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Hemolytic potential of miltefosine is dependent on cell concentration: Implications for in vitro cell cytotoxicity
1Instituto de Física, Universidade Federal de Goiás, Goiânia, GO, Brazil.
Abstract:
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 5×10(9) cells/mL to ~2.9 μM for ~5×10(6) 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.
Insights
Miltefosine
Area of Science:
- Biophysics
- Pharmacology
- Cell Biology
Background:
- Miltefosine exhibits diverse therapeutic activities, including antiparasitic, antibacterial, antifungal, and antitumor effects.
- The precise mechanism of action for miltefosine remains incompletely understood.
- Previous studies highlight cell concentration-dependent effects of miltefosine, but the underlying biophysical basis is unclear.
Purpose of the Study:
- To elucidate the cell concentration-dependent hemolytic mechanism of miltefosine.
- To quantify key biophysical parameters governing miltefosine's interaction with cell membranes.
- To assess the hemolytic potential of miltefosine at clinically relevant concentrations.
Main Methods:
- Hemolysis assays were performed across a range of cell concentrations in phosphate-buffered saline (PBS).
- A mathematical model incorporating membrane-water partition coefficient (K), and intracellular (cm) and aqueous (cw) miltefosine concentrations was developed.
- Erythrocyte membrane-plasma partition coefficient (Log KM/P) was determined using whole blood hemolysis measurements.
Main Results:
- Miltefosine's hemolytic potential is significantly dependent on cell concentration, with lower concentrations required for 50% hemolysis at lower cell densities.
- Best-fit biophysical parameters were determined as log K=4.68, cm=110.8 mM, and cw=2.3 μM.
- Clinically relevant plasma concentrations of miltefosine (~90 μg/mL) resulted in minimal hemolysis (2.8%), with significant hemolysis (>5%) observed only at doubled concentrations.
Conclusions:
- Miltefosine-induced hemolysis is driven by drug accumulation in the cell membrane, leading to membrane damage at critical concentrations.
- The cell concentration-dependent activity of miltefosine, observed in hemolysis and Leishmania inhibition, is attributable to membrane partitioning.
- The biophysical parameters provide a quantitative framework for understanding miltefosine's membrane interactions and predicting its in vivo effects.
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