COX-2 Assays Research Articles

Cyclooxygenase-1 and Cyclooxygenase-2 Selectivity of Widely Used Nonsteroidal Anti-Inflammatory Drugs

Purpose: Both isoforms of cyclo-oxygenase, COX-1 and COX-2, are inhibited to varying degrees by all of the available nonsteroidal anti-inflammatory drugs (NSAIDs). Because inhibition of COX-1 by NSAIDs is linked to gastrointestinal ulcer formation, those drugs that selectively inhibit COX-2 may have less gastrointestinal toxicity. We measured the extent to which NSAIDs and other anti-inflammatory or analgesic drugs inhibit COX-1 and COX-2 in humans. Subjects and Methods: Aliquots of whole blood from 16 healthy volunteers were incubated ex vivo with 25 antiinflammatory or analgesic drugs at six concentrations ranging from 0 (control) to 100 μM (n = 5 for each). Blood was assayed for serum-generated thromboxane B 2 synthesis (COX-1 assay) and for lipopolysaccharide-stimulated prostaglandin E 2 synthesis (COX-2 assay). In addition, gastric biopsies from the same volunteers were incubated with each drug ex vivo and mucosal prostaglandin E 2 synthesis measured. Results: Inhibitory potency and selectivity of NSAIDs for COX-1 and COX-2 activity in blood varied greatly. Some NSAIDs (eg, flurbiprofen, ketoprofen) were COX-1 selective, some (eg, ibuprofen, naproxen) were essentially nonselective, while others (eg, diclofenac, mefenamic acid) were COX-2 selective. Inhibitory effects of NSAIDs on gastric prostaglandin E 2 synthesis correlated with COX-1 inhibitory potency in blood ( P <0.001) and with COX-1 selectivity ( P <0.01), but not with COX-2 inhibitory potency. Even COX-2 “selective” NSAIDs still had sufficient COX-1 activity to cause potent inhibitory effects on gastric prostaglandin E 2 synthesis at concentrations achieved in vivo. Conclusion: No currently marketed NSAID, even those that are COX-2 selective, spare gastric COX activity at therapeutic concentrations. Thus, all NSAIDs should be used cautiously until safer agents are developed.

Amentoflavone fromBiophytum sensitivumand its Effect on COX-1/COX-2 Catalysed Prostaglandin Biosynthesis

Amentoflavone (I3', II8-biapigenin) was isolated from the roots of Biophytum sensitivum DC. (Oxalidaceae) and proved to be a selective inhibitor of cyclooxygenase (COX)-1 catalysed prostaglandin biosynthesis when tested in vitro with an IC (50) value of 12.4 microM (standard: indomethacin, IC (50) = 1.1 microM). Doses of up to 37 microM showed only a slight inhibition in the corresponding COX-2 assay. Quantification of amentoflavone was carried out by reversed phase HPLC in methanolic and aqueous extracts of the roots, stems and leaves. Highest amounts of amentoflavone were detected in methanolic extracts of roots and stems (0.26-0.35%), while considerably lower amounts were detected in the corresponding water extracts.

Development of a radiochemical cyclooxygenase-1 and -2 in vitro assay for identification of natural products as inhibitors of prostaglandin biosynthesis.

A radiochemical enzyme assay for studying cyclooxygenase (COX)-catalyzed prostaglandin biosynthesis in vitro was optimized with respect to both COX-1 and COX-2 activity. The assay can be used to assess the relative selectivity of plant-derived inhibitors on COX-1 and COX-2 Assay conditions were optimized for both enzymes with respect to concentration of cofactors (l-epinephrine, reduced glutathione, and hematin), activation time (enzyme and cofactors), reaction time, and pH. Moreover, the kinetic parameters, Km and Kcat, of both enzymes were estimated. Five COX inhibitors were used to validate the assay, indomethacin, aspirin, naproxen, ibuprofen, and the arylsulfonamide NS-398, all with different COX selectivity and time dependency. Time-dependent inhibition was determined by comparing the inhibition, with and without preincubation of enzyme and inhibitor. Two flavonoids, (+)-catechin and quercitrin, were examined with respect to inhibition of COX-catalyzed prostaglandin biosynthesis. (+)-Catechin showed equal inhibitory effects on the two enzymes. Quercitrin was found to be inactive toward both COX-1- and COX-2-catalyzed prostaglandin biosynthesis. The optimization procedure resulted in a considerable reduction of the amount of enzyme required for adequate prostglandin biosynthesis and a reliable method suited to evaluate natural products on inhibition of COX-2-catalyzed prostaglandin biosynthesis, as well as on COX-1.

Effect of structural modification of enol-carboxamide-type nonsteroidal antiinflammatory drugs on COX-2/COX-1 selectivity.

Meloxicam (5), an NSAID in the enol-carboxamide class, was developed on the basis of its antiinflammatory activity and relative safety in animal models. In subsequent screening in microsomal assays using human COX-1 and COX-2, we discovered that it possessed a selectivity profile for COX-2 superior to piroxicam and other marketed NSAIDs. We therefore embarked on a study of enol-carboxamide type compounds to determine if COX-2 selectivity and potency could be dramatically improved by structural modification. Substitution at the 6- and 7-positions of the 4-oxo-1,2-benzothiazine-3-carboxamide, alteration of the N-methyl substituent, and amide modification were all examined. In addition we explored several related systems including the isomeric 3-oxo-1,2-benzothiazine-4-carboxamides, thienothiazines, indolothizines, benzothienothiazines, naphthothiazines, and 1,3- and 1,4-dioxoisoquinolines. While a few examples were found with greater potency in the COX-2 assay, no compound tested had a better COX-2/COX-1 selectivity profile than that of 5.