NPC 15199 (N-(Fluorenyl-9-methoxycarbonyl)leucine) was administered daily (10 or 100 mg/kg, s.c.) for 7 days

Fatty acid cyclooxygenase activity and 5-lipoxygenase activity were assessed using whole cell assays. Cyclooxygenase activity was measured by incubating arachidonic acid with 3T3 fibroblasts and measuring prostaglandin E2 synthesis. 5-Lipoxygenase was measured in blood from CD-1 mice. To 1 ml of Hanks' balanced salt solution was added 10 p1 of blood. After 5 min, 0.3 mkCi (1 Ci = 37 GBq) of [3H]arachidonic acid was added, followed by 1 mkM ionomycin. Incubation was for 5 min at 37C, followed by centrifugation at 10,000 x g for 10 sec. Supernatant (250 mk1) was injected onto a reverse-phase HPLC column (HP Hypersil ODS 5 mkM, 200 x 4.6 mm) at room temperature and eluted with at linear gradient from water/ acetonitrile/trifluoroacetic acid (60:40:0.01) to water/ acetonitrile/trifluoroacetic acid (10:90:0.01) at 2 ml/min. [3H]labeled leukotriene C4 was quantitated using an in-line liquid scintillation detector (Radiomatic Instruments and Chemical, Tampa, FL). Test compounds (N-(FLUORENYL-9-METHOXYCARBONYL)LEUCINE) were added to the blood suspension at the beginning of the 5-min incubation before addition of radiolabeled substrate. Plasma corticosterqne was quantitated by radioimmunoassay