Cannabinoids have recently been shown to induce the expression of the cyclooxygenase-2 (COX-2) isoenzyme in H4 human neuroglioma cells. Using this cell line, the present study investigates the contribution of the second messenger ceramide to this signaling pathway. Incubation of cells with the endocannabinoid analog R(ϩ)-methanandamide (R(ϩ)-MA) was associated with an increase of intracellular ceramide levels. Enhancement of ceramide formation by R(ϩ)-MA was abolished by fumonisin B 1 , a ceramide synthase inhibitor, whereas inhibitors of neutral sphingomyelinase (spiroepoxide, glutathione) and serine palmitoyltransferase (L-cycloserine, ISP-1) were inactive in this respect. R(ϩ)-MA caused a biphasic activation of the p38 and p42/44 mitogen-activated protein kinases (MAPKs), with phosphorylation peaks occurring after 15-min and 4-to 8-h treatments, respectively. Inhibition of ceramide synthesis with fumonisin B 1 was associated with a suppression of R(ϩ)-MAinduced delayed phosphorylations of p38 and p42/44 MAPKs and subsequent COX-2 expression. The involvement of ceramide in COX-2 expression was corroborated by findings showing that C 2 -ceramide and neutral sphingomyelinase from Bacillus cereus caused concentration-dependent increases of COX-2 expression that were suppressed in the presence of 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)imidazol (SB203580, a p38 MAPK inhibitor) or 2Ј-amino-3Ј-methoxyflavone (PD98059, a p42/44 MAPK activation inhibitor). In contrast, dihydro-C 2 -ceramide being used as a negative control did not induce MAPK phosphorylation and COX-2 expression. Collectively, our results demonstrate that R(ϩ)-MA induces COX-2 expression in human neuroglioma cells via synthesis of ceramide and subsequent activation of p38 and p42/44 MAPK pathways. Induction of COX-2 expression via ceramide represents a hitherto unknown mechanism by which cannabinoids mediate biological effects within the central nervous system.

The isoxazol derivative leflunomide [N-(4'-trifluoromethylphenyl)-5-methylisoxazole-4-carboxamide] is an inhibitor of de novo pyrimidine synthesis used for the treatment of rheumatoid artrithis. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of leflunomide and its active metabolite, A77 1726, in human plasma. The analytes were separated using a mobile phase, consisting of acetonitrile, water and formic acid (40/59.8/0.2, v/v), at a flow rate of 0.5 mL/min, and UV detection at 261 nm. The retention times for A77 1726, leflunomide and warfarin (internal standard) were 8.2, 16.2 and 12.2 min, respectively. The validated quantification range of the method was 0.05-100 micro g/mL for leflunomide and 0.1-100 micro g/mL for A77 1726. The developed procedure was applied to assess steady-state plasma concentrations of A77 1726 in patients with rheumatoid arthritis treated with 10 or 20 mg leflunomide per day.