Reconstituted high density lipoprotein enriched with the polyene antibiotic amphotericin B

Abstract

The polyene antibiotic amphotericin B (AMB) is an effective antifungal agent whose therapeutic potential is limited by poor aqueous solubility and toxicity toward host tissues. Addition of apolipoprotein A-I to a multilamellar phospholipid vesicle dispersion containing 20% (w/w) AMB induces the formation of reconstituted high density lipoprotein (rHDL), with solubilization of the antibiotic. Density gradient ultracentrifugation resulted in flotation of the complexes to a density of 1.16 g/ml, and negative stain electron microscopy revealed a population of disk-shaped particles. Native gradient polyacrylamide gel electrophoresis indicated a particle diameter of z8.5 nm. Absorbance spectroscopy provided evidence for AMB integration into the lipid milieu. AMB-rHDLs were potent inhibitors of Saccharomyces cerevisiae growth, yielding 90% growth inhibition at ,1 mg/ml yeast culture. In studies with pathogenic fungal species, similar growth inhibition characteristics were observed. Compared with AMB-deoxycholate micelles, AMB-rHDL displayed greatly attenuated red blood cell hemolytic activity and decreased toxicity toward cultured hepatoma cells. In in vivo studies in immunocompetent mice, AMB-rHDLs were nontoxic at 10 mg/kg, and they showed efficacy in a mouse model of candidiasis at concentrations as low as 0.25 mg/kg. These results indicate that AMB-rHDLs constitute a novel formulation that effectively solubilizes the antibiotic and elicits strong in vitro and in vivo antifungal activity with no observed toxicity at therapeutic

Peptide stabilized amphotericin B nanodisks

“…4F AMB-ND were similar in morphology to apoA-I AMB-ND reported earlier [9], displaying a population of particles with diameter of ~12 nm (range 6-24 nm) (Figure 4). …”

“…ND particles were prepared essentially as described by Oda et al [9]. Briefly, 10 mg DMPC was dissolved in chloroform: methanol (3:1 v/v) and dried under a stream of nitrogen gas, coating the vessel wall with the phospholipid.…”

“…Recently we described a novel lipid formulation of the water insoluble antibiotic amphotericin B (AMB), termed nanodisks (ND), comprised of a nanometer scale disk shaped phospholipid bilayer stabilized by recombinant human apolipoprotein A-I (apoA-I) [9]. ND are formed by a reaction between amphipathic apolipoproteins and phospholipid vesicles to which AMB has been added.…”

“…AMB was stably incorporated into ND particles at a concentration of 2.5 mg AMB per 10 mg phospholipid. Formulation of AMB into apolipoprotein stabilized ND preserves the potent biological activity of AMB against yeast and pathogenic fungi yet attenuates toxicity of this antibiotic toward red blood cells and cultured hepatoma cells [9]. AMB-ND display in vivo efficacy in mice infected with the pathogenic fungi, Candida albicans [9] as well as the protozoal parasite, Leishmania major [8].…”

“…In the process of characterizing AMB-ND and evaluating their therapeutic potential in treatment of systemic fungal infections [9] or leishmaniasis [8], it was recognized that the apolipoprotein component of the ND particle represents a limiting factor. Whereas apolipoproteins serve key functions in facilitating ND formation and as a structural component of the product particles, we hypothesized that peptides represent a potential alternative to recombinant apolipoprotein.…”