6-ketoprostaglandin-f1-alpha and teriflunomide

1. The immunosuppressive and anti-inflammatory drug leflunomide has several sites of action, although its precise mode of action is unknown. 2. 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. 3. 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 PGE2 (A771726, IC50 3.5 microg ml-1) than of NO2 (A771726, IC50 380 microg ml-1). At high concentrations (>300 microg ml-1) A771726 also exhibited the expression of COX-2 and iNOS proteins. 4. In A549 cells treated for 24 h with interleukin-1beta, to induce COX-2, A771726 potently inhibited PGE2 synthesis (IC50 0.13 microg ml-1). In the same cells, A771726 was notably less active (IC50, 52 microg ml-1) at inhibiting the formation of PGE2 stimulated by exposure to 30 microM arachidonic acid. 5. In a human whole blood assay, measuring the accumulation of TxB2 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 IC50 values of 31 and 185 microg ml-1; for A771726 the corresponding values were 40 and 69 microg ml-1. 6. Pre-treatment of rats with leflunomide or A771726 (10 mg kg-1, i.p.) inhibited the plasma accumulation of 6-keto-PGF1alpha but not NO2/NO3 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-PGF1alpha which was inhibited only by higher doses of A771726 (50 mg kg-1, i.p.). 7. 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.

Topics: 6-Ketoprostaglandin F1 alpha; Aniline Compounds; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acid; Blotting, Western; Cell Survival; Crotonates; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dinoprostone; Enzyme Induction; Enzyme Inhibitors; Humans; Hydroxybutyrates; In Vitro Techniques; Isoenzymes; Isoxazoles; Leflunomide; Macrophages; Male; Membrane Proteins; Mice; Nitric Oxide; Nitric Oxide Synthase; Nitriles; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Wistar; Substrate Specificity; Toluidines

1. Exposure of tissues to endotoxin (LPS) and/or cytokines leads to the induction of both inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2). It has previously been reported that there is 'cross-talk' between these two systems. However, such previous studies have been limited by the availability of highly selective inhibitors. Here we have investigated the interactions between iNOS and COX-2 in vivo using 1400W, an iNOS-selective inhibitor, and celecoxib, a COX-2-selective inhibitor. 2. Infusion of LPS to rats for 6 h caused a time-dependent increase in the plasma concentrations of 6 keto-prostaglandin F1alpha (6 keto-PGF1alpha) and nitrite/nitrate (NO2/NO3), consistent with the induction of iNOS and COX-2. Bolus injection of arachidonic acid (AA) at t=6 h resulted in a further increase of circulating levels of 6 keto-PGF1alpha in LPS-treated animals. 3. Treatment of rats with 1400W or the non-selective NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) inhibited the increase in plasma NO2/NO3 but were both without effect on the plasma concentration of 6 keto-PGF1alpha before or after AA. 4. Treatment with the non-steroidal anti-inflammatory drugs (NSAIDs), A771726 or diclofenac, or with celecoxib significantly reduced the increase in circulating 6 keto-PGF1alpha caused by LPS, and the large increase in 6 keto-PGF1alpha following injection of AA. None of the COX inhibitors affected the increase in plasma NO2/NO3. Dexamethasone, however, significantly inhibited both the increase in 6 keto-PGF1alpha and the increase in NO2/NO3. 5. In conclusion, the use of selective inhibitors does not support the concept of cross talk in vivo between iNOS and COX-2.

Topics: 6-Ketoprostaglandin F1 alpha; Amidines; Analysis of Variance; Aniline Compounds; Animals; Anti-Inflammatory Agents, Non-Steroidal; Benzylamines; Celecoxib; Crotonates; Cyclooxygenase 2; Dexamethasone; Diclofenac; Enzyme Inhibitors; Hydroxybutyrates; Isoenzymes; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitriles; Nitrites; Nitrogen Dioxide; omega-N-Methylarginine; Prostaglandin-Endoperoxide Synthases; Pyrazoles; Rats; Rats, Wistar; Sulfonamides; Toluidines