COX Assay

COX-1 and COX-2 activities were measured using purified ovine COX-1 and human recombinant COX-2 with colorimetric COX inhibitor screening kits obtained from Cayman Chemical Co. Test compounds or vehicle were incubated for 30 minutes at 25°C with diluted enzyme in a 96-well half-area nonbinding plate. Sulindac sulfide (SS) and celecoxib (CC) were used as positive controls for COX-1 and COX-2 inhibition, respectively. The activities of COX-1 and COX-2 activities were measured, after the addition of arachidonic acid, by the formation of prostaglandin, measured at 420 nm as specified by the manufacturer. For these assays, it is important to determine for each compound the concentrations needed for a linear response: for COX-1 inhibition, a linear dose response was observed for both SS and MCI-100; for COX-2 inhibition, the assay linearity was not as good with either SS or MCI-100, but since MCI-100 showed negligible (0.1%) activity compared with CC, the slight lack of linearity was not relevant. Each experimental condition was performed in triplicates on a 96-well plate.