Amphotericin B liposome and preparation method thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to amphotericin B liposome and a preparation method thereof.The preparation method comprises the steps that S1, amphotericin B, a film-forming material, an antioxidant and an acidified organic solvent are evenly mixed and dried to obtain fat powder; s2, the fat powder is subjected to hydration, homogenization, filtration, incubation and freeze drying in sequence; the incubation temperature is 60 to 70 DEG C, and the incubation time is 2 to 12 hours. The liposome prepared by the method provided by the invention is a round small single-chamber liposome with a uniform form; through detection, the amphotericin B lipid compound has the compounding rate of more than or equal to 99%, the particle size is small, the average particle size is less than or equal to 100nm, the particle size after redissolving is 50-150nm, the toxicity is low (the potassium release value is high), and the amphotericin B lipid compound can be prepared into low-toxicity freeze-dried powder injection for injection.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an amphotericin B liposome and its preparation method. Background Technology

[0002] Amphotericin B is a polyene antifungal drug, considered the most effective treatment for deep fungal infections and the first-line clinical treatment for invasive fungal infections. Currently, there is no suitable alternative, and it remains regarded as the "gold standard" for treating fungal infections. It works by specifically binding to ergosterol in the fungal cell membrane through its hydrophobic polyene structure, leading to membrane integrity disruption and the formation of transmembrane ion channels, allowing intracellular small molecules (such as K+) to pass through. + Na + H + This leads to electrolyte leakage and osmotic imbalance, causing the outflow of cell contents, which ultimately results in the lysis and death of fungal cells.

[0003] Amphotericin B is poorly absorbed after oral and intramuscular injection and causes significant local irritation; therefore, its primary route of administration in clinical practice is intravenous injection. However, amphotericin B has low solubility (less than 0.01 mg / mL in water) and poor lipid solubility. Therefore, traditional amphotericin B injections use sodium deoxycholate as a solubilizer, mixed with amphotericin B at a molar ratio of 1:2 to solubilize and prepare the injection solution. Although amphotericin B binds to ergosterol in fungal cell membranes, it also partially binds to cholesterol in mammalian cell membranes. Especially when amphotericin B is administered in its free form, it directly enters the bloodstream and is excreted by the kidneys, leading to serious adverse reactions such as nephrotoxicity and hemolysis. Traditional amphotericin B injections can cause acute erythrocyte hemolysis and severe renal tubular toxicity even at therapeutic doses. The nephrotoxicity of amphotericin B is dose-dependent; reducing the dosage can decrease nephrotoxicity. Formulating amphotericin B into a liposome formulation can maintain the efficacy of amphotericin B while significantly reducing the dosage and toxicity, resulting in good tolerability and high efficacy.

[0004] Currently, the commercial production process of amphotericin B liposomes is relatively complex, and their in vitro potassium release advantage is not significant, leading to an increased probability of adverse reactions in patients. Therefore, there is an urgent need to conduct in-depth research on the preparation process of amphotericin B liposomes and propose a novel method that reduces both process complexity and toxicity. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides an amphotericin B liposome and its preparation method, which reduces process complexity while lowering toxicity.

[0006] On one hand, the present invention provides a method for preparing amphotericin B liposomes, comprising the steps of: S1, Amphotericin B, film-forming material, antioxidant and acidified organic solvent are mixed and dried to obtain lip powder; S2, the lipid powder is sequentially hydrated, homogenized, filtered, incubated and freeze-dried; The incubation temperature is 60-70℃, and the incubation time is 2-12 hours.

[0007] The term "film-forming material" refers to the materials that make up liposomes, mainly including phospholipid compounds and sterol compounds.

[0008] The phospholipid compound is any one or a mixture of two or more of egg yolk lecithin (EPC), soybean lecithin (SPC), hydrogenated soybean phosphatidylcholine (HSPC), and sodium distearate phosphatidylglycerol (DSPG-Na) in any proportion, preferably hydrogenated soybean phosphatidylcholine (HSPC) and sodium distearate phosphatidylglycerol (DSPG-Na).

[0009] The sterol compound may be one or more of cholesterol, dihydrocholesterol, ergosterol, stigmasterol, sitosterol, and fungiol, with cholesterol being preferred.

[0010] The antioxidants mentioned above mainly include one or more of α-tocopherol and its derivatives (such as α-tocopherol succinate), ascorbate palmitate, propyl gallate, butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT), with α-tocopherol being preferred.

[0011] In some embodiments, the molar ratio of the amphotericin B, the film-forming material, and the antioxidant is 1:(5-15):(0.01-0.1).

[0012] In some embodiments, the film-forming material comprises cholesterol and phospholipids, and the antioxidant comprises α-tocopherol. The molar ratio of amphotericin B, cholesterol, phospholipids, and α-tocopherol is 1:(1-5):(5-10):(0.01-0.1), a preferred molar ratio is 1:(2-3):(6-9):(0.02-0.05), and the most preferred molar ratio is 1:2.5:7:0.03.

[0013] Acidification involves adding an acidic substance to an organic solvent. The acidic substance may be an inorganic acid, such as hydrochloric acid, sulfuric acid, or phosphoric acid. The acid can be prepared into a 0.1-5 mol / L, preferably 2.5 mol / L, hydrochloric acid solution according to conventional methods in the art.

[0014] The organic solvent is any one or a mixture of two or more of the following in any proportion: dichloromethane, chloroform, methanol, ethyl acetate, acetone, n-butanol, 2,5-dimethylfuran (DMF), dimethyl sulfoxide (DMSO), and dimethyl ether (DME). Dichloromethane and methanol are preferred.

[0015] In some embodiments, the ratio of the amphotericin B to the acidified organic solvent is 5-50 mg / mL.

[0016] The amount of organic solvent used is sufficient to fully dissolve amphotericin B, cholesterol, and phospholipids, resulting in a concentration of amphotericin B of 5-50 mg / mL, preferably 10-30 mg / mL. The volume ratio of dichloromethane to methanol in the organic solvent is 0.1-10:1, preferably 1:1.

[0017] The drying process can be conventional in the art, and the goal is to obtain the dried powder.

[0018] This invention discovers that incubation can improve the stability of liposomes and reduce their toxicity, and based on this discovery, proposes a novel method for preparing amphotericin B liposomes. The incubation process allows for precise control of temperature and time. Toxicity in this invention refers to: 1) the significant nephrotoxicity of free amphotericin B when it enters the bloodstream and is excreted by the kidneys; and 2) its tendency to induce potassium ion leakage from erythrocytes, causing various infusion reactions such as chills and shivering. To address this, this invention introduces potassium release as a toxicity evaluation indicator. Potassium release is defined as the sample concentration corresponding to the release of 50% potassium ions (potassium ion release after cell rupture), similar to LD50; therefore, higher potassium release corresponds to lower toxicity. This invention found that after incubation under appropriate conditions, potassium release from liposomes significantly increases, i.e., toxicity significantly decreases.

[0019] Commonly used phospholipids such as HSPC have a phase transition temperature of 52-55℃. Furthermore, literature reports that amphotericin B degradation accelerates above 60℃; therefore, conventional processes typically raise the temperature to approximately 55℃. Existing technologies do not include incubation; the only similar process is hydration, which usually lasts less than 1 hour. Prolonged incubation also significantly increases the degradation rate of amphotericin B. In contrast, this invention sets an incubation temperature of 60-70℃ after liposome formation, slightly above the phase transition temperature, for 2-12 hours. This not only improves the stability and quality of the drug formulation but also reduces toxicity. This demonstrates that temperature can mediate a mechanism for reducing toxicity, and this mechanism requires sufficient time to fully develop.

[0020] Furthermore, the incubation temperature is 62-67°C.

[0021] Preferably, the incubation temperature is 65°C.

[0022] Through extensive testing, this invention has found that the optimal incubation temperature is 65°C, which is also the incubation temperature with the lowest toxicity. The tests also showed that if the temperature differs from 65°C by 2-3°C, potassium release will decrease by about 40%, which significantly increases toxicity.

[0023] Preferably, the incubation time is 5-6 hours.

[0024] Based on the determination of incubation temperature, this invention, through extensive experiments, also discovered that incubation time has a significant impact on toxicity. Overall, toxicity is low after incubation of 2-12 hours; however, toxicity shows a trend of initial decrease followed by increase. From a theoretical perspective, it is highly likely that at least two processes exist simultaneously: one is the process of increasing toxicity, which is slow initially and rapid later; the other is the process of decreasing toxicity, which is rapid initially and slow later. Therefore, the toxicity is lowest during the middle of the incubation period, 5-6 hours.

[0025] Furthermore, the pH of the acidified organic solvent is 1.5-2.5.

[0026] This invention can also control impurities by acidifying the organic solvent. Impurity control has two main aspects: first, maintaining total impurities at a low level; and second, selectively reducing the content of impurities that affect subsequent processes. As a preferred embodiment, the total impurities are lowest when the pH of the acidified organic solvent is 2.0, and the content of impurity 3 (an impurity with a retention time of 0.78 at 383 nm) is also lowest. Although this impurity can be converted into a pharmaceutically active substance later, its presence will affect subsequent processes; therefore, acidification control can simultaneously reduce total impurities and accelerate the process flow.

[0027] Furthermore, hydration is performed using a medium, wherein the ratio of amphotericin B to the medium is 2-8 mg / mL. It is understood in this invention that hydration can be carried out by mixing and stirring the liposome powder with the medium. Mixing amphotericin B with the medium in the above-mentioned ratio can promote liposome formation and accelerate the process.

[0028] The hydration medium is typically an aqueous medium, usually containing buffer components that regulate and control the pH of the hydration system. The total volume of the medium ensures that the concentration of amphotericin B in the medium is 2-8 mg / mL, preferably 3-5 mg / mL. Further, the medium includes a first medium and a second medium. In one embodiment of the invention, the hydration includes: mixing the lipid powder with the first medium, stirring for 0.5-12 hours, and then mixing with the second medium; the first medium accounts for 40-60% of the total volume of the medium.

[0029] Another key aspect of this invention is that by first adding a first medium that accounts for 40-60% of the total volume, there is no need to use a buffer solution or adjust the pH.

[0030] When subsequent lyophilization is involved, the medium usually also contains lyophilization protectants, which are one or more of the following: sugars (such as glucose, mannose, sucrose, trehalose, lactose, etc.), sugar alcohols (such as sorbitol, etc.), polymers (such as polyvinylpyrrolidone, polyethylene glycol, etc.), buffer solutions, and pH adjusters (phosphate buffer, citrate, succinate, etc.), with sucrose and disodium succinate being preferred.

[0031] The hydration temperature is 40-60℃, preferably 45-55℃.

[0032] The hydration time is 0.5-12 hours, preferably 1-6 hours.

[0033] Furthermore, in one embodiment of the present invention, the medium comprises methyl-β-cyclodextrin, L-histidine, and water-soluble chitosan.

[0034] Regarding the composition of the medium, considering the effects of the chemical structures of each component and the synergistic effects between components, after extensive experiments, this invention found that most commonly considered stability-enhancing reagents and reagent combinations actually reduce the stability of the prepared liposomes compared to sucrose and disodium succinate. Only the combination of methyl-β-cyclodextrin, L-histidine, and water-soluble chitosan has a significant stability-enhancing effect, and the three have a synergistic effect. Preferably, the weight ratio of methyl-β-cyclodextrin, L-histidine, and water-soluble chitosan is (1-5):(1-5):1, and the concentration of water-soluble chitosan in the medium is preferably 0.5-3 g / L, which can achieve a better effect in improving the stability of liposomes.

[0035] The homogenization process resulted in an average particle size of less than 100 nm for amphotericin B liposomes.

[0036] The homogenization pressure is 300-1200 bar, preferably 500-1000 bar.

[0037] The homogenization is performed 1-12 times, preferably 2-6 times.

[0038] Preferably, the steps include: S1, Amphotericin B, cholesterol, phospholipids, and α-tocopherol were added to a 2.5 mol / L hydrochloric acid-acidified dichloromethane / methanol mixed solvent in a molar ratio of 1:2.5:7:0.03. The mixture was stirred and dissolved at 30-40℃ to obtain a mixed solution. The mixed solution was then spray-dried to obtain a lip powder. The volume ratio of dichloromethane to methanol in the dichloromethane / methanol mixed solvent was 1:1. S2, the lipid powder is added to a buffer solution comprising 40% of the total volume, the buffer solution comprising sucrose and disodium succinate, and hydrated at 55°C for 4 hours; the buffer solution is replenished, and the sample is homogenized 4 times at a pressure of 900 bar and a temperature of 50°C; after homogenization, the sample is filtered through a 0.22 μm filter membrane; incubated at 65°C for 5 hours; and freeze-dried.

[0039] On the other hand, the present invention provides amphotericin B liposomes prepared by the method provided by the present invention.

[0040] The above discloses the preferred method for preparing amphotericin B liposomes of the present invention. The prepared liposomes are small, uniformly shaped, spherical, single-chambered liposomes. Testing shows that the amphotericin B lipid complex has a composite rate ≥99%, a small particle size (average particle size ≤100 nm), a particle size of 50-150 nm after reconstitution, low toxicity (potassium release K50 ≥4.0), low total impurity content, and low content of impurities interfering with the process. The amphotericin B liposomes prepared by the above method can be used as a lyophilized powder for injection.

[0041] In summary, this invention has the following beneficial technical effects: This invention discovers that incubation can reduce toxicity, and based on this discovery, proposes a novel method for preparing amphotericin B liposomes. Extensive experiments have yielded the incubation temperature and time with the lowest toxicity. This invention also discovers that different acidification methods affect impurities; by controlling acidification, total impurities can be reduced simultaneously, accelerating the process. This invention discovers that by first adding a first medium accounting for 40-60% of the total volume for hydration, no further pH adjustment is needed. Based on the above research, an optimal method for preparing injectable amphotericin B liposomes has been obtained. Furthermore, it has been found that by combining incubation with a specially formulated medium for hydration and incubation, the release rate of the prepared liposomes in simulated gastric juice is significantly reduced, allowing more liposomes to enter the intestine for absorption and bioavailability. The release cycle of the prepared liposomes in simulated vaginal fluid is significantly increased, thus prolonging the duration of amphotericin B's efficacy. Attached Figure Description

[0042] Figure 1 : Cryotransmission electron microscopy image of the amphotericin B liposomes of the present invention. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0044] Example 1: Preparation and testing of amphotericin B liposomes Weigh 10.00 g of amphotericin B, 16.80 g of distearate phosphatidylglycerol (DSPG), 0.13 g of α-tocopherol, 42.60 g of hydrogenated soybean phosphatidylcholine (HSPC), and 10.40 g of cholesterol (CHOL), and place them in a mixed solvent of dichloromethane and methanol acidified with 2.5 mol / L hydrochloric acid. Dissolve by stirring at 30 °C to obtain a mixed solution. The volume ratio of dichloromethane to methanol is 1:1, the total volume is 640 mL, and the amount of 2.5 mol / L hydrochloric acid used is 8 mL. Spray dry the above mixed solution to obtain a lipid powder.

[0045] The hydration medium was a buffer solution (pH 7.8) containing sucrose and disodium succinate, with sucrose accounting for 7% by mass and disodium succinate accounting for 0.2% by mass, for a total volume of 2000 mL. The above-mentioned lipid powder was added to the hydration medium at 40% of the total volume and hydrated at 55 °C for 4 h. After the hydrated solution was replenished with buffer solution (to make up the remaining 60% volume), it was homogenized 4 times at a homogenization pressure of 900 bar and a temperature of 50 °C. After homogenization, the sample was filtered through a 0.22 μm filter membrane, incubated at 65 °C for 5 h, and then freeze-dried.

[0046] The amphotericin B liposomes obtained by freeze-drying are lyophilized powder injections for injection. After being mixed and dissolved with an injection solution (such as sterile water for injection), they can be used to obtain amphotericin B liposome injection solution for further dilution. They can also be used for oral or vaginal administration.

[0047] like Figure 1 The prepared liposomes were uniformly shaped, spherical, small, single-chambered liposomes. Testing showed that the amphotericin B lipid complex had a composite rate ≥99%, and the particle size was small, with an average particle size ≤100 nm. The particle size after reconstitution was 50-150 nm. (Particle size was measured by transmission electron microscopy.) Example 2: Incubation Experiment The present invention also found that incubation before freeze-drying, especially incubation under certain conditions, can reduce the toxicity of amphotericin B liposomes. Here, toxicity refers to the fact that amphotericin B can bind to ergosterol on the cell membrane, causing cell rupture and releasing potassium ions from the cell. This embodiment illustrates a series of experiments on incubation during the development of the present invention to reveal the role of incubation in the preparation of amphotericin B liposomes and to determine suitable incubation conditions.

[0048] A series of experiments were conducted according to the method in Example 1, with the only difference being the adjustment of the incubation step. A preparation method without incubation was used as a control (the group whose incubation temperature and time were not recorded in Table 1). The experimental groups adjusted the incubation temperature and incubation time respectively. The amphotericin B liposomes prepared in each group were subjected to toxicity tests, with "potassium release" as the detection index. The value of potassium release corresponds to the sample concentration corresponding to the release of 50% potassium ions (potassium ions released after cell rupture). Therefore, the higher the potassium release, the lower the toxicity of the corresponding amphotericin B liposome. The specific detection method is as follows: a solution containing approximately 1 mg of amphotericin B per 1 mL was prepared using amphotericin B liposome buffer for injection as the test solution; an appropriate amount of the test solution was accurately measured, and the test solution was diluted to a concentration of 100 μg / mL with amphotericin B liposome buffer for injection, and then serially diluted 11 times by 2-fold with amphotericin B liposome buffer for injection. Accurately measure 64 μL of each of the above concentration solutions and place them in separate 1.5 mL centrifuge tubes. Add 96 μL of washed rat red blood cell sample, seal, and incubate at 36±1℃ for 4 h. After incubation, centrifuge the samples at 1000g for 10 min at 4℃, collect the supernatant, and determine the potassium ion concentration using an ion-selective electrode method.

[0049] The toxicity test results for each group are shown in Table 1.

[0050] Table 1: Toxicity test results for each group

[0051] Based on the above experimental results, it can be seen that incubation can effectively reduce toxicity compared to the method without incubation. However, different incubation temperatures and incubation times have significant differences in their effects on toxicity. The incubation temperature is preferably 60-70℃, and the incubation time is preferably 2-12h. Among these, the incubation temperature has a very significant effect on toxicity, with 65℃ being the optimal value. A fluctuation of 2℃ around 65℃ can lead to a difference of about 40% in potassium release value. Based on the optimal incubation temperature, an incubation time of 5-6h is the most preferred.

[0052] Example 3: Tests on impurities controlled by different acidification methods This invention also controls impurities through acidification. The preparation of amphotericin B liposomes involves various impurities, primarily affecting two aspects: first, some impurities may be converted into active substances in subsequent processes. Although this does not affect the purity and activity of the product, it consumes time in these processes; second, high impurity content will correspondingly reduce the drug content and efficacy. Therefore, it is necessary to control impurities to save processing time and improve efficacy. This embodiment lists experiments related to acidification and impurity control conducted according to this invention to reveal the impact of acidification on impurities.

[0053] A series of experiments were conducted following the method described in Example 1. The volume ratio of dichloromethane to methanol was 1:1, with a total volume of 640 mL. The only difference was the acidification of the mixed solvent of dichloromethane and methanol, which was divided into three groups: T1, acidified with 7 mL of 2.5 mol / L hydrochloric acid, with a pH of approximately 2.5; T2, acidified with 8 mL of 2.5 mol / L hydrochloric acid, with a pH of approximately 2.0; and T3, acidified with 9 mL of 2.5 mol / L hydrochloric acid, with a pH of approximately 1.5. Impurities were detected using high-performance liquid chromatography (HPLC). The impurities were mainly unknown, so they were named according to their retention time. Detection was performed at 303 nm and 383 nm. It was found that the impurities detected at 303 nm were all present in extremely low concentrations, so the impurities detected at 383 nm were primarily considered and designated as impurities 1 through 8. Impurity tracking analysis revealed that impurity 3 would be converted into an active substance in subsequent processes. Although this did not affect the purity and activity of the product, it required time in the subsequent processes. Finally, the total amount of impurities was integrated and recorded as total impurities. The specific detection method is as follows: Solution preparation: Citric acid solution: Weigh 4.2g of citric acid, add 1000mL of water, dissolve, mix well, and filter (can be prepared in equal proportions).

[0054] Citric acid solution (pH 4.7): Take citric acid solution and adjust the pH to 4.7 ± 0.01 with concentrated ammonia.

[0055] Citric acid solution (pH 3.9): Take citric acid solution and adjust the pH to 3.9 ± 0.01 with concentrated ammonia.

[0056] Mobile phase A: Measure 1200 mL of citric acid solution (pH 4.7), 520 mL of acetonitrile, and 260 mL of methanol, mix well, and sonicate for 10 min to obtain the mobile phase A.

[0057] Mobile phase B: Measure 200 mL of citric acid solution (pH 3.9), 680 mL of acetonitrile, and 120 mL of methanol, mix well, and sonicate for 10 min to obtain the mobile phase.

[0058] Blank solvent: namely methanol.

[0059] Dilute hydrochloric acid: Take 23.4 mL of hydrochloric acid and place it in a 100 mL volumetric flask. Dilute with water to the mark and shake well (can be prepared in equal proportions).

[0060] Ammonium acetate solution: Dissolve 1g of ammonium acetate in 100mL of water and mix well (it can be prepared in equal proportions).

[0061] Methanol-ethanol mixed solution: Measure 10 mL of anhydrous methanol and 40 mL of anhydrous ethanol, and mix well (can be prepared in equal proportions).

[0062] Control solution a: Accurately weigh approximately 20 mg of amphotericin B reference standard into a 50 mL volumetric flask, dissolve in 15 mL of DMSO, and dilute to the mark with blank solvent within 2 hours, then mix well. Then accurately measure 5 mL of the above solution into a 25 mL volumetric flask, dilute to the mark with blank solvent, and mix well (amphotericin B concentration is 0.8 μg / mL).

[0063] Control solution b: Accurately measure 1 mL of control solution a into a 100 mL volumetric flask, and dilute to the mark with blank solvent (use within 24 hours of preparation; amphotericin B concentration is 0.8 μg / mL).

[0064] Control solution d: Accurately weigh about 10 mg of amphotericin B reference standard into a 50 mL volumetric flask, dissolve it in 5 mL of DMSO, then add 35 mL of a methanol-ethanol mixture within 2 hours, add 0.1 mL of dilute hydrochloric acid, shake well and let stand for 2.5 hours, then add 10 mL of ammonium acetate solution and shake well (for system suitability).

[0065] System suitability solution: Accurately weigh approximately 106 mg of amphotericin B liposome reference powder, place it in a 50 mL volumetric flask, add methanol to approximately 80% of the volume, shake for approximately 20 min, and then dilute to the mark with methanol.

[0066] Test solution: Take approximately 106 mg of the powder from the content variation section, place it in a 50 mL volumetric flask, add approximately 80% methanol, shake for 20 min, dilute to the mark with methanol, and mix well. Prepare two parallel solutions.

[0067] The chromatographic conditions are shown in Table 2.

[0068] Table 2: Chromatographic conditions

[0069] The detection results of each impurity and total impurities under different acidification conditions are shown in Table 3.

[0070] Table 3: Detection results of various impurities and total impurities under different acidification conditions

[0071] RRT refers to the retention time corresponding to each impurity. Impurity 3 is the key impurity for reducing process time. The content of impurity 3 in group T2 is the lowest, and the total impurities in group T2 are also the lowest.

[0072] Example 4: Hydration Test Conventional hydration involves adding lipid powder to an acidic buffer solution, adjusting the pH to 4-6 depending on the substrate. Another key aspect of this invention is the pre-addition of a specific proportion of buffer solution at a lower pH for hydration, simultaneously promoting the conversion of impurities 3 into more active ingredients. After hydration, the medium is added for homogenization and subsequent steps. This method eliminates the need for pH adjusters, avoiding issues like excessively high local acid or alkali concentrations and multiple pH adjustments, thus simplifying the process. This embodiment includes experiments related to hydration to demonstrate the simplification effect of this approach.

[0073] A series of experiments were conducted according to the method of Example 1, with the only difference being the hydration steps. The conventional hydration method (i.e., adding the medium all at once, the medium being the same as the buffer solution in Example 1) was used as the control, and the experimental group was used for adding the medium sequentially. The medium added first was called the first medium, and its percentage of the total volume was recorded. The total volume of the medium in the control and experimental groups was the same.

[0074] Encapsulation efficiency, particle size, and stability were tested. Comprehensive analysis revealed that the control group exhibited good encapsulation efficiency, particle size, and stability, but impurity 3 exceeded expected levels. For the experimental groups, adding 40-60% of the medium first (without preparing acidic buffer or adjusting pH) resulted in good encapsulation efficiency, particle size, stability, and low levels of impurity 3. Other experimental groups showed incomplete hydration. Therefore, adding 40-60% of the medium first for hydration, then replenishing the medium before homogenization, filtration, incubation, and lyophilization can effectively reduce localized high acid / base concentrations and repeated pH adjustments, as well as reagent waste.

[0075] Example 5: Test of hydration medium As a preferred embodiment, another key aspect of this invention lies in the innovative composition of the hydration medium, which greatly improves the stability of amphotericin B liposomes and can be used to prepare amphotericin B formulations for oral or vaginal administration. This embodiment provides relevant experiments to illustrate the above key points.

[0076] Unlike the amphotericin B liposomes for injection in Examples 1-4, the amphotericin B liposomes prepared in this example using the hydration medium exhibit higher stability in simulated gastric and vaginal fluids due to changes in surface charge and membrane properties, making them more suitable for preparing amphotericin B formulations for oral or vaginal administration. A series of experiments were conducted following the method in Example 1, the difference being the composition of the hydration medium. Using Example 1 as a control, the composition of the hydration medium in the experimental groups was adjusted by adding the following components to phosphate buffer (pH 7.8): T1, containing 5 g / L sucrose and 7.5 g / L trehalose; T2, containing 5 g / L mannitol and 2.5 g / L L-histidine; T3, containing 2.5 g / L methyl-β-cyclodextrin and 2.5 g / L... L-histidine and 1 g / L water-soluble chitosan (CAS: 9012-76-4); 2 mg of the lyophilized powder from the control and experimental groups was dissolved in 10 mL of sterile water for injection, and 10 times the volume of commercially available simulated gastric fluid and simulated vaginal fluid were added for drug release testing. The temperature was maintained at 37°C and shaken. Samples were taken at 30 min, 1 h, 2 h and 4 h after the start of the experiment, and the content of amphotericin B was detected according to the detection method in Example 3. The total release amount of amphotericin B was calculated according to the sampling ratio, and the release rate was calculated as follows: release rate = total mass of amphotericin B in simulated gastric fluid or simulated vaginal fluid / total mass of amphotericin B corresponding to the lyophilized powder. The results are shown in Tables 4 and 5.

[0077] Table 4: Detection results of simulated gastric fluid drug release

[0078] Table 5: Detection results of drug release from simulated vaginal fluid

[0079] The experimental results show that there was no significant difference in drug release in simulated gastric fluid compared to the control, T1, and T2. However, the release rate of T3 was significantly lower than that of the former at different time points. Usually, the formulation can be emptied from the stomach within 2 hours, but a large number of liposomes are still retained to enter the intestine and release the active ingredient to produce the drug effect. In simulated vaginal fluid, there was no significant difference in drug release compared to the control, T1, and T2. The drug was almost completely released at 2 hours, while T3 was almost completely released at 4 hours, which prolongs the antibacterial time of the drug.

[0080] In the course of this invention research, the following comparative experiments D1, D2, and D3 were also conducted. D1 was based on T3 without methyl-β-cyclodextrin, D2 was based on T3 without L-histidine, and D3 was based on T3 without water-soluble chitosan. 2 mg of the lyophilized powder from the control and comparative experiments D1, D2, and D3 was dissolved in 10 mL of sterile water for injection. Ten times the volume of commercially available simulated gastric fluid and simulated vaginal fluid were added for drug release testing. The temperature was maintained at 37°C and shaken. Samples were taken at 30 min, 1 h, 2 h, and 4 h after the start of the experiment, and the content of amphotericin B was detected according to the detection method in Example 3. The total release amount of amphotericin B was calculated according to the sampling ratio, and the release rate was calculated as follows: Release rate = Total mass of amphotericin B in simulated gastric fluid or simulated vaginal fluid / Total mass of amphotericin B corresponding to the lyophilized powder. The results are shown in Tables 6 and 7.

[0081] Table 6: Detection results of simulated gastric fluid drug release

[0082] Table 7: Detection results of drug release from simulated vaginal fluid

[0083] The above experimental results show that when any two of methyl-β-cyclodextrin, L-histidine, and water-soluble chitosan are used as components in the hydration medium, the drug release of the resulting liposomes is not significantly different from that of the control. However, T3 shows that the release rate at different time points is significantly lower than that of the former, indicating that methyl-β-cyclodextrin, L-histidine, and water-soluble chitosan have a synergistic effect when used as components in the hydration medium.

[0084] Preliminary analysis suggests that the effects are due to the chemical structures of the three substances and are related to processes such as hydration, incubation, and freeze-drying. Possible mechanisms include: 1) synergistic inhibition of membrane leakage: methyl-β-cyclodextrin aids in membrane defect repair, L-histidine regulates membrane potential, and water-soluble chitosan acts as a physical barrier; 2) inhibition of oxidative degradation and enzymatic damage: methyl-β-cyclodextrin encapsulates unsaturated fatty acids, inhibiting oxidative degradation; L-histidine inhibits enzyme activity by regulating microenvironment pH and chelates metals to inhibit metal-mediated oxidation; and water-soluble chitosan acts as a physical barrier against enzymes; 3) inhibition of freeze-drying damage: all three substances maintain membrane fluidity and prevent ice crystal penetration during freeze-drying. Further investigation is needed to determine the specific mechanisms.

[0085] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent modifications or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing liposome-encapsulated amphotericin B, characterized by, The preparation method comprises the following steps: S1, mixing amphotericin B, film forming material, antioxidant and acidified organic solvent, and drying to obtain a lipid powder; S2, sequentially performing hydration, homogenization, filtration, incubation and freeze-drying on the lipid powder; The temperature of the incubation is 60-70℃, and the time of the incubation is 2-12h.

2. The production method according to claim 1, wherein The temperature of the incubation is 62-67℃.

3. The production method according to claim 1, wherein The time of the incubation is 5-6h.

4. The production method according to any one of claims 1 to 3, characterized by, The molar ratio of the amphotericin B, film forming material and antioxidant is 1: (5-15) : (0.01-0.1).

5. The production method according to any one of claims 1 to 3, wherein The pH of the acidified organic solvent is 1.5-2.

5.

6. The production method according to any one of claims 1 to 3, wherein The ratio of the amphotericin B to the acidified organic solvent is 5-50mg / mL.

7. The production method according to any one of claims 1 to 3, wherein The ratio of the amphotericin B to the medium is 2-8mg / mL.

8. The production method according to claim 7, wherein The medium comprises a first medium and a second medium; the hydration comprises mixing the lipid powder with the first medium, stirring for 0.5-12h, and then mixing with the second medium; the first medium accounts for 40-60% of the total volume of the medium.

9. The production method according to claim 8, wherein The medium comprises methyl-β-cyclodextrin, L-histidine and water-soluble chitosan.

10. Amphotericin B liposome prepared by the preparation method in any one of claims 1-9.

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