Concentration of Antifungal Agents within Host Cell Membranes: a New Paradigm Governing the Efficacy of Prophylaxis

Abstract

Posaconazole prophylaxis has proven highly effective in preventing invasive fungal infections, despite relatively low serum concentrations. However, high tissue levels of this agent have been reported in treated patients. We therefore hypothesized that the intracellular levels of antifungal agents are an important factor in determining the success of fungal prophylaxis. To examine the effect of host cell-associated antifungals on the growth of medically important molds, we exposed cells to antifungal agents and removed the extracellular drug prior to infection. Epithelial cells loaded with posaconazole and its parent molecule itraconazole, but not other antifungals, were able to inhibit fungal growth for at least 48 h and were protected from damage caused by infection. Cell-associated posaconazole levels were 40-to 50-fold higher than extracellular levels, and the drug was predominantly detected in cellular membranes. Fungistatic levels of posaconazole persisted within epithelial cells for up to 48 h. Therefore, the concentration of posaconazole in mammalian host cell membranes mediates its efficacy in prophylactic regimens and likely explains the observed discrepancy between serum antifungal levels and efficacy.

Understanding antifungal prophylaxis with posaconazole in hematology patients: an evolving bedside to bench story

“…Notably, the posaconazole-treated cells were found to resist infection by A. fumigatus 14,18 The protective effect of cell-associated posaconazole persisted for at least 48 h after removal of free drug. 18 Measurement of posaconazole kinetics in epithelial cells demonstrated that this prolonged post-antifungal effect was due to the persistence of high levels of drug within the epithelial cells, 18 thus providing an explanation for the prolonged post-antifungal effect reported in animal studies of posaconazole. 12 In addition, cellular fractionation experiments and localization studies using fluorophore-conjugated posaconazole have demonstrated that this hydrophobic antifungal accumulates only within membrane compartments of host cells, rather than throughout the whole cell.…”

“…12 In addition, cellular fractionation experiments and localization studies using fluorophore-conjugated posaconazole have demonstrated that this hydrophobic antifungal accumulates only within membrane compartments of host cells, rather than throughout the whole cell. 18,19 Because membranes represent only a small fraction (<10%) of the volume of eukaryotic cells, the actual membrane concentration of posaconazole is at least 10-fold higher than prior estimates of total cellular concentration. Thus the membrane concentrations of posaconazole can reach levels as high as 400-fold greater than those found in the serum.…”

“…Indeed, the addition of serum to posaconazole-loaded cells does not impair their ability to inhibit the growth of A. fumigatus in vitro. 18 In addition, the highly hydrophobic nature of posaconazole would predict that, within the vascular compartment, this agent would easily flux between plasma proteins and the more hydrophobic cell membranes of fungi. This hypothesis is also supported by in vitro experimental data in which the observed antifungal effect of posaconazole in the presence of human serum is much greater than would have been predicted based on the free drug concentrations.…”

Latest Trends in Fungal Epidemiology Inform Treatment Choices and Stewardship Initiatives

“…After previous research had suggested that intracellular levels of posaconazole may be more relevant to therapeutic efficacy than serum levels [24], Campoli et al observed that posaconazole was predominantly found in host cell membranes in concentrations sufficient to inhibit growth and prevent fungal damage [25]. Subsequent research, presented at ECCMID, showed that during infection, posaconazole transferred from host cell membranes to fungal membranes and was concentrated within the endoplasmic reticulum (ER) [26].…”

Antifungal stewardship considerations for adults and pediatrics

“…95 It has been found that posaconazole penetrates alveolar cells and monocytes in concentrations that far exceed the blood, and this finding may be the explanation for why posaconazole is effective in preventing IFI despite low serum levels. 102 With regard to therapeutic efficacy, a target concentration of at least 1 mg/L has been proposed and is based on the study by Walsh et al that showed improved clinical response rates with higher posaconazole levels in patients with established IFI on salvage therapy. 103 It is unknown how feasible it would be to attain and/or maintain such a level in practice.…”