Hemolytic potential of miltefosine is dependent on cell concentration: Implications for in vitro cell cytotoxicity assays and pharmacokinetic data

• The hemolytic potential of miltefosine is hematocrit dependent.
• IC50values in cytotoxicity assays are dependent on cell concentration.
• In erythrocyte membranes, 50% hemolysis was observed for 111 mM miltefosine.

Miltefosine possesses antiparasitic, antibacterial, antifungal and antitumor activities; however, its mechanism of action is not well established. In the current work, the miltefosine concentrations required to achieve 50% hemolysis in PBS were shown to vary from 600 μM using 5 × 109 cells/mL to ~ 2.9 μM for ~ 5 × 106 cells/mL. This cell concentration-dependent hemolytic potential was described using an equation that included the membrane–water partition coefficient (K) and miltefosine concentrations in the cell membrane (cm) and aqueous medium (cw) as variables. The best-fit values for the 50% hemolysis data were log K = 4.68, cm = 110.8 mM, and cw = 2.3 μM. Hemolysis measurements in whole blood were used to determine the erythrocyte membrane–plasma partition coefficient of miltefosine (Log KM/P = 1.77). Additionally, miltefosine concentration in whole blood was found to be ~ 86% of that in plasma. Previously reported clinical pharmacokinetics data indicate that the plasma concentration of miltefosine peaks at ~ 90 μg/mL when treating visceral leishmaniasis. Using this concentration, which corresponds to ~ 77 μg/mL miltefosine in whole blood, we found only 2.8% hemolysis. Significant hemolysis (5.4%) was observed only after doubling the concentration to 180 μg/mL. Recently reported data indicate that miltefosine inhibitory concentrations in Leishmania are also dependent on cell concentration. The biophysical parameters assessed in the current study indicated that this type of response is associated with the accumulation of the drug in the cell membrane, which becomes damaged when critical drug concentrations are reached.

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