Illustrator 2015

In vitro culture of E. multilocularis (isolate H95) metacestodes in co-culture with Reuber rat hepatoma cells was performed as described previously (Stadelmann et al., 2010 (link)). All compounds were prepared as 20 mM stocks in DMSO. The metacestode vesicle damage was assessed by phosphoglucose isomerase (PGI) assay as described previously (Stadelmann et al., 2010 (link)). In short, in vitro cultured metacestode vesicles of approximately 4 mm in size were extensively washed in PBS and taken up in an equal volume of DMEM without phenol red (Bioswisstec, Schaffhausen, Switzerland), including penicillin/streptomycin (100 U/mL, Thermo Fisher Scientific, Zug, Switzerland). Parasites were distributed into a 48 well-plate at 1 mL per well and mefloquine or derivatives were added to a final concentration of 10, 20, 30, and 40 μM. 0.1% Tx-100 served as a positive control, DMSO only as a solvent control. Samples were prepared in triplicates. PGI-assays were carried out after 5 and 12 days (Stadelmann et al., 2010 (link)). Active mefloquine derivatives were further tested at concentrations ranging from 40 to 1.25 μM in a 1:2 serial dilution and parasite damage by PGI-assay was assessed as described previously (Stadelmann et al., 2010 (link)). Calculations were performed in Microsoft Excel (2010), and final figures were prepared in Adobe Illustrator 2015.1.0.

Mefloquine concentrations were modeled using a standard two-compartment pharmacokinetic model with first-order absorption. Mean mefloquine concentrations and a mean dose of 2.04 mg were used for calculations. Primary parameters were the absorption rate constant k_a, the apparent clearance after extravascular administration CL/F, the intercompartment clearance CL_d/F, and the apparent volumes of the central and peripheral compartment V₁/F and V₂/F. A secondary parameter was the terminal elimination half-life T_½. Expected steady-state minimum (C_min) and maximum (C_max) concentrations were derived by simulating continued mefloquine dosing. Pharmacokinetic calculations were done using Phoenix WinNonlin 7.0 (Certara, Princeton, NJ, USA) and Figures prepared in Microsoft Excel (2010) and in Adobe Illustrator 2015.1.0.

HPLC was performed as described previously with adaptations (Ingram et al., 2013 (link)). Mefloquine concentrations were analyzed on an Ultimate 3000 System (Dionex, Reinach, Switzerland) with an EC 250/4 Nucleodur 100-5 C18ec (Macherey Nagel) and UV detection at 284 nm. The mobile phase consisted of 35% methanol, 25% acetonitrile, and 40% potassium dihydrogen phosphate (pH 3.9). Column temperature was 25 °C, flow rate constant at 1 mL/min and each run was 10 min. The recorded peaks were annotated according to the retention times of known standards. Stability of samples was assessed with the help of the internal quinine standard. Mefloquine plasma concentrations were quantified based on internal calibration of the peak area to the internal standard quinine and calculated with a linear calibration curve in the software Chromeleon Ultimate 3000 (Dionex, CA, USA) and Microsoft Excel 2010. Further calculations and figures were prepared in SigmaPlot Version 14, and in Adobe Illustrator 2015.1.0.