Effects of aggregation and solvent on the toxicity of amphotericin B to human erythrocytes

Abstract

In aqueous suspensions of amphotericin B (AmB), a polyene antibiotic and antifungal agent, three forms of AmB coexist: monomers, water-soluble oligomers, and non-water-soluble aggregates. The toxicity of the water-soluble self-associated form of AmB compared with that of the non-water-soluble self-associated form was tested by measuring induction of K+ leakage from human erythrocytes, using different suspensions containing the antibiotic and phosphate-buffered saline. These suspensions were obtained from various stock solutions of the antibiotic in dimethyl formamide or dimethyl sulfoxide. Their circular dichroism spectra around 340 nm, indicative of the degree of AmB self-association, were strongly dependent on the concentration of organic solvent in the suspensions. The nonsoluble self-associated form was separated from the water-soluble form by centfugation. The nonsoluble form was favored by a high concentration ofAmB of the stock solution.The kinetics of AmB-induced K+ leakage from human erythrocytes also appeared to be strongly dependent on the AmB concentration of the stock solution being much weaker with concentrated stock solutions. It was concluded that the only form of AmB toxic to human erythrocytes is the water-soluble self-associated form (in contrast with fungal cells on which the monomeric form is also active). This result may be important in the design of new less toxic AmB derivatives and in the understanding of the mechanism of action of liposomal AmB.Amphotericin B (AmB) remains the drug of choice for treating most systemic fungal infections. The recent introduction of liposomal AmB formulations with lower toxicity to patients (see, for instance, reference 5), has increased the clinical interest in this drug. However, its mechanism of action is still imperfectly understood. Inducing permeability to K+ is thought to be the first event following the addition of AmB to cells and is therefore an indicator of its activity. In order to understand the mechanism of its selectivity between fungal (ergosterol-containing membranes) and mammalian (cholesterol-containing membranes) cells, several studies have been performed from the functional point of view with mammalian erythrocytes (see references 2 [and references therein], 6, 26, and 32) as well as from the molecular point of view (3,29,30,33). In a previous study (3), we were able to show that AmB induces permeability to K+ in cholesterol-containing membranes, particularly in erythrocytes only when AmB is in the self-associated form in the incubation medium. The threshold of concentration beyond which AmB self-association starts therefore appeared to be a determining factor in the activity of the drug. This point is very important for the design of new less toxic derivatives of AmB. It can also be a clue for the mechanism by which the selectivity of the drug is increased when it is incorporated into liposomes.However, a more detailed analysis of the toxicity of the self-associated form was necessary, because it is well-known (23) that two self-assoc...

Structural insights on biologically relevant cationic membranes by ESR spectroscopy

“…The use of AmB is limited by several side effects, including nephrotoxicity and hemolytic activity; therefore much effort has been made to develop formulations able to reduce its toxicity and increase its therapeutic effect (Brajtburg and Bolard 1996;Torrado et al 2008). Both the therapeutic and toxic effects of AmB seem to be related to its state of aggregation, and the monomeric state of AmB is usually ascribed as the less toxic state (LamyFreund et al 1989;Bolard et al 1991;Barwicz and Tancrede 1997;Barwicz et al 1992;Legrand et al 1992). In this context, sonicated DODAB bicelles have been found to be able to solubilize mainly monomeric AmB molecules (Vieira and Carmona-Ribeiro 2001) and display low nephrotoxicity (Lincopan et al 2005).…”

Eficiência terapêutica das formulações lipídicas de anfotericina B

“…O DOC é empregado para solubilização da AB, entretanto o sistema não é homogê-neo, podendo apresentar em sua constituição três formas diferentes: monomérica, oligomérica e agregados de AB-DOC (Legrand et al, 1992) misturados com micelas puras de DOC (Lamy-Freund et al, 1991). Existe equilíbrio, portanto, entre essas formas e suas proporções dependem da diluição empregada (Legrand et al, 1992).…”

Novel targeting using nanoparticles: an approach to the development of an effective anti-leishmanial drug-delivery system

“…Only this product has been reported to cause a change in the UV-Vis spectrum, with a very broad peak appearing around 329 nm and decreased intensities at 405, 364, and 382 nm, when the monomeric state is converted to the aggregate state. 58 A close association is believed to exist between AmpB's state of aggregation and its toxicity. 58 The mechanism of action proposed for the toxic effect of this drug is derived from its interaction with sterols in bilayer membranes, such as cell walls, causing either the formation of pores in the membrane, leading to cellular destruction or to the inhibition of membrane repair.…”