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. 2000 Jun;44(6):1598-603.
doi: 10.1128/AAC.44.6.1598-1603.2000.

Mild heating of amphotericin B-desoxycholate: effects on ultrastructure, in vitro activity and toxicity, and therapeutic efficacy in severe candidiasis in leukopenic mice

E W van Etten  1 , W van Vianen, P Roovers, P Frederik
Affiliations

Mild heating of amphotericin B-desoxycholate: effects on ultrastructure, in vitro activity and toxicity, and therapeutic efficacy in severe candidiasis in leukopenic mice

E W van Etten et al. Antimicrob Agents Chemother. 2000 Jun.

Abstract

Heated (20 min at 70 degrees C) amphotericin B-desoxycholate (hAMB-DOC) was further characterized, as was another formulation obtained after centrifugation (60 min, 3000 x g), hcAMB-DOC. Conventional AMB-DOC consisted of individual micelles (approximately 4 nm in diameter) and threadlike aggregated micelles, as revealed by cryo-transmission electron microscopy. For both hAMB-DOC and hcAMB-DOC, pleiomorphic cobweb structures were observed with a mean particle size of approximately 300 nm as determined by laser diffraction. The potent antifungal activity of AMB-DOC against Candida albicans is not reduced by heating. Effective killing of C. albicans (>99.9% within 6 h) was obtained at 0.1 mg/liter with each of the AMB formulations. For AMB-DOC, hAMB-DOC, and hcAMB-DOC, cation release ((86)Rb(+)) from C. albicans of > or =50% was observed at 0.8, 0.4, and 0.4 mg/liter, respectively. After heating of AMB-DOC, toxicity was reduced 16-fold as determined by red blood cell (RBC) lysis. For AMB-DOC, hAMB-DOC, and hcAMB-DOC, hemolysis of > or =50% was observed at 6.4, 102.4, and 102.4 mg/liter, respectively. In contrast, AMB-DOC and its derivates showed similar toxicities in terms of cation release from RBC. For AMB-DOC, hAMB-DOC, and hcAMB-DOC, cation release ((86)Rb(+)) of > or =50% was observed at 1.6, 0.8, and 0.8 mg/liter, respectively. In persistently leukopenic mice with severe invasive candidiasis, higher dosages of both hAMB-DOC and hcAMB-DOC were tolerated than those of conventional AMB-DOC (3 versus 0.8 mg/kg of body weight, respectively), resulting in significantly improved therapeutic efficacy. In conclusion, this new approach of heating AMB-DOC may be of great value for further optimizing the treatment of severe fungal infections.

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Figures

FIG. 1
FIG. 1
Cryo-transmission electron micrographs of small threadlike micelles of AMB-DOC (arrows) (A), pleiomorphic cobweb structures of hAMB-DOC (arrow) (B), and hcAMB-DOC, which has an appearance similar to that of hAMB-DOC (arrow) (C). Regions of higher density near hole edges indicate thicker ice. Bars = 50 nm (A and C) and 60 nm (B).
FIG. 1
FIG. 1
Cryo-transmission electron micrographs of small threadlike micelles of AMB-DOC (arrows) (A), pleiomorphic cobweb structures of hAMB-DOC (arrow) (B), and hcAMB-DOC, which has an appearance similar to that of hAMB-DOC (arrow) (C). Regions of higher density near hole edges indicate thicker ice. Bars = 50 nm (A and C) and 60 nm (B).
FIG. 2
FIG. 2
(A) Effects of AMB-DOC and its derivates on ion fluxes in yeast cells (C. albicans). The release of 86Rb+ caused by various concentrations of AMB-DOC (●), hAMB-DOC (▴), and hcAMB-DOC (▾) was measured as described in Materials and Methods. Data points are means of triplicate determinations. (B) Effects of AMB-DOC and its derivates on ion fluxes (closed symbols) and hemolysis (open symbols) of erythrocytes. The release of 86Rb+ and the hemolysis induced by various concentrations of AMB-DOC (● and ○), hAMB-DOC (▴ and ▵), and hcAMB-DOC (▾ and ▿) were determined as described in Materials and Methods. Data points are means of triplicate determinations.
FIG. 3
FIG. 3
Effect of early (A) or delayed (B) treatment with AMB-DOC or its derivates on survival rates of persistently leukopenic mice with severe invasive candidiasis (Kaplan-Meier plot). Leukopenic mice were inoculated i.v. at time zero with 3 × 104 CFU of C. albicans. (A) Groups of 10 animals each were treated i.v. 6 h after C. albicans inoculation with a single dose of AMB-DOC (●), hAMB-DOC (▴), or hcAMB-DOC (▾) at 0.8 mg of AMB/kg. (B) Mice were treated 16 h after C. albicans inoculation with a single dose of AMB-DOC (●) at 0.1 mg of AMB/kg or with hAMB-DOC (▴) or hcAMB-DOC (▾), each at 0.6 mg of AMB/kg. Controls were treated with placebo (5-DW) (■). #, P ≤ 0.05 versus placebo-treated mice; ∗, P ≤ 0.001 versus placebo-treated mice.
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