to 2.5. Then add sodium hydroxide. After the mixture becomes turbid, add hydrochloric acid again. Repeat this process three times to clear the mixture. Then add 5g HSPC (hydrogenated soybean phosphatidylcholine) and 1.1g cholesterol to the mixture. Continue stirring to obtain an orange-yellow drug-phospholipid mixed solution.

[0059] The mixture of dichloromethane and methanol in a 1:1 mass ratio is used to obtain the product.

[0060] S2. The mixed solution and the freeze-drying protection solution are sheared at high speed (2000 r / min) for 10 min to form lipid vesicles. Then, the mixture is aerated for 100 min and pre-frozen. Finally, it is freeze-dried under low pressure to obtain raw material powder.

[0061] The freeze-drying protective solution is obtained by mixing freeze-drying protectant and water at a mass ratio of 1:5. Sucrose is selected as the freeze-drying protectant, and the amount of freeze-drying protectant added is 13 times the mass of amphotericin B.

[0062] The gas source for aeration was nitrogen, with an aeration flow rate of 100 L / min and an aeration time of 100 min. The pre-freezing parameters were as follows: first, pre-freezing at -10℃ under positive pressure (1.05 atmospheres) for 1 hour, followed by pre-freezing at -55℃ under normal pressure for 1 hour. The low-pressure freeze-drying process consisted of five stages: the first stage involved treatment at -65℃ and 12 Pa vacuum for 10 minutes; the second stage involved treatment at -30℃ and 12 Pa vacuum for 12 hours; the third stage involved treatment at 5℃ and 12 Pa vacuum for 2 hours; the fourth stage involved treatment at 10℃ and 12 Pa vacuum for 24 hours; and the fifth stage involved treatment at 20℃ and 12 Pa vacuum for 24 hours to complete the low-pressure freeze-drying process and obtain the raw material powder.

[0063] S3. The raw material powder from step S2 is reconstituted with a 20% sucrose solution. The amount of sucrose solution added is three times the mass of the raw material powder. The pH is adjusted to between 2 and 5, and the mixture is hydrated for 1 hour. Then, the pH is adjusted to 5 by sodium succinate and sodium hydroxide. After homogenization, the mixture is extruded to the target particle size of 60-120 nm. After incubation at 40°C for 2 hours, the mixture is filtered to remove bacteria and then freeze-dried to obtain amphotericin B liposomes.

[0064] Example 3

[0065] A method for preparing amphotericin B liposomes includes the following steps:

[0066] S1. Add 1.7g DSPG (distearylphosphatidylglycerol), 1.0g amphotericin B, and 12.8mg DL-α-tocopherol to a mixed organic solvent of dichloromethane and methanol. Stir until the mixture is clear, then add hydrochloric acid to adjust the pH to 2-2.5. Then add sodium hydroxide. After the mixture becomes turbid, add hydrochloric acid again. Repeat this process three times to clear the mixture. Then add 4.26g HSPC (hydrogenated soybean phosphatidylcholine) and 1.04g cholesterol to the mixture and continue stirring to obtain an orange-yellow drug-phospholipid mixed solution.

[0067] The mixture of dichloromethane and methanol in a 1:1 mass ratio is used to obtain the product.

[0068] S2. The mixed solution and the freeze-drying protection solution are sheared at high speed (2000 r / min) for 10 min to form lipid vesicles. Then, the mixture is aerated for 150 min and pre-frozen. Finally, it is freeze-dried under low pressure to obtain raw material powder.

[0069] The freeze-drying protective solution is obtained by mixing freeze-drying protectant and water at a mass ratio of 1:3. Sucrose is selected as the freeze-drying protectant, and the amount of freeze-drying protectant added is 13 times the mass of amphotericin B.

[0070] The gas source for aeration was nitrogen, with an aeration flow rate of 1 L / min and an aeration time of 150 min. The pre-freezing parameters were as follows: first, treatment at 10℃ and 1.3 atmospheres for 5 hours, followed by treatment at -35℃ and atmospheric pressure for 10 hours for pre-freezing. The low-pressure freeze-drying process consisted of five stages: the first stage involved treatment at -25℃ and 20Pa vacuum for 60 minutes; the second stage involved treatment at -10℃ and 20Pa vacuum for 48 hours; the third stage involved treatment at 15℃ and 20Pa vacuum for 24 hours; the fourth stage involved treatment at 30℃ and 20Pa vacuum for 5 hours; and the fifth stage involved treatment at 60℃ and 20Pa vacuum for 5 hours to complete the low-pressure freeze-drying process and obtain the raw material powder.

[0071] S3. The raw material powder from step S2 is reconstituted with a 20% sucrose solution. The amount of sucrose solution added is 4 times the mass of the raw material powder. The pH is adjusted to between 4 and 5, and the mixture is hydrated for 1 hour. Then, the pH is adjusted to 6 by sodium succinate and sodium hydroxide. After homogenization, the mixture is extruded to the target particle size of 60-120 nm. After being kept at 70°C for 0.5 hours, it is filtered to remove bacteria and then freeze-dried to obtain amphotericin B liposomes.

[0072] Example 4

[0073] A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that in step S1, amphotericin B and DL-α-tocopherol are added to a mixed organic solvent of dichloromethane and methanol, then hydrochloric acid is added to acidify and adjust the pH to 2, and then DSPG (distearate phosphatidylglycerol) is added. The mixture is stirred until the lipids are completely dissolved, and then sodium hydroxide is added. After the mixture becomes turbid, hydrochloric acid is added. The remaining operations are the same as in Example 1.

[0074] Example 5

[0075] A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that in step S1, DSPG (distearylphosphatidylglycerol) and amphotericin B are added to a mixed organic solvent of dichloromethane and methanol, then hydrochloric acid is added to acidify and adjust the pH to 2, then DL-α-tocopherol is added, followed by sodium hydroxide. After turbidity is observed, hydrochloric acid is added again, and the process is repeated three times. The remaining operations are the same as in Example 1.

[0076] Example 6

[0077] A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that DSPG (distearate phosphatidylglycerol), HSPC (hydrogenated soybean phosphatidylcholine), amphotericin B and DL-α-tocopherol are added to a mixed organic solvent of dichloromethane and methanol, acidified with hydrochloric acid, repeatedly dissolved with sodium hydroxide, and then cholesterol is added. The remaining operations are the same as in Example 1.

[0078] Example 7

[0079] A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that the aeration treatment is not performed in step S2.

[0080] Example 8

[0081] A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that in step S1, DSPG (distearate phosphatidylglycerol) and amphotericin B and DL-α-tocopherol are added to a mixed organic solvent of dichloromethane and methanol, stirred until the mixture is clear, and then hydrochloric acid is added to adjust the pH to 2. Then HSPC and cholesterol are added directly without adding sodium hydroxide or repeated dissolution.

[0082] Example 9

[0083] A method for preparing amphotericin B liposomes, which is carried out according to the method in Example 1, except that DL-α-tocopherol is not added to the raw materials.

[0084] Example 10

[0085] A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that after aeration in step S2, the liposomes are first pre-frozen at -45°C for 7 hours, and then subjected to low-pressure freeze-drying.

[0086] Example 11

[0087] A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that the specific parameters for the low-pressure freeze-drying treatment in step S2 are as follows:

[0088] The first stage involves processing at -45℃ and a vacuum of 1 Pa for 30 minutes; the second stage involves processing at 0℃ and a vacuum of 1 Pa for 30 hours; the third stage involves processing at 10℃ and a vacuum of 1 Pa for 30 hours; and the fourth stage involves processing at 20℃ and a vacuum of 1 Pa for 20 hours to complete the low-pressure freeze-drying process and obtain the raw material powder.

[0089] Example 12

[0090] A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that the specific parameters for the low-pressure freeze-drying treatment in step S2 are as follows:

[0091] The first stage involves processing at -45℃ and a vacuum of 1 Pa for 30 minutes; the second stage involves processing at 5℃ and a vacuum of 1 Pa for 40 hours; and the third stage involves processing at 15℃ and a vacuum of 1 Pa for 40 hours to complete the low-pressure freeze-drying process and obtain the raw material powder.

[0092] Comparative Example 1

[0093] A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that the raw material powder is obtained by spray drying after aeration treatment in step S2.

[0094] Comparative Example 2

[0095] A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that after aeration in step S2, the raw material powder is obtained by rotary evaporation at 65°C for 3 hours.

[0096] Comparative Example 3

[0097] A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that in step S2, after aeration treatment, no pre-freezing treatment is performed, and low-pressure freeze drying is performed directly.

[0098] Comparative Example 4

[0099] A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that the pre-freezing treatment and lyophilization treatment in step S2 are carried out under normal pressure.

[0100] Performance testing

[0101] The encapsulation efficiency and polymer dispersion index (PDI) of the amphotericin B liposomes prepared in the examples and comparative examples of this application were tested, and the test results are shown in Table 1 below.

[0102] Table 1:

[0103]

[0104] In addition, the organic solvent residue of the amphotericin B liposomes in the embodiments of this application was detected, and the content was less than 0.0001%. The lyophilization method for removing organic solvents in this application has a good effect on the removal of organic solvents. In addition, the particle size of the amphotericin B liposomes in Example 1 of this application was measured, and it was less than 100 nm. Referring to the test results in Table 1 above, the PDI of the amphotericin B liposomes prepared in the embodiments of this application was less than 0.3. The liposome product has qualified particle size and uniform particle size, and it has a higher encapsulation efficiency. Combining the test results of Examples 1 and 4, in Example 1, amphotericin B, DL-α-tocopherol, and DSPG were added simultaneously, while in Example 4, amphotericin B and DL-α-tocopherol were added, followed by acidification before adding phospholipids. It can be seen that the acidification of the mixed system before adding phospholipids may help the phospholipids dissolve and disperse, resulting in better encapsulation efficiency and a lower polymer dispersion coefficient in the final liposomes. Combining the test results of Example 5, adding phospholipids and amphotericin B first, then adding hydrochloric acid, and then adding DL-α-tocopherol resulted in a reduced effect. In Example 6, adding phospholipid raw materials, amphotericin B, and DL-α-tocopherol together, followed by acidification with hydrochloric acid, resulted in a reduced encapsulation efficiency. It is evident that the order of phospholipid raw materials, DL-α-tocopherol, and repeated acidification and dissolution operations affects the formation and distribution of phospholipid structure, thereby affecting the encapsulation efficiency of the final liposome product.

[0105] Referring to the test results of Examples 1 and 7, when no aeration treatment was performed in step S2, the encapsulation efficiency of the liposome product decreased while the dispersion coefficient increased. Combining this with the test results of Example 8, when only hydrochloric acid was added for acidification without the addition of sodium hydroxide and repeated dissolution, the encapsulation efficiency of the liposome product decreased while the dispersion coefficient increased. Repeated dissolution helps to fully bind the drug and phospholipids. Referring to the test results of Example 9, the encapsulation efficiency decreased without the addition of DL-α-tocopherol (vitamin E). As an antioxidant, it can reduce the occurrence of lipid peroxidation, especially the effect of subsequent aeration treatment, which helps to maintain the integrity and stability of the liposome membrane and improve the encapsulation efficiency. Combining this with the test results of Examples 10-12, it can be seen that when the two-stage pre-freezing treatment and five-step low-pressure freeze-drying treatment in this application are used, the liposomes obtained have a higher encapsulation efficiency and a lower dispersion coefficient, and the particle size is more uniform.

[0106] Referring to the test results of Example 1, Comparative Examples 1 and 2, compared with traditional spray drying or rotary evaporation methods for removing organic solvents, the encapsulation efficiency of the liposomes obtained in this application is higher. Combining this with the test results of Comparative Example 3, when low-pressure freeze-drying was performed directly without pre-freezing treatment in Comparative Example 3, the encapsulation efficiency was significantly reduced, and the dispersion coefficient was also increased. Furthermore, the residual organic solvent content increased by 8%, while in Comparative Example 4, when freeze-drying was carried out at atmospheric pressure, the residual organic solvent content increased by 5.6%. The removal effect of organic solvents was significantly reduced, and the encapsulation efficiency also decreased further. In addition, the freeze-drying technique used in this application solves the problem of reduced removal efficiency of organic solvents due to azeotropic effects, compared with conventional spray drying or rotary evaporation.

[0107] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing amphotericin B liposomes, characterized in that, Includes the following steps: S1. Mix amphotericin B with liposome raw materials and organic solvents to obtain a mixed solution; S2. After shearing the mixed solution and the freeze-drying protective solution to form lipid vesicles, the mixture is then pre-frozen and then freeze-dried under low pressure to obtain the raw material powder. S3. Dissolve, hydrate, homogenize, extrude, filter, and freeze-dry the raw material powder from step S2 to obtain amphotericin B liposomes; the pre-freezing parameters for step S2 are: First, treat at atmospheric pressure or positive pressure of 1.05-1.3 atmospheres and 0±10℃ for 1-5 hours, then treat at atmospheric pressure and -45±10℃ for 1-10 hours for pre-freezing treatment; The low-pressure freeze-drying process involves the following five stages, with specific parameters as follows: The first stage involves treatment at -45±20℃ and a vacuum of 1-20Pa for 10-60 minutes; the second stage involves treatment at -20±10℃ and a vacuum of 1-20Pa for 12-48 hours; the third stage involves treatment at 10±5℃ and a vacuum of 1-20Pa for 2-24 hours; the fourth stage involves treatment at 20±10℃ and a vacuum of 1-20Pa for 5-24 hours; and the fifth stage involves treatment at 40±20℃ and a vacuum of 1-20Pa for 5-24 hours. After the mixed solution and the freeze-drying protective solution are sheared to form lipid vesicles in step S2, they are further aerated. The gas source for the aeration treatment is air or inert gas, the aeration flow rate is 1-100 L / min, and the aeration time is 100-150 min. In step S1, the organic solvent used is a mixed solution of dichloromethane and methanol; The liposome raw material in step S1 includes at least phospholipid compounds and also excipients. The phospholipid compounds include a mixture of distearate phosphatidylglycerol and hydrogenated soybean phospholipids. The excipients include cholesterol and DL-α-tocopherol; The specific operation of step S1 is as follows: add distearate phosphatidylglycerol, amphotericin B and DL-α-tocopherol to a mixed organic solvent of dichloromethane and methanol, stir until the mixture is clear, adjust the pH to 2-2.5, then add alkali, add acid again after turbidity, and repeat the process three times to clear the system. Then add hydrogenated soybean lecithin and cholesterol to the system and continue stirring to obtain a mixed solution. In step S1, the mass ratio of amphotericin B to distearate phosphatidylglycerol is 1:(1.5-1.7), the amount of DL-α-tocopherol added is 1-1.8 wt% of the amount of amphotericin B added, the amount of hydrogenated soybean lecithin added is 4-5 times the mass of amphotericin B, and the mass ratio of amphotericin B to cholesterol added is 1:(0.9-1.1). The specific operation of step S3 is as follows: the raw material powder in step S2 is reconstituted with sucrose solution, the pH is adjusted to between 2.5 and 5.0, hydrated, and then the pH is adjusted to 5-6 by sodium succinate and sodium hydroxide. After high pressure homogenization, it is extruded to the target particle size, and then kept at 40-70℃ for 0.5-2h for sterilization by filtration. Finally, it is freeze-dried to obtain amphotericin B liposomes.

2. The method for preparing amphotericin B liposomes according to claim 1, characterized in that: The pre-freezing parameters in step S2 are: First, it is treated at normal pressure and 0℃ for 2 hours, and then at normal pressure and -45℃ for 5 hours for pre-freezing treatment; The low-pressure freeze-drying process involves the following five stages, with specific parameters as follows: The first stage involves treatment at -45℃ and a vacuum of 1 Pa for 30 minutes; the second stage involves treatment at -10℃ and a vacuum of 1 Pa for 20 hours; the third stage involves treatment at 10℃ and a vacuum of 1 Pa for 20 hours; and the fourth stage involves treatment at 20℃ and a vacuum of 1 Pa for 20 hours. The fifth stage involved processing at 40°C and a vacuum of 1 Pa for 20 hours.

3. The method for preparing amphotericin B liposomes according to claim 1, characterized in that: The freeze-drying protection solution in step S2 is obtained by mixing freeze-drying protectant and water at a mass ratio of 1:(2-5). The freeze-drying protectant is selected from one or more of sucrose, glucose, mannitol, trehalose, and maltose. The amount of freeze-drying protectant added is 12-15 times the mass of amphotericin B.

4. Amphotericin B liposomes prepared by the preparation method according to any one of claims 1-3.

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