For cutaneous blood flow measurements in vivo, SD ratswere dosed orally with either PF-04418948. Approximately 1 h 25 min post dose, at the approximate peak of PF-04418948 exposure, rats were anaesthetized, their abdomen was shaved, baseline scanning laser Doppler recordings were taken over a 2.5 × 2.5 cm area of the abdomen every 5 min for a total of 35 min. Following these baseline recordings, 10 µL of 10, 30, 100 or 1000 µg·mL−1s.c. of butaprost (10% ethanol + 90% saline) was injected into the centre of the scan area. Laser Doppler recordings were taken for a further 60 min. Escalating oral doses of PF-04418948 (1-10 mg/kg) reduced the peak and AUC butaprost-induced cutaneous blood flow response in a dose-dependent fashion.

Recombinant CHO cells, expressing EP2 receptor were suspended in DMEM at 1 × 10^6 cells*mL−1. Stocks of PGE2, BW245C and PF-04418948 (4 mM) were prepared in 100% DMSO and diluted in compound buffer (PBS; 2.5% (v/v) DMSO, 0.15% (v/v) pluronic F-127) to 2 µM final assay concentration. PGE2 was further diluted to 5 nM in PBS. PF-04418948 stock was serially diluted in 100% DMSO. Compounds were diluted 1:40 in compound buffer, and 5 µL transferred to a Lumitrac 200 white plate. Prepared cells (5 uL) were added and incubated at 37°C for 30 min. Agonist (5 uL) was added and incubated for 90 min at 37°C. The plates were then placed at -80°C to lyse the cells. Plates were thawed at 37°C for 15 min. Pre-warmed DiscoveRx assay reagents were added according to the manufacturer's protocol. Plates were incubated in the dark for 4-20 h and read on a plate reader with visible light. The agonist assay protocol was as the antagonist protocol, except that 5 µL compound buffer was used instead of the agonist stimulation. PF-04418948 exhibited antagonistic activity with IC50 of 16 nM.