Determination of the Relative Toxicity of Amphotericin B Formulations: A Red Blood Cell Potassium Release Assay

Abstract

Amphotericin B remains the drug of choice for the treatment of life-threatening, systemic fungal infections. Nevertheless, the use of amphotericin B in the traditional deoxycholate formulation (d-AmB) is limited by severe toxic side effects. Several relatively new formulations have recently received regulatory approval: amphotericin B colloidal dispersion (ABCD), amphotericin B lipid complex (ABLC), and liposomal amphotericin B (AmBisome). We quantitate the level of reduction of intrinsic toxicity of these formulations over d-AmB using a red blood cell potassium release assay, employing a variety of blood sources and incubation times. We also examine the practice of diluting d-AmB in the parenteral nutrition product Intralipid. Overall, the propensity in each formulation of amphotericin B to partition into the red cell membrane during incubation is measured by determining the concentration of amphotericin B required to achieve 50% potassium release. The concentrations leading to 50% potassium release were in the order AmBisome ABLC ABCD >> d-AmB d-AmB/Intralipid and reflect inversely the degree to which each formulation makes amphotericin B available to mammalian, cholesterol-containing membranes. To evaluate intrinsic efficacy in an analogous way we investigated the availability of amphotericin B to partition into ergosterol-containing membranes by carrying out potassium release experiments using Candida albicans fungal cells instead of red blood cells. In contrast to the results obtained with red cells, the concentrations required to achieve potassium release from fungal cells were essentially identical for all formulations. It can be concluded that the intrinsic, physicochemical component of therapeutic index improvement in these formulations follows the order AmBisome ABLC ABCD >> d-AmB d-AmB/Intralipid.