A771726, the active metabolite of leflunomide, directly inhibits the activity of cyclo‐oxygenase‐2 in vitro and in vivo in a substrate‐sensitive manner

The immunosuppressive and anti‐inflammatory drug leflunomide has several sites of action, although its precise mode of action is unknown. Here we show in vitro and in vivo that leflunomide and/or its active metabolite A771726, inhibit the activity of cyclo‐oxygenase (COX) at doses below those that affect protein expression. In J774.2 macrophages treated with endotoxin for 24 h to induce COX‐2 and iNOS, leflunomide and A771726 inhibited more potently the accumulation of PGE 2 (A771726, IC 50 3.5 μg ml −1 ) than of NO 2 (A771726, IC 50 380 μg ml −1 ). At high concentrations (>300 μg ml −1 ) A771726 also exhibited the expression of COX‐2 and iNOS proteins. In A549 cells treated for 24 h with interleukin‐1β, to induce COX‐2, A771726 potently inhibited PGE 2 synthesis (IC 50 0.13 μg ml −1 ). In the same cells, A771726 was notably less active (IC 50 , 52 μg ml −1 ) at inhibiting the formation of PGE 2 stimulated by exposure to 30 μ M arachidonic acid. In a human whole blood assay, measuring the accumulation of TxB 2 in response to calcium ionophore as a measure of COX‐1 activity and in response to incubation with bacterial endotoxin as a measure of COX‐2 activity, leflunomide inhibited COX‐1 and COX‐2 with IC 50 values of 31 and 185 μg ml −1 ; for A771726 the corresponding values were 40 and 69 μg ml −1 . Pre‐treatment of rats with leflunomide or A771726 (10 mg kg −1 , i.p.) inhibited the plasma accumulation of 6‐keto‐PGF 1α but not NO 2 /NO 3 following infusion of endotoxin. Injection of a bolus of arachidonic acid following 6 h infusion of endotoxin caused a marked acute rise in plasma 6‐keto‐PGF 1α which was inhibited only by higher doses of A771726 (50 mg kg −1 , i.p.). In conclusion, leflunomide via A771726 can directly inhibit the activity of COX, an effect that appears blunted both by increases in substrate supply and possibly by plasma binding. Only at much higher drug levels does leflunomide and/or A771726 inhibit the induction of COX‐2 or iNOS proteins.British Journal of Pharmacology (1999) 127 , 1589–1596; doi: 10.1038/sj.bjp.0702708