Differential inhibition of human prostaglandin endoperoxide H synthases-1 and -2 by nonsteroidal anti-inflammatory drugs.

We developed an in vitro expression system for accurate kinetic analyses of the inhibition of the human prostaglandin H synthase isozymes (hPGHS-1 and -2) by nonsteroidal anti-inflammatory drugs (NSAIDs). Assays of instantaneous inhibition in which enzyme, 10 microM arachidonate, and an NSAID were mixed simultaneously were used to determine apparent affinities of 14 common NSAIDs for hPGHS-1 and hPGHS-2. All NSAIDs except salicylate had appreciable apparent affinities (IC50 < or = 100 microM) for hPGHS-1. Most NSAIDs also exhibited appreciable affinities toward hPGHS-2, but three prominent exceptions were indomethacin, piroxicam and phenylbutazone. We subsequently performed measurements of time-dependent inhibition in which either (a) enzyme and an NSAID were preincubated before substrate was added to initiate the reactions or (b) recovery of activity after time-dependent inhibition was measured using intact cells preincubated with various NSAIDs. Indomethacin, flurbiprofen, meclofenamate and diclofenac, but not ibuprofen, piroxicam or phenylbutazone, caused time-dependent inhibition of both hPGHS-1 and -2 in vitro. For cells pretreated with flurbiprofen or meclofenamate, hPGHS-2 activities, but not hPGHS-1 activities, were recovered relatively rapidly; with indomethacin, recoveries of hPGHS-1 and hPGHS-2 activities were both slow. hPGHS-2 is thought to be the target of NSAIDs acting as anti-inflammatory agents. However, our results indicate that neither measurements of affinities of NSAIDs for hPGHS-2 conducted in vitro with 10 microM arachidonate nor measurements of time-dependent inhibition of hPGHS-2 always predict whether a compound (e.g., piroxicam or phenylbutazone) has anti-inflammatory activity in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)