International Journal of Pharmaceutics 447 (2013) 38–46 Contents lists available at SciVerse ScienceDirect International Journal of Pharmaceutics jo ur n al homep age: www.elsevier.com/locate/ijpharm Hemolytic and pharmacokinetic studies of liposomal and particulate amphotericin B formulations Dolores R. Serrano a , Leticia Hernández b , Laura Fleire a , Iban González-Alvarez c , Ana Montoya b , María P. Ballesteros a , María A. Dea-Ayuela c,d , Guadalupe Miró b , Francisco Bolás-Fernández d , Juan J. Torrado a,∗ a Farmacia y Tecnologia Farmaceutica, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramon y Cajal, 28040 Madrid, Spain b Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense, Madrid, 28040 Madrid, Spain c Departamento de Farmacia, Facultad de Ciencias de la Salud, Universidad Cardenal Herrera-CEU, Moncada, 46113 Valencia, Spain d Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense, Plaza Ramon y Cajal, 28040 Madrid, Spain a r t i c l e i n f o Article history: Received 2 January 2013 Received in revised form 13 February 2013 Accepted 14 February 2013 Available online 21 February 2013 Keywords: Amphotericin B Pharmacokinetics Hemolysis Aggregation state Albumin microparticles Passive targeting a b s t r a c t Amphotericin B (AmB) is a very effective antifungal and antiparasitic drug with a narrow therapeutic window. To improve its efficacy/toxicity balance, new controlled release formulations have been devel- oped based on different encapsulation systems, aggregation states and particle sizes modifications. The kinetics of the hemolytic process was studied not only to characterize the toxicity of different formula- tions but also as an indicator of drug release. Pharmacokinetic studies in beagle dogs were carried out with those formulations that exhibited the least hemolytic toxicity: liposomal formulation (AmBisome ® ), poly-aggregated AmB and encapsulated particulate AmB formulation. A novel poly-aggregated AmB for- mulation proved to be comparable in terms of low hemolytic activity with the marketed gold standard formulation: AmBisome ® . Its pharmacokinetic profile, characterized by a smaller area under the curve and larger volume of distribution, was markedly different from AmBisome ® , resulting in a cost-effective alternative for the treatment of leishmaniasis which can enhance the AmB passive target by the uptake by the cells of the reticulo-endothelial system. Effects of different variables such as type of formulation, dose, microencapsulation, anesthesia and dog’s healthy state on AmB pharmacokinetics were studied. © 2013 Elsevier B.V. All rights reserved. 1. Introduction AmB is a potent antifungal and antiparasitic drug acting by bind- ing preferentially and selectively to ergosterol present in fungal and bacterial membranes compared to cholesterol localized in mammal Abbreviations: AmB, Amphotericin B; RBCs, red blood cells; RES, reticulo- endothelial system; FD, non-microencapsulated free dimeric AmB formulation; F, Fungizone ® ; HF, heated-Fungizone; MD, microencapsulated dimer AmB; FP, non- microencapsulated free poly-aggregated AmB formulation; MP, microencapsulated poly-aggregated AmB; DLS, dynamic light scattering; TEM, transmission electron microscopy; SEM, scanning electron microscopy; PBS, phosphate buffer solution; Abs, absorbance; K d , degradation constant; R, correlation coefficient; NNN medium, Novy-MacNeal-Nicolle medium; AUC, area under the plasma concentration versus time curve; AUMC, area under the first moment curve; Cmax, concentration at time zero; , terminal phase elimination rate constant; t 1/2 , terminal elimination half- life; Cl, body clearance; Vss , volume of distribution at steady state; Varea, volume of distribution or volume area; MRT, mean residence time; ts , mean residence time in systemic circulation; tp, mean residence time in peripheral tissues. ∗ Corresponding author at: Departamento de Farmacia y Tecnología Farmacéutica, Facultad de Farmacia, Universidad Complutense, Plaza Ramón y Cajal S/N, 28040, Madrid, Spain. Tel.: +34 913941620; fax: +34 913941736. E-mail address: torrado1@farm.ucm.es (J.J. Torrado). membranes and by disrupting them. The molecular interaction of AmB with the natural components of the membranes and the role of different proteins and lipids in this action is complex and has impor- tant consequences in its activity and toxicity (Brajtburg and Bolard, 1996; Cheron et al., 2003; Hartsel et al., 2001; Torrado et al., 2008). After AmB formulations intravenous administration, interaction of AmB with RBCs causes their lysis leading to anaemia. Thus, safety of novel AmB delivery strategies such as micelles, microspheres, microemulsions, liposomes, nanoparticles and others can be stud- ied by comparing their hemolysis potential. However, hemolysis assays used by various research groups vary significantly (see Table 1 in supplementary data). Additionally using human to animal ori- gin blood leads to differences, as human red blood cells are more resistant compared to rat making it difficult to compare the safety of novel formulations (Fukui et al., 2003; Knopik-Skrocka et al., 2003). Additionally, studying the hemolysis over various time points is more meaningful when controlled release formulations are under investigation as release from various formulations differs over time and this is not accounted if only one time point is studied (Adams et al., 2003; Adams and Kwon, 2003). These studies are vital to predict the safety of the in vitro formulations that can progress to preclinical studies. Furthermore, due to the low solubility of AmB 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.02.038