Reformulation of Fungizone by PEG-DSPE Micelles: Deaggregation and Detoxification of Amphotericin B

Abstract

Purpose Fungizone® (AmB-SD), amphotericin B solubilized by sodium deoxycholate, contains a highly aggregated form of the antifungal agent that causes dose-limiting renal toxicity. With the aim of reducing the formulation’s toxicity by co-delivering monomeric amphotericin B (AmB) and sodium supplementation, we deaggregated AmB-SD with FDA-approved excipient PEG-DSPE in 0.9% NaCl-USP. Herein, we describe a reformulated AmB-SD with PEG-DSPE micelles that results in a less toxic drug with maintained antifungal activity. Methods We compared the aggregation state and particle size of AmB-SD alone or combined with PEG-DSPE micelles. In vitro hemolytic activity and in vivo renal toxicity were measured to determine the toxicity of different formulations. In vitro antifungal assays were performed to determine differences in efficacy among formulations. Results PEG-DSPE micelles in saline deaggregated AmB-SD. Deaggregated AmB-SD exhibited significantly reduced in vitro and in vivo toxicity. In vitro antifungal studies showed no difference in minimum inhibitory and fungicidal concentrations of AmB-SD combined with PEG-DSPE relative to the drug alone. Conclusions Reformulation of AmB-SD with PEG-DSPE micelles in saline facilitates co-delivery of monomeric AmB and sodium supplementation, potentially reducing the dose-limiting nephrotoxicity of AmB-SD. Ease of preparation and commercially available components lead us to acknowledge its potential for clinical use.

Antifungal Efficacy of an Intravenous Formulation Containing Monomeric Amphotericin B, 5-Fluorocytosine, and Saline for Sodium Supplementation

“…A similar degree of aggregation was observed at 24 and 48 hours. These data are consistent with results from prior evaluations of mAmB-90 alone (28). …”

“…At each time point, the average monomer to aggregate ratio of AmB-SD combined with 5-FC was less than 0.3, indicating a highly aggregated state (data not shown). Previously, we determined that the IV/I ratio of AmB-SD alone was less than 0.20 (28), so here we report a slightly less aggregated state in the presence of 9 mg/ml 5-FC. PEG-DSPE micelles deaggregated AmB-SD in normal saline containing 5-FC (Table I).…”

“…Hemolytic activity did not exceed 10% at any tested concentration when 5-FC (50 and 100 μg/ml) was combined with 1, 6, and 10 μg/ml AmB as mAmB-90. The hemolytic activity of mAmB-90 with 5-FC is comparable to that of mAmB-90 alone in the same concentration range (28). At 6 and 10 ug/ml, AmB-SD with 100 μg/ml 5-FC exhibited at least 10 times the hemolytic activity measured from either combination of mAmB-90 and 5-FC (p <0.0001).…”

“…From DLS measurements, we show that AmB-SD precipitates in the presence of 5-FC in saline (Figure 3), resulting in micron-sized particles that could cause an embolism. However, deaggregation of AmB and a particle size of less than 30 nm were maintained when our reformulation of AmB-SD was combined with 5-FC (28). …”

“…Previously, we combined these strategies and described a simple method of preparing AmB-SD with micelles of the FDA-approved PEG-lipid poly(ethylene glycol)-distearoylphosphatidylethanolamine (PEG-DSPE) in saline. AmB-SD in a 1:90 mole ratio with PEG-DSPE micelles in saline (mAmB-90) allowed for co-delivery of monomeric AmB and sodium supplementation, a combination that caused significantly less renal toxicity than AmB-SD alone in rats dosed with 2 mg/kg AmB for three days (28). …”