A liposome preparation co-loading amphotericin b-posaconazole and a preparation method and application thereof

Abstract

The application belongs to the technical field of pharmaceutical preparations, and relates to a liposome preparation for co-carrying amphotericin B and posaconazole as well as a preparation method and application thereof. The liposome preparation comprises amphotericin B, posaconazole, liposomes, an antioxidant and a freeze-drying protective agent. The mass ratio of amphotericin B to posaconazole is 1-3:1, and the mass ratio of amphotericin B to the liposomes, the antioxidant and the freeze-drying protective agent is 1:5-6:0.01-0.1:16-20. Through a specific formula ratio and method, the application co-encapsulates amphotericin B and posaconazole in the liposomes, and improves the solubility of the two drugs. The obtained liposomes have uniform particle size distribution and good stability, better exert the synergistic antifungal infection effect of amphotericin B and posaconazole, effectively solve the problems of low bioavailability and significant toxic and side effects of the two drugs, and provide a safe, effective and stable new preparation for the combined treatment of clinical deep fungal infections.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, and relates to a liposomal formulation co-loaded with amphotericin B-posaconazole, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Amphotericin B is a polyene antifungal antibiotic that exerts its potent and broad-spectrum antifungal effect by binding to ergosterol in the fungal cell membrane, forming microporous channels that cause leakage of essential intracellular substances. It is considered the "gold standard" treatment for deep fungal infections. However, amphotericin B has poor water solubility and serious toxic side effects, including hepatotoxicity, nephrotoxicity, hematologic toxicity, adverse infusion reactions, and electrolyte disturbances, especially nephrotoxicity, which greatly limits its clinical application. To reduce its toxic side effects, Gilead Sciences (formerly Nexstar) used lipid technology to modify the formulation of amphotericin B, successfully developing injectable amphotericin B liposomes (trade name AmBisome). This formulation effectively reduces the toxic side effects of amphotericin B and has significant clinical advantages, and has been widely used globally for over 30 years. Nevertheless, single-component amphotericin B liposomes still face the problem of drug resistance in clinical applications.

[0004] Posaconazole is a second-generation triazole antifungal drug with a broad antibacterial spectrum and strong antifungal activity. It has good therapeutic effects on various deep fungal infections, including invasive Aspergillus, Fusarium, yeast, mycobacteria, and Coccidioides. However, posaconazole is lipophilic and has a high protein binding rate, resulting in low bioavailability of oral suspensions. While delayed-release tablets and injections improve bioavailability, their targeting is poor, and long-term administration can lead to accumulation in the body, resulting in significant hepatotoxicity and side effects. In particular, the use of sulfobutyl betacyclodextrin as an excipient in the injection formulation to increase posaconazole solubility has a certain degree of nephrotoxicity.

[0005] The combined use of amphotericin B and triazole antifungal drugs has become a research hotspot in the treatment of invasive fungal infections in recent years. Posaconazole, due to its broad-spectrum antifungal activity and low resistance rate, shows synergistic potential when used in combination with amphotericin B in antifungal therapy. However, current technology lacks a solution for co-encapsulating amphotericin B and posaconazole in the same liposome delivery system; simultaneously, technology for simultaneously encapsulating two antifungal drugs with different mechanisms of action in the same liposome system remains a gap. Although existing technologies have methods for simultaneously encapsulating multiple antifungal compounds in liposome compositions, the membrane structure of liposomes differs significantly from conventional liposomes, resulting in substantial differences in in vivo release behavior, stability, and formulation processes. Therefore, developing a liposome co-loaded with amphotericin B and posaconazole to improve the stability of both and achieve a synergistic antifungal effect has significant practical application value. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a liposomal formulation co-loaded with amphotericin B and posaconazole, its preparation method, and its application. Through a specific formulation ratio and method, amphotericin B and posaconazole are co-encapsulated in liposomes, improving the solubility of both drugs. The resulting liposomes exhibit uniform particle size distribution and good stability, better leveraging the synergistic antifungal effects of amphotericin B and posaconazole. This effectively solves the problems of low bioavailability and significant toxic side effects of the two drugs, providing a safe, effective, and stable new formulation for the combined treatment of deep fungal infections in clinical practice.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: In one aspect, a liposomal formulation co-loaded with amphotericin B-posaconazole includes amphotericin B, posaconazole, liposomes, an antioxidant, and a lyophilization protectant; wherein the mass ratio of amphotericin B to posaconazole is 1~3:1, and the mass ratio of amphotericin B to liposomes, antioxidant, and lyophilization protectant is 1:5~6:0.01~0.1:16~20.

[0008] In a second aspect, a method for preparing the liposome formulation described in the first aspect of the present invention includes the following steps: Amphotericin B, posaconazole, liposome membrane material for liposome preparation, and antioxidant were added to an organic solvent. The pH was adjusted by adding a pH adjuster, the mixture was stirred to dissolve, and the organic solvent was evaporated to obtain a lipid membrane. Dissolve the freeze-drying protectant in water to prepare a freeze-drying aqueous solution; The lipid membrane was hydrated using lyophilized aqueous solution to obtain a crude liposome suspension; The crude liposome suspension was sheared and emulsified, then homogenized by microfluidic jet, followed by incubation, sterilization, filtration, and freeze-drying to obtain the final product. During the homogenization process, the homogenization pressure was 6000~8000 psi.

[0009] Thirdly, the use of the liposome formulation described in the first aspect of the present invention in the preparation of an antifungal infection drug.

[0010] The beneficial effects of this invention are as follows: 1. The liposomal formulation co-loaded with amphotericin B-posaconazole provided by this invention can exert a synergistic effect and reduce toxic side effects. Experiments show that by co-loading amphotericin B-posaconazole into liposomes, this invention enables amphotericin B and posaconazole to produce a synergistic effect, more effectively enhancing the antifungal effect, reducing drug hepatotoxicity and nephrotoxicity, and improving medication safety.

[0011] 2. The preparation method provided by this invention has high encapsulation efficiency and good stability. By combining thin-film dispersion with shear emulsification and microfluidic homogenization processes, the encapsulation efficiency of the obtained liposomes is ≥95%, the particle size is controlled within 120~150 nm, PDI≤0.2, the particle size distribution is narrow, and the particle size does not change significantly under accelerated conditions.

[0012] 3. The preparation method provided by this invention is simple and easy to industrialize. The core processes of this preparation method are only formulation, shearing, and homogenization, requiring no special equipment, and the steps are concise, making it easy to scale up production. The freeze-dried formulation has good stability, facilitating transportation and clinical use. Detailed Implementation

[0013] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0014] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0015] The liposomes described in this invention refer to (micro) spherical vesicles with a closed bilayer structure formed by the self-assembly of cholesterol and phospholipids dispersed in an aqueous phase.

[0016] Due to the low bioavailability and significant toxic side effects of amphotericin B and posaconazole, this invention proposes a liposomal formulation co-loaded with amphotericin B-posaconazole, its preparation method, and its application.

[0017] A typical embodiment of the present invention provides a liposomal formulation co-loaded with amphotericin B-posaconazole, comprising amphotericin B, posaconazole, liposomes, an antioxidant, and a lyophilization protectant; wherein the mass ratio of amphotericin B to posaconazole is 1~3:1, and the mass ratio of amphotericin B to liposomes, antioxidant, and lyophilization protectant is 1:5~6:0.01~0.1:16~20.

[0018] In some embodiments, the liposomes are composed of a combination of cholesterol and phospholipids, wherein the phospholipids are one or more of hydrogenated soybean phosphatidylcholine, sodium distearate phosphatidylglycerol, and egg yolk lecithin.

[0019] In some embodiments, the antioxidant is DL-α-tocopherol.

[0020] In some embodiments, the freeze-drying protectant is sucrose, trehalose, or mannitol.

[0021] In some embodiments, a pH adjuster is further included, the pH adjuster being selected from one or more of hydrochloric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, citric acid, and sodium citrate.

[0022] In some embodiments, the liposome particle size is 120~150nm, PDI≤0.2, and encapsulation efficiency≥95%.

[0023] Another embodiment of the present invention provides a method for preparing the above-mentioned liposome formulation, comprising the following steps: Amphotericin B, posaconazole, liposome membrane material for liposome preparation, and antioxidant were added to an organic solvent. The pH was adjusted by adding a pH adjuster, the mixture was stirred to dissolve, and the organic solvent was evaporated to obtain a lipid membrane. Dissolve the freeze-drying protectant in water to prepare a freeze-drying aqueous solution; The lipid membrane was hydrated using lyophilized aqueous solution to obtain a crude liposome suspension; The crude liposome suspension was sheared and emulsified, then homogenized by microfluidic jet, followed by incubation, sterilization, filtration, and freeze-drying to obtain the final product. During the homogenization process, the homogenization pressure was 6000~8000 psi.

[0024] In some embodiments, the organic solvent is a mixture of chloroform and methanol. Specifically, the volume ratio of chloroform to ethanol is 1:2 to 3.

[0025] In one embodiment, a pH adjuster is used to adjust the pH to 3.6-4.4.

[0026] In some embodiments, the temperature for removing the organic solvent is 35~45°C.

[0027] In some embodiments, the temperature for hydrating the lipid membrane is 40–60°C. This condition is beneficial for accelerating the hydration rate.

[0028] Shearing in shear emulsification refers to the process by which strong mechanical force generated by mechanical equipment (such as a shear emulsifier) ​​breaks immiscible liquids (such as oil and water) into tiny droplets and disperses them uniformly. In some embodiments, during shear emulsification, the shearing speed is 10,000 to 15,000 rpm and the shearing time is 20 to 30 minutes.

[0029] In some embodiments, the homogenization process is repeated 5 to 7 times.

[0030] A third embodiment of the present invention provides the application of the above-mentioned liposome preparation in the preparation of antifungal infection drugs.

[0031] In some embodiments, the fungus is Candida albicans.

[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0033] Example 1 A liposomal formulation co-loaded with amphotericin B-posaconazole has the following formulation as shown in Table 1.

[0034] Table 1. Formulation Prescription

[0035] Its preparation method includes the following steps: (1) Add amphotericin B, posaconazole, hydrogenated soybean phosphatidylcholine, cholesterol, distearate phosphatidylglycerol sodium, and DL-α-tocopherol to 50 L of organic solvent (chloroform to methanol volume ratio 1:2.5). Add sodium dihydrogen phosphate and disodium hydrogen phosphate to adjust the pH to 4.0 and stir until completely dissolved. Rotary evaporate in a 40 °C water bath (60 rpm) until a uniform and transparent lipid film is formed.

[0036] (2) Dissolve sucrose in water for injection to a concentration of 9% (w / v), filter and sterilize for later use. Take the above solution, preheat to 40°C, add it to the lipid membrane, and hydrate in a 40°C water bath for 30 min to obtain a crude liposome suspension.

[0037] (3) The crude liposomes were emulsified under high shear at 12,000 rpm for 25 min, and then homogenized 6 times at 7,000 psi using a microfluidic homogenizer. The homogenized suspension was incubated at room temperature for 30 min, filtered through a 0.22 μm polyethersulfone filter for sterilization, dispensed into vials, and freeze-dried (freeze-drying process: pre-freeze at -45℃ for 3 h, dry once at -20℃ for 20 h, and dry twice at 30℃ for 6 h) to obtain the freeze-dried formulation of co-loaded amphotericin B-posaconazole liposomes.

[0038] Example 2 A liposomal formulation co-loaded with amphotericin B-posaconazole has the following formulation as shown in Table 2.

[0039] Table 2 Formulation Prescription

[0040] Its preparation method includes the following steps: (1) Add amphotericin B, posaconazole, egg yolk lecithin, cholesterol, distearate phosphatidylglycerol sodium, and DL-α-tocopherol to 45 L of organic solvent (chloroform to methanol volume ratio 1:2). Add sodium citrate and sodium citrate to adjust the pH to 4.0 and stir until completely dissolved. Rotary evaporate in a 45°C water bath (60 rpm) until a uniform and transparent lipid film is formed.

[0041] (2) Dissolve trehalose in water for injection to a concentration of 9% (w / v), filter and sterilize for later use. Take the above solution, preheat to 55°C, add it to the lipid membrane, and hydrate in a 55°C water bath for 30 min to obtain a crude liposome suspension.

[0042] (3) The crude liposomes were emulsified under high shear at 15,000 rpm for 20 min, and then homogenized 5 times at 8,000 psi using a microfluidic homogenizer. The homogenized suspension was incubated at room temperature for 30 min, filtered through a 0.22 μm polyethersulfone filter for sterilization, dispensed into vials, and freeze-dried (freeze-drying process: pre-freeze at -45℃ for 3 h, dry once at -20℃ for 20 h, and dry twice at 30℃ for 6 h) to obtain the freeze-dried formulation of co-loaded amphotericin B-posaconazole liposomes.

[0043] Example 3 A liposomal formulation co-loaded with amphotericin B-posaconazole has the following formulation as shown in Table 3.

[0044] Table 3 Formulation Prescription

[0045] Its preparation method includes the following steps: (1) Add amphotericin B, posaconazole, hydrogenated soybean phosphatidylcholine, cholesterol, egg yolk lecithin, distearate phosphatidylglycerol sodium, and DL-α-tocopherol to 55 L of organic solvent (chloroform to methanol volume ratio 1:3), add 1 mol / L hydrochloric acid solution to adjust to pH 4.0, and stir until completely dissolved. Rotary evaporate in a 35℃ water bath (60 rpm) until a uniform and transparent lipid film is formed.

[0046] (2) Dissolve mannitol in water for injection to a concentration of 9% (w / v), filter and sterilize for later use. Take the above solution, preheat to 60°C, add it to the lipid membrane, and hydrate in a 60°C water bath for 30 min to obtain a crude liposome suspension.

[0047] (3) The crude liposomes were emulsified under high shear at 10,000 rpm for 30 min, and then homogenized 7 times at 6,000 psi using a microfluidic homogenizer. The homogenized suspension was incubated at room temperature for 30 min, filtered through a 0.22 μm polyethersulfone filter for sterilization, dispensed into vials, and freeze-dried (freeze-drying process: pre-freeze at -45℃ for 3 h, dry once at -20℃ for 20 h, and dry twice at 30℃ for 6 h) to obtain the freeze-dried formulation of co-loaded amphotericin B-posaconazole liposomes.

[0048] Comparative Example 1 The formulation is the same as in Example 1. Amphotericin B liposome formulation (excluding posaconazole) and posaconazole liposome formulation (excluding amphotericin B) are prepared separately, lyophilized, and then physically mixed at a 1:1 mass ratio. The preparation processes for amphotericin B liposome formulation and posaconazole liposome formulation are the same as in Example 1.

[0049] Comparative Example 2 The formulation is the same as in Example 1, but the homogenization pressure of the microjets in the preparation method is 5000 psi and the number of cycles is 7.

[0050] Performance testing: The encapsulation efficiency, particle size before and after lyophilization, and PDI of the liposome formulations prepared in the examples and comparative examples of this application were tested, and the test results are shown in Table 1 below.

[0051] Table 1. Performance Comparison Data Between Examples and Comparative Examples

[0052] Table 1 shows that the liposome formulations co-loaded with amphotericin B-posaconazole in Examples 1 to 3 of this invention have higher encapsulation efficiency. The experiment proves that the homogenization pressure and number of homogenization cycles have a significant impact on the quality of the liposome formulations.

[0053] Example 1 was placed under the conditions of 40°C and 75% relative humidity for 6 months. Samples were taken at 0, 1, 3, and 6 months respectively to determine the encapsulation efficiency, particle size and PDI. The measurement results are shown in Table 2.

[0054] Table 2 Stability Data of Example 1

[0055] The results in Table 2 show that under the accelerated conditions of 40°C and 75% relative humidity, the particle size and encapsulation efficiency of the amphotericin B-posaconazole co-loaded liposome preparation of the present invention remain basically unchanged, with good stability.

[0056] In vitro synergistic antifungal activity study: The checkerboard microdilution method was adopted, with Candida albicans ( Candida albicans ) as the test strain, to determine the minimum inhibitory concentration (MIC) of Example 1, Comparative Example 1, amphotericin B single-drug liposome, and posaconazole single-drug liposome, and calculate the fractional inhibitory concentration index (FICI). FICI ≤ 0.5 indicates synergism, 0.5 < FICI ≤ 1 indicates addition, and > 1 indicates antagonism. The results are shown in Table 3 below.

[0057] Table 3 MIC Values and FICI Evaluation of Each Sample against Candida albicans

[0058] The results in Table 3 indicate that when amphotericin B and posaconazole are used in combination through a co-loaded liposome preparation, there is a synergistic effect between the two, which can more effectively inhibit the growth of Candida albicans, reduce the severity of deep fungal infection, and thus exhibit better effects in the treatment of deep fungal infections than administration of a single drug or a physical mixture.

[0059] Preliminary in vivo safety evaluation: Kunming mice (20±2 g, half male and half female) were randomly divided into 5 groups: blank control group (normal saline), Example 1 group (amphotericin B 10 mg / kg + posaconazole 10 mg / kg), AmBisome group (amphotericin B 10 mg / kg), amphotericin B deoxycholate group (AmB-DOC, 1 mg / kg, the dose was reduced due to high toxicity), and posaconazole injection group (10 mg / kg). A single administration was performed via tail vein, and blood was collected on the 3rd day after administration to measure serum creatinine (Cr) and alanine aminotransferase (ALT). The results are shown in Table 4; Table 4 Serum Biochemical Indicators of Mice in Each Group

[0060] Note: *P<0.05 compared with the blank control group.

[0061] The results in Table 4 show that the creatinine and ALT levels in the mice in Example 1 group were not significantly different from those in the blank control group, and were comparable to those in the AmBisome group, but significantly lower than those in the AmB-DOC and posaconazole injection groups, demonstrating that the co-loaded liposome formulation of the present invention significantly reduced drug nephrotoxicity and hepatotoxicity.

[0062] In summary, the co-loaded amphotericin B-posaconazole liposome formulation provided by this invention has high encapsulation efficiency, good stability, significant synergistic antifungal activity, and low toxicity. Moreover, the preparation process is simple, suitable for industrial production, and has broad clinical application prospects.

[0063] 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 liposomal formulation co-loaded with amphotericin B-posaconazole, characterized in that, It includes amphotericin B, posaconazole, liposomes, antioxidants, and lyophilization protectants; wherein the mass ratio of amphotericin B to posaconazole is 1~3:1, and the mass ratio of amphotericin B to liposomes, antioxidants, and lyophilization protectants is 1:5~6:0.01~0.1:16~20.

2. The liposome formulation as described in claim 1, characterized in that, The liposomes are composed of cholesterol and phospholipid compounds; wherein the phospholipid compounds are one or more of hydrogenated soybean phosphatidylcholine, distearate phosphatidylglycerol sodium, and egg yolk lecithin.

3. The liposome formulation as described in claim 1, characterized in that, The antioxidant is DL-α-tocopherol.

4. The liposome formulation as described in claim 1, characterized in that, The freeze-drying protectant is sucrose, trehalose, or mannitol.

5. The liposome formulation as described in claim 1, characterized in that, It also includes a pH adjuster selected from one or more of hydrochloric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, citric acid, and sodium citrate.

6. The liposome formulation as described in claim 1, characterized in that, The liposomes have a particle size of 120~150nm, a PDI of ≤0.2, and an encapsulation efficiency of ≥95%.

7. A method for preparing the liposome formulation according to claim 1, characterized in that, Includes the following steps: Amphotericin B, posaconazole, liposome membrane material for liposome preparation, and antioxidant were added to an organic solvent. The pH was adjusted by adding a pH adjuster, the mixture was stirred to dissolve, and the organic solvent was evaporated to obtain a lipid membrane. Dissolve the freeze-drying protectant in water to prepare a freeze-drying aqueous solution; The lipid membrane was hydrated using lyophilized aqueous solution to obtain a crude liposome suspension; The crude liposome suspension was sheared and emulsified, then homogenized by microfluidic jet, followed by incubation, sterilization, filtration, and freeze-drying to obtain the final product. During the homogenization process, the homogenization pressure was 6000~8000 psi.

8. The preparation method according to claim 7, characterized in that, The organic solvent is a mixture of chloroform and methanol; Alternatively, adjust the pH to 3.6-4.4 using a pH adjuster; Alternatively, the temperature for removing organic solvents is 35~45℃; Alternatively, the temperature for hydrating lipid membranes is 40~60℃; Alternatively, during the shear emulsification process, the shearing speed is 10,000~15,000 rpm and the shearing time is 20~30 min; Alternatively, the homogenization process can be repeated 5 to 7 times.

9. The use of a liposome formulation according to any one of claims 1 to 6 in the preparation of an antifungal drug.

10. The application as described in claim 9, characterized in that, The fungus in question is Candida albicans.

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