In vitro activity: Carprofen (S and R stereoisomers) inhibits canine COX2 with IC50 of 0.102 microM for the racemic mixture, the inhibition is primarily attributable to the S enantiomer (IC50, 0.0371 microM), which is approximately 200-fold more potent than the R enantiomer (IC50, 5.97 microM). Carprofen binds to human serum albumin (HSA) by both fluorescence and equilibrium dialysis (ED) methods is characterized by two sets of association constants [K1 = 5.1 μM (fluorescence) and 3.7 μM (ED), K2 = 37 μM (fluorescence) and 13 μM (ED)]. Carprofen primarily binds to site II, the benzodiazepine site, while the low affinity site of carprofen is site I, the warfarin site, the carboxyl group of carprofen is found to play an important role especially in the high affinity binding of carprofen to HSA.

Cell Assay: Cruciate ligament cells are used and incubated with DMEM supplemented with 10% FCS for 24 hours to synchronize cell cycles. The cell cultures are then preincubated without (control) or with a nonselective COX inhibitor (acetylsalicylic acid) or a preferential COX-2 inhibitor (Carprofen, meloxicam, or robenacoxib) to ssess whether NSAIDs prevented apoptosis when the cells are subsequently incubated with SNP. For all cell cultures except those designated as controls, 1 of 3 concentrations of 1 of the 4 NSAIDs (10, 100, or 200 μg of acetylsalicylic acid/mL; 0.1, 1, or 10 μg of Carprofend/mL; 0.1, 1, or 10 μg of meloxicame/mL; or 0.1, 1, or 10 μg of robenacoxibf/mL) is added to the culture media of each cell culture, and the cells are incubated for 2 hours.