Healthy subjects received a single oral dose (1-120 mg), and healthy subjects and type 2 diabetic patients received a loading dose and thereafter once-daily doses (0.5-16 mg) of ragaglitazar for 6 and 20 days

Normal human urothelial (NHU) cells were grown as non-immortal lines in vitro and exposed to ragaglitazar. Growth of NHU cell cultures in the presence of PPAR agonists was assessed using the thiazolyl blue tetrazolium (MTT; Sigma Aldrich) dye reduction assay. Briefly, NHU cells were seeded overnight in 96-well flat-bottom Primaria® tissue culture plates at a density of 210^4 cells/ml. PPAR agonist (ragaglitazar) were added 24 h after seeding and tested over a range of concentrations. Growth medium and drugs were re-applied every 3 days and cell proliferation was assessed over a period of 8 days, unless otherwise stated. At selected time points, MTT (500 g/ml; 200 mkl volume) was added to each well and incubated for 4 h at 37 C. Following this period, MTT was aspirated from each well and MTT-formazan crystals were solubilized by addition of DMSO (spectrophotometric grade; 200 mkl). Absorbance at 570 nm of each sample well was measured using an automated plate reader.