Amphotericin B release rate is the link between drug status in the liposomal bilayer and toxicity

Yuri Svirkin; Jaeweon Lee; Richard Marx; Seongkyu Yoon; Nelson Landrau; Md Abul Kaisar; Bin Qin; Jin H Park; Khondoker Alam; Darby Kozak; Yan Wang; Xiaoming Xu; Jiwen Zheng; Benjamin Rivnay

doi:10.1016/j.ajps.2022.04.007

Amphotericin B release rate is the link between drug status in the liposomal bilayer and toxicity

Asian J Pharm Sci. 2022 Jul;17(4):544-556. doi: 10.1016/j.ajps.2022.04.007. Epub 2022 Jun 8.

Authors

Affiliations

¹ Landrau Scientific Innovations, Leominster, MA, United States of America.
² University of Massachusetts Lowell, Lowell, MA, United States of America.
³ Office of Research and Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United States of America.
⁴ Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United States of America.
⁵ Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, U.S. Food and Drug Administration, Silver Spring, MD, United States of America.

Abstract

Amphotericin B (AmB) is an amphiphilic drug commonly formulated in liposomes and administered intravenously to treat systemic fungal infections. Recent studies on the liposomal drug product have shed light on the AmB aggregation status in the bilayer, which heat treatment (curing) modifies. Although toxicity was found related to aggregation status - loose aggregates significantly more toxic than tight aggregates - the precise mechanism linking aggregation and toxicity was not well understood. This study directly measured drug release rate from various AmB liposomal preparations made with modified curing protocols to evaluate correlations among drug aggregation state, drug release, and in vitro toxicity. UV-Vis spectroscopy of these products detected unique curing-induced changes in the UV spectral features: a ∼25 nm blue-shift of the main absorption peak (λ_max) in aqueous buffer and a decrease in the OD₃₄₆/OD₃₂₂ ratio upon thermal curing, reflecting tighter aggregation. In vitro release testing (IVRT) data showed, by applying and fitting first-order release kinetic models for one or two pools, that curing impacts two significant changes: a 3-5-fold drop in the overall drug release rate and a ten-fold decrease in the ratio between the loosely aggregated and the tightly aggregated, more thermodynamically stable drug pool. The kinetic data thus corroborated the trend independently deduced from the UV-Vis spectral data. The in vitro toxicity assay indicated a decreased toxicity with curing, as shown by the significantly increased concentration, causing half-maximal potassium release (TC₅₀). The data suggest that the release of AmB requires dissociation of the tight complexes within the bilayer and that the reduced toxicity relates to this slower rate of dissociation. This study demonstrates the relationship between AmB aggregation status within the lipid bilayer and drug release (directly measured rate constants), providing a mechanistic link between aggregation status and in vitro toxicity in the liposomal formulations.

Keywords: Aggregation Status; Amphotericin B; Drug Release; In Vitro Toxicity; Liposomes; UV–Vis Spectrum.