Supplementary Materials
Pharmacokinetic profile of GSK1521498
Animals
Adult male Long-Evans rats (Charles River Laboratories, Inc., Raleigh, NC), weighing
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
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
± 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.
Drug
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
(2-8 ºC).
Surgery
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.
Materials and Methods
The dose formulation was removed from storage and brought to room temperature (2025°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
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
HPLC/MS/MS analysis. The lower limit of quantification for GSK1521498 was 10 ng/mL
using a 25 μL portion of rat plasma.
Results
Fig.1: Mean 24-h plasma concentration/time profile in rats (n=3) following GSK1521498
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.
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 chocolateflavoured 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.
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.
The aversion/preference tests were conducted in the absence of drug treatment. Data are
presented as mean + SEM.