Male rats or pregnant female rats (Day 18 of gestation) were treated with 3 mg/kg [3H] Ticagrelor (111.4 MBq/mg). Rats were humanely euthanized with carbon dioxide at the designated times post-dose. Immediately prior to euthanizing the rat, a whole blood sample (0.5 mL) was collected into heparinized tubes by venesection of a tail vein

and aliquots removed for blood radioactivity analysis. Each rat was immediately frozen and embedded in a block of methyl cellulose. Sagittal sections (30 μm) were prepared, freeze-dried and applied to phosphor screens along with a series of calibration standards containing known concentrations of radioactivity. BIBF 1120 mouse Ceritinib cell line After 7 days of exposure, the radioactivity present in various organs and tissues were determined using the Cyclone Storage Phosphor system (Packard; Meriden, CT). Blood sample radioactivity was quantified in scintilant for 5 minutes, together with representative

blank and standard vials using liquid scintillation analyzer with automatic quench correction using an external standard method. Ticagrelor and a major active metabolite (AR-C124910) were evaluated at 10 μM in more than 300 receptor, enzyme and electrophysiological assays (Ricerca Biosciences LLC) including dopamine D1, D2L and D4.2 receptors as well as the dopamine transporter using in vitro radioligand binding assays and methodologies described 2-hydroxyphytanoyl-CoA lyase in the literature [12], [17], [18], [20], [24], [44] and [43]. Human recombinant CHO-K1 cells were used for the dopamine transporter and D4.2 receptor, whereas human recombinant CHO cells were used for Dopamine D1 and D2L receptors. The radiolabelled ligands were [3H] SCH-23390, [3H] spiperone [3H] dopamine and [126]RTI-55 for the D1, D2L and D4.2 receptors and dopamine transporter, respectively. The data were calculated as a percentage inhibition of specific binding at the test concentration of 10 μM. Assays with significant inhibition (>50% effect) at 10 μM were followed up with concentration-response

curves. In the case of the dopamine transporter, a concentration-response curve was generated using ten ascending concentrations in half log10 intervals enabling calculation of the inhibitory concentration fifty percent (IC50), inhibition constant (Ki) and Hill coefficient (nH). IC50 values were determined by a non-linear, least squares regression analysis using the MathIQ™ software (ID Business Solutions Ltd., UK). This software was also used to calculate nH. The Ki value was calculated using the Cheng-Prusoff equation [9]. These assays were repeated four times in order to generate a robust estimate of affinity. The rat ovariectomized in vivo assay was a modification of Brott et al.