1 Reactive oxygen species (ROS) have been proposed to play a role in the side e ects of the immunosuppressive drug cyclosporin A (CsA). 2 The aim of this study was to investigate whether cytochrome P-450 (CYP) dependent metabolism of CsA could be responsible for ROS generation since it has been suggested that CsA may in¯uence the CYP system to produce ROS. 3 We show that CsA (1 ± 10 mM) generated antioxidant-inhibitable ROS in rat aortic smooth muscle cells (RASMC) using the¯uorescent probe 2,7-dichloro¯uorescin diacetate. 4 Using cytochrome c as substrate, we show that CsA (10 mM) did not inhibit NADPH cytochrome P-450 reductase in microsomes prepared from rat liver, kidney or RASMC. 5 CsA (10 mM) did not uncouple the electron¯ow from NADPH via NADPH cytochrome P-450 reductase to the CYP enzymes because CsA did not inhibit the metabolism of substrates selective for several CYP enzymes that do not metabolize CsA in rat liver microsomes. 6 CsA (10 mM) did not generate more radicals in CYP 3A4 expressing immortalized human liver epithelial cells (T5-3A4 cells) than in control cells that do not express CYP 3A4. 7 Neither diphenylene iodonium nor the CYP 3A inhibitor ketoconazole were able to block ROS formation in rat aortic smooth muscle or T5-3A4 cells. 8 These results demonstrate that CYP enzymes do not contribute to CsA-induced ROS formation and that CsA neither inhibits NADPH cytochrome P-450 reductase nor the electron transfer to the CYP enzymes.

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The immunosuppressive drug cyclosporin A (CsA) depresses neutrophil oxidative burst which may lead to an increased susceptibility to infection in transplant patients. Using specific CsA analogues we investigated the mechanism of inhibition of the oxidative burst and evaluated short and long‐term effects of CsA on dimethylsulphoxide‐differentiated HL‐60 neutrophils. A biphasic pattern was observed: a 4 h pre‐treatment with CsA (1 μM) diminished the fMLP induced [Ca2+]c rise, reactive oxygen species (ROS) production, and β‐glucuronidase release by about 40%, whereas a 20 h pre‐treatment increased these responses by about 1.5 fold. [MeVal4]CsA, which binds with high affinity to cyclophilin but inhibits the interaction of the CsA‐cyclophilin complex with calcineurin, blocked the stimulation observed with CsA after a 20 h incubation but did not alter the CsA effects after a 4 h pre‐treatment. PSC 833 (1 μM), a potent multi drug resistance transporter (MDR) inhibitor, diminished ROS production to the same extent as a 4 h CsA incubation but was ineffective after a 20 h pre‐treatment. An involvement of MDR as a basis for CsA or PSC 833 action was ruled out based on the results of the calcein retention assay. [3H]CsA uptake showed that CsA and [MeVal4]CsA, but not CsH or PSC 833 were strongly taken up and retained by the cells. In conclusion, the reduction of the responses after 4 h appear to be due to a primary reduction of calcium signalling, while the enhanced responses after 20 h may be due to calcineurin inhibition. British Journal of Pharmacology (1998) 124, 1774–1780; doi: