In the model of inflammatory pain subcutaneous injection of complete Freund's adjuvant (CFA) into the plantar surface of the hind paw produced a significant decrease in mechanical paw weight threshold (PWT) at 5 days post injection. PF-3845 [1 – 30 mg/kg, oral administration (p.o),] caused a dose-dependent inhibition of mechanical allodynia with a minimum effective dose (MED) of 3 mg/kg (rats were analyzed at 4 hr post-dosing with PF-3845).

GDH-coupled FAAH assays were performed in 96-well UV clear-bottom microplates in a total volume of 200 μl per well at 25°C. 20 μl of 50% DMSO and 20 μl of oleamide at variable concentrations in 75% ethanol and 25% DMSO were added to a 140-μl reaction mixture containing final concentrations of 48 mM NaPi (pH 7.4), 150 μM NADH, 3 mM α-ketoglutarate, 2 mM ADP, 1 mM EDTA, and 30 units/ml GDH. After the resulting mixture was mixed in a plate vortex, 20 μl of 100 nM (apparent concentration) hFAAH, rFAAH, or h/rFAAH in 20 mM NaPi (pH 7.8) and 1% Triton X-100 was added to initiate the reaction. Absorbance at 340 nm was collected over a period of 90 min with readings taken in 15-s intervals by using a SpectraMax microplate spectrophotometer equipped with Softmax Pro software (Molecular Devices). Kinact/Ki for PF-3845 was 12.600 M-1s-1