Ammediol

Polyvinylpyrrolidone (PVP K30) (Duchefa Farma B.V., Haarlem, The Netherlands), hydroxypropyl cellulose SLFP and LFP (HPC-SLFP and HPC-LFP) were obtained from Nisso HPC (Nisso Chemical Europe GmbH, Düsseldorf, Germany). Bovine intestinal alkaline phosphatase (BIAP), PEG 1500, ammediol, and HEPES were obtained from Sigma-Aldrich (St. Louis, MO, USA). Kollidon VA64 was purchased from BASF Pharma, Ludwigshafen, Germany. Eudragit L100 was obtained from Pharma Excipients, Steinhausen, Switzerland and D-mannitol was purchased from VWR Chemicals BDH, Radnor, PA, USA. The molecular weight (Da) and viscosity (mPa·s) of Kollidon VA64, Eudragit S100, Eudragit L100, HPC-SLFP and HPC-LFP are 45,000–70,000/below 10, 135,000/50–200, 135,000/50–200, 100,000/3.0–5.9 and 140,000/6.0–10.0, respectively. Inulin 4 kDa was a generous gift from Sensus (Roosendaal, The Netherlands). Eudragit S100 was a gift from Evonik Operation GmbH (Essen, Germany). Macrogolum 6000 (PEG6000) and talc were obtained from BUFA (IJsselstein, The Netherlands). Croscarmellose sodium (AcDiSol) was obtained from FMC BioPolymer (Philadelphia, PA, USA).

The cysticidal activity of DT was determined by the alamarBlue™ assay and confirmed visually by inverted microscopy. Cysts of A. castellanii Neff were prepared as previously described [25 (link)]. Briefly, trophozoites were transferred from PYG medium based cultures (trophozoite medium) to Neff’s encystment medium (NEM; 0.1 M KCl, 8 mM MgSO₄·7H₂O, 0.4 mM CaCl₂·2H₂O, 1 mM NaHCO₃, 20 mM ammediol (2-amino-2-methyl-1,3-propanediol; Sigma Aldrich, Madrid, Spain), pH 8.8 at 25 °C) and were cultured in this medium with gentle shaking for a week in order to obtain mature cysts. After that, mature cysts were harvested and washed twice using PYG medium.
A serial dilution of the tested molecules was made in PYG. The in vitro susceptibility assay was performed in sterile 96-well microtiter plates (Corning™). To these wells, containing 50 µL of drug dilutions, 5 × 10⁴ mature cysts of Acanthamoeba/mL were added. After 7 d of incubation with the drugs, the plate was centrifuged at 3000 rpm for 10 min. The supernatant was removed and replaced with 100 µL of fresh PYG medium in each well. Finally, 10 μL of the Alamar Blue Assay Reagent (Biosource, Europe, Nivelles, Belgium) was placed into each well, and the plates were then incubated for 144 h at 28 °C. The emitted fluorescence was periodically examined with an Enspire microplate reader (PerkinElmer, Waltham, MA, USA) at 570/585 nm.