Category: Formulation and Quality
Purpose: Liposomal amphotericin B (AmpB), AmBisome®, is an antifungal product used worldwide with annual sales of $420 million in 2018. AmBisome® has a rather complex physical structure that AmpB forms stable ionic complex with the lipid bilayer, which imparts high stability and low toxicity in systemic circulation . Both composition and manufacturing process define the physical structure of AmBisome®, while inability to reproduce precise AmBisome® structure will result in faster drug release and higher toxicity in vitro and in vivo . Herein we established several analytical methods for the characterization of liposomal AmpB, and carried out the quality comparison of AmBisome® and two generic products approved in India (Phosome® and Amphonex®). We also compared an in-house prepared AmpB liposomal formulation (35:48-7) to demonstrate the utility of established methodologies to discriminate poorly performing generic AmBisome® products.
Methods: Liposomal AmpB 35:48-7 was prepared by our collaborator using a high-pressure homogenization process . Quantification of AmpB was carried out using high performance liquid chromatography (HPLC). Size distribution of liposomes was measured using both dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) system. Drug release was measured using a USP-4 apparatus CE7 Smart (Sotax AG). The media was 10 mM HEPES containing 5% sucrose, 0.01% sodium azide and 5% γ-cyclodextrin (pH 7.4). Different formulations were placed in Float-A-Lyzer (300 kDa MWCO) and the media was perfused in a closed-loop with a flow rate of 16 mL/min and temperature set at 55 °C. In vitro toxicity was evaluated using a red blood cells (RBCs) hemolysis study and the hemolysis ratio was determined by measuring the potassium release from broken RBCs. Washed RBCs were incubated with different AmpB formulations at a series of determined concentration for 12 h at 37 °C, and the potassium concentration was measured using a potassium ion selective electrode (Thermo Scientific).
Results: Characterization of different formulations was shown in Table 1. The detected AmpB concentration of Phosome® and Amphonex® was 10%-15% lower than that of AmBisome®. All the formulations showed similar size distribution of around 100 nm. The particle concentration of Phosome® was slightly lower than AmBisome® and Amphonex®, while that of 35:48-7 was much lower than the other products. Phosome® and Amphonex® showed similar drug release profiles to AmBisome®, that achieved around 60% of drug release within 24 h (Fig. 1A). Faster drug release was observed for 35:48-7, with a F2 value of about 37 compared to AmBisome®. As for the in vitro toxicity, K50 value was defined as the concentration of AmpB that induced 50% of potassium release. Free AmpB solution exhibited high toxicity with a K50 of 0.40 ± 0.03 μg/mL, and the liposomal formulation greatly reduced the toxicity. No significant difference of hemolysis ratio was observed among AmBisome®, Phosome® and Amphonex® (Fig. 1B), and 35:48-7 showed a much higher toxicity with a significantly lower K50.
Conclusion: In this study, we established several analytical methodologies for characterization of liposomal AmpB. These methods could be used to compare similarity between generic products and AmBisome® as well as to guide manufacturing process optimization for generic products.
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