Effect of Aggregation on the Kinetics of Autoxidation of the Polyene Antibiotic Amphotericin B

Abstract

We have previously studied the autoxidation of the polyene antibiotic amphotericin B (AB). In this paper we describe the dependence of the kinetics of autoxidation on the aggregation state of the antibiotic. Autoxidation, which is involved in drug inactivation and has been suggested to play a role in the mechanism of drug action, was assessed through the reaction of formed radicals with the spin label Tempol (2,2,6,6-tetramethyl-4-hydroxy-A/-oxylpiperidine) by following the loss of the electron spin resonance signal, as previously described, and by oxygen consumption. Two types of AB (I and II) were used, the former being obtained by further purification of the latter. The kinetics of autoxidation were compared for aggregates formed by the antibiotic. Differences in aggregation state for both type I and type II AB were observed between monomeric, borax-complexed, and preparations in water containing variable proportions of dimethyl sulfoxide (DMSO) by optical absorption and circular dichroism (CD) spectra. On the other hand, although the suspensions of type I and type II AB in water-10% DMSO did not differ in their optical properties, they could be distinguished by quasielastic light scattering experiments, type II yielding smaller aggregates. It is proposed that the lack of difference in optical and CD spectra are due to the similarity of the microenvironments in both aggregates. In contrast, the borax complex of both type I and type II AB yielded similar optical and CD spectra and quasielastic light scattering behavior, indicating that complexation led to similar aggregates. Whereas monomeric type I and type II AB displayed similar autoxidation kinetics, the aggregates formed in water-10% DMSO reacted at different rates, with type II yielding slower kinetics. Transition metal ion chelating agents had little, but equivalent, or no effects on the kinetics of autoxidation. The borax complexes of type I and type II AB displayed similar kinetics. Oxygen consumption measurements were in agreement with the electron spin resonance results. Aggregation properties could be related to the mechanism of action and/or toxicity of the antibiotic.