Comparative EPR spectroscopy analysis of amphotericin B and miltefosine interactions with Leishmania, erythrocyte and macrophage membranes

Abstract

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to study the interactions of amphotericin B (AmB) with the plasma membrane of Leishmania (L.) amazonensis promastigotes, human erythrocytes and J774.A1 murine macrophages, in comparison with reported and novel data for miltefosine (MIL). One of the objectives of this work is to look for the relationships between the activities of these two drugs in the Leishmania parasite with their changes in the cell membrane. A spin-labeled stearic acid inserted into the cell membranes showed strong interactions with putative AmB/sterol complexes, characterized by reductions in molecular dynamics. The concentration of the drugs in the plasma membrane that reduced the cell population by 50%, and the membrane-water partition coefficient of the drugs, were assessed. These biophysical parameters enabled estimates of possible therapeutic concentrations of these two drugs in the interstitial fluids of the tissues to be made. AmB displayed higher affinity for the plasma membrane of L. amazonensis than for that of the macrophage and erythrocyte, denoting a preference for a membrane that contains ergosterol. AmB also demonstrated higher hemolytic potential than MIL for measurements on erythrocytes in both PBS and whole blood. For MIL, the EPR technique detected membrane changes induced by the drug in the same concentration range that inhibited the growth of parasites, but in the case of AmB, an 8-fold higher concentration of the IC50 was necessary to observe a reduction in membrane fluidity, suggesting a better localized effect of AmB on the membrane. Taken together, the results demonstrate that the antiproliferative and cytotoxic effects of both drugs are associated with changes in cell membranes.