Supplementary Materials s6

Supplementary Materials s6

<p>Supplementary Materials</p><p>Pharmacokinetic profile of GSK1521498</p><p>Animals</p><p>Adult male Long-Evans rats (Charles River Laboratories, Inc., Raleigh, NC), weighing</p><p>311-338 g at the beginning of the experiment, were held 3/cage or less in polycarbonate solid-bottomed cages with Bed-O’Cobbs™ (The Andersons, Maumee, OH, USA) or Alpha</p><p>Dri™ (Shepherd Specialty Papers, Inc. Kalamazoo, Michigan, USA), under a reversed 12h light/dark cycle. Environmental controls were maintained at 70 ± 10°F (temperature) and 55</p><p>± 30% humidity. Food (LabDiet™ brand Certified Rodent Diet 5002) and water were allowed ad libitum through completion of the study. The study was conducted in accordance with the US Animal Welfare Act, as amended. </p><p>Drug</p><p>GSK1521498 was supplied by Chemical Development, GSK. A 4 mg/mL stock solution of GSK1521498 in a vehicle containing acidified hydroxypropyl beta cyclodextrin was prepared. The stock solution was diluted to 1 mg/mL (expressed in terms of free base) with a phosphate buffer and filtered through a 0.20 micron filter prior to administration. Dilutions were prepared 1 day prior to each dose administration and stored at refrigerated temperature</p><p>(2-8 ºC).</p><p>Surgery</p><p>A femoral vein of 3 rats was surgically cannulated and the animals were allowed to recover from surgery for at least 36 hours prior to dosing. After surgery, rats were housed individually in metabolism cages. Each animal was fitted with a harness and tether to protect the dosing and blood sampling cannulas. </p><p>Materials and Methods</p><p>The dose formulation was removed from storage and brought to room temperature (20-</p><p>25°C) the morning prior to each day of dosing. Rats (n=3) received a single intraperitoneal injection of the GSK1521498 (3 mg/kg expressed in terms of parent compound) in an injection volume of 3 mL/Kg. Venous blood samples (approximately 0.3 mL) were collected into syringes via the femoral vein cannula and were transferred to tubes containing K2</p><p>EDTA at 5, 15 and 30 minutes and 1, 2, 4, 8, and 24 hours after dosing. Rat plasma samples were analyzed for GSK1521498 using a method based on protein precipitation, followed by</p><p>HPLC/MS/MS analysis. The lower limit of quantification for GSK1521498 was 10 ng/mL using a 25 μL portion of rat plasma.</p><p>Results</p><p>Fig.1: Mean 24-h plasma concentration/time profile in rats (n=3) following GSK1521498</p><p>3mg/kg IP administration. Near-peak exposures are observed within 30 minutes of dosing. Effects of GSK1521498 and NTX on responding for food under continuous reinforcement.</p><p>Fig.2: On the top (A), effect of GSK1521498 on food-taking under a fixed-ratio 1 schedule of reinforcement (n=6 males, grey bars and n=6 females, white bars). On the bottom (B), effect of NTX on food-taking under a fixed-ratio 1 schedule of reinforcement (n=6 males, grey bars and n=6 females, white bars). Data shown are mean (+SEM) number of chocolate- flavoured food pellets eaten in 1h session. * p<0.05, **p<0.01 compared with vehicle treated animals. Effects of GSK1521498 and NTX on place aversion or preference conditioning.</p><p>Fig.3: Time spent on the infusion-paired side of a two-compartment apparatus 1 day before conditioning sessions (Baseline) and 1 day after the 2 conditioning sessions (Test) (n=9 per group). During the conditioning phase, the rats were injected with GSK1521498 0, 1 or 3 mg/kg (IP) 30 min before the session (on the top, A) or NTX 0, 1 or 3 mg/kg (SC) 10 min before the session (on the bottom, B) and confined in one of the compartments for 30 min.</p><p>The aversion/preference tests were conducted in the absence of drug treatment. Data are presented as mean + SEM.</p>

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