Factors Affecting Enhanced Permeation of Amphotericin B Across Cell Membranes and Safety of Formulation

• Published in: AAPS PharmSciTech 2016-08, Vol.17 (4), p.820-828
• Main Authors: Adhikari, Kajiram, Buatong, Wilaiporn, Thawithong, Ekawat, Suwandecha, Tan, Srichana, Teerapol
• Format: Article
• Language: English
• Subjects: Amphotericin B - chemistry; Amphotericin B - metabolism; Biochemistry; Biomedical and Life Sciences; Biomedicine; Biotechnology; Cell Line; Cell Membrane - metabolism; Chemistry, Pharmaceutical - methods; Cholesterol - chemistry; Cholesterol - metabolism; Deoxycholic Acid - chemistry; Deoxycholic Acid - metabolism; Drug Combinations; Ergosterol - chemistry; Ergosterol - metabolism; HEK293 Cells; Humans; Lipid Bilayers - metabolism; Lipids - chemistry; Permeability; Pharmacology/Toxicology; Pharmacy; Phospholipids - chemistry; Phospholipids - metabolism; Research Article
• ISSN: 1530-9932; 1530-9932
• DOI: 10.1208/s12249-015-0406-x

The aim of this study was to determine amphotericin B (AmB) permeation across lipid bilayer membranes mounted on Transwell® and to observe the phagocytosis of the AmB and the AmB-lipid formulations by alveolar macrophage (AM) cell lines using a fluorescence microscope. The lipid bilayer membranes were prepared from phospholipid and ergosterol as well as phospholipid and cholesterol in a ratio (67:33 mol%). AmB-lipid formulations were prepared from AmB incorporated with four lipid derivatives during a lyophilization process. In vitro cytotoxicity studies were carried out on kidney cells by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The levels of nitric oxide production by AMs exposed to these AmB-lipid formulations were determined by the Griess reaction. Phagocytosis of the AmB-lipid formulations was carried out using AM cells. The lipid bilayer membranes and AmB-lipid formulations were successfully prepared. In vitro cytotoxicity results showed less toxicity to kidney cells than pure AmB, and a 1,000-fold less production of nitric oxide by NR8383 cell lines was obtained when compared to lipopolysaccharide. Permeation results were two- to fivefold higher than for pure AmB in the ergosterol containing lipid bilayer and two- to fourfold higher than AmB in the cholesterol containing compositions, both of which were enough to kill the fungi according to their MICs and MFCs. AM phagocytosed the AmB-lipid formulations. We suggest that these products especially the AmB-sodium deoxycholate sulfate are potential candidates for targeting AM cells for the treatment of invasive pulmonary aspergillosis.