Tetrahydrouridine (THU)

Cell experiment:

Cell growth for pancreatic and lung carcinoma cell lines is carried out using the colorimetric methylene blue assay in 96-well plates at a density of 5,000 cells/well. Cells are either exposed or not exposed to Tetrahydrouridine (100 µM), counting the first 12 hrs as Day 0. Mean values are calculated from three different wells in triplicates for four days[1].

Animal experiment:

Mice[2] CD-1 mice (male 30-38 g and female 24-31g) from are individually housed in polycarbonate cages suspended on stainless steel racks with SaniChip certified hardwood bedding. Mice are assigned to four dose groups and a vehicle control group. Animals are gavaged with DAC or its vehicle 1 hour ± 5 minutes after administration of THU or its vehicle at a dose volume of 10 mL/kg. The DAC doses are selected based on the range finding study in which the mice tolerated six oral doses (2x/week) of 0.1, 0.2 and 0.4 mg/kg DAC in combination with a fixed dose of 167 mg/kg THU. A fixed Tetrahydrouridine dose (500 mg/m2) and the optimal timing between Tetrahydrouridine and DAC administration (60 min) are selected. Conversion of milligrams per body surface area dose in mice into milligrams per kilogram body weight dose estimation is based on Michaelis constant (km) values for mice obtained from US Food and Drug Administration published guidelines. In brief, the mouse dose in milligrams per body surface area (500 mg/m2) is divided by the km of 3 to convert the dose to milligrams per kilogram body weight (167 mg/kg).

References:

[1]. Funamizu N, et al. Tetrahydrouridine inhibits cell proliferation through cell cycle regulation regardless of cytidine deaminase expression levels. PLoS One. 2012;7(5):e37424.
[2]. Terse P, et al. Subchronic oral toxicity study of decitabine in combination with tetrahydrouridine in CD-1 mice. Int J Toxicol. 2014 Mar-Apr;33(2):75-85.