Measurement of differential inhibition of COX-1 and COX-2 and the pharmacology of selective inhibitors.
Since the discovery of a second isoenzyme of cyclooxygenase (COX)-2, it has been hypothesized that the antiinflammatory effects of non-steroidal antiinflammatory drugs (NSAIDs) are dependent on their inhibition of COX-2, whereas inhibition of constitutive COX-1 is responsible for their gastric and renal side effects as well as for inhibition of platelet activation. Consequently, a large number of in vitro assays have been developed to characterize the COX-1 and COX-2 inhibitory activities of NSAIDs in order to look for compounds with preferential inhibition of COX-2. Depending on the test system, however, the experimental conditions may vary greatly, thereby affecting the outcome. These important variables include the source of enzymes COX-1 and COX-2 (human or animal); the cell system used (intact normal cells or transfected cell lines); the method of enzyme preparation (purified enzymes, microsomal or whole cell assays); the COX-2-inducing agent; the source of arachidonic acid and its concentration; the incubation time with drug, inducing agent or arachidonic acid; and the protein concentration in the medium. Depending on the test system employed for defining the IC(50) for COX-1 and COX-2 inhibition, the resultant ratio between these activities may lead to divergent and confusing comparisons. We review the various in vitro test systems available for the measurement of COX-1 and COX-2 inhibition. The use of human recombinant enzymes and the human whole blood assay are examined in detail. The relevance of these test systems to in vivo animal models of inflammation and the side effects of NSAIDs (e.g., gastric mucosal and renal damage) is assessed. Finally, the results of clinical studies on the effect of inhibiting COX-1 and COX-2 activity is summarized.
Pairet
M
M
Boehringer Ingelheim Pharma KG, Biberach/Riss, Germany.
van Ryn
J
J