The structures of the DIP-compound are shown in supplementary figure S1 and have originally been published by Wenzler et. al..
DIP-02, MW 447.37 (MolPort, Riga, Latvia)
DIP-03 = CASIN, MW 342.87 (MolPort, Riga, Latvia)
DIP-07 = Desloratadine, a tricyclic H1-antihistamine, MW 310.83 (MolPort, Riga, Latvia)
DIP-19, MW 439.42 (MolPort, Riga, Latvia)
Carvedilol, diazepam and amodiaquine as internal standards (IS) were purchased from Sigma-Aldrich (Buchs, Switzerland). See the Supplementary Information for details.
Female NMRI mice (Charles River, France) that weighed between 20 and 25 g at the start of the experiment were used for the efficacy and snapshot PK studies.
Efficacy in a T. brucei mouse model.
Mice were infected intraperitoneally (i.p.) with 104 T. b. rhodesiense STIB900 1 day prior to compound treatment. A control group was infected, but remained untreated. Experimental groups of 2 mice were treated with the test compounds and tail blood was sampled of each individual mouse for microscopic qualitative assessment of parasitaemia as depicted in figure 1A. Mice were euthanised if no clear reduction in parasitaemia was detected compared to the untreated controls. The reference drug pentamidine cures this model at 3×4 mg kg-1 or 4×1 mg kg-1 i.p.
Snapshot PK experiment
2 mice were used per compound; the samples were pooled for each timepoint. Baseline blood samples (50 µl) were taken from the tail of an uninfected mouse one the day prior to compound administration. Compounds were injected at doses of 150 or 50 mg kg-1 i.p., and blood samples were taken over a period of 8 h. DIP-03 and DIP-07 were sampled at 10 min, 30 min, 1 h, 3 h and 8 h after the compounds were injected; DIP-19 at 10 min, 30 min, 2 h, 4 h and 8 h (Fig. 1B). Blood compound concentrations were measured by UPLC MS/MS, see supplementary information.
Plasma Protein Binding
Mouse plasma protein binding was assessed at Bienta, Ukraine. The assay was performed in ready-to-use 96-well dialysis units (HTD96b dialyzer). Each individual well unit consisted of 2 chambers separated by a vertically aligned dialysis membrane strip with molecular weight cut-offs (MWCO) from 12 to 14 kDa. The compounds were tested at 1 μM in mouse plasma (MPH07-0500). Verapamil was used as reference compound (data not shown).