Despite increasing information about the mechanism of action of cyclosporine A (CsA) ,

Recent reports have shown that cyclosporine (CsA)-treated patients have abundant calcineurin phosphatase (CN) activity in vivo despite CsA blood concentrations that would be completely inhibitory in vitro. We sought to determine whether this disparity was a result of altered distribution of CsA in culture medium (CM) compared with whole blood (WB). CN activity was measured in peripheral blood leukocytes (PBL) that had been exposed in vitro to CsA in either WB or CM. Cells from both groups were also stimulated with OKT3 to determine the effect of CsA on the induction of IFN-gamma synthesis. CsA accumulation in PBL was determined by liquid scintillation counting of PBL exposed to 3H-CsA. The IC50 for CsA inhibition of CN activity was significantly lower for PBL in CM (2 micrograms/L) compared with PBL in WB (102 micrograms/L, P < or = 0.005). Likewise, for CsA inhibition of IFN-gamma induction, the IC50 was 18 micrograms/L for PBL in CM compared with 690 micrograms/L for PBL in WB (P < or = 0.005). The shift in IC50 was accompanied by a 10-fold increase in 3H-CsA in PBL in CM compared with PBL in WB. We conclude that PBL exposed to CsA in CM accumulate significantly more CsA than PBL in WB. The result is that CsA inhibition of CN activity and cytokine induction appears at least an order of magnitude greater than its true effect in biologic fluids. This disparity is due to partitioning of CsA to irrelevant CsA binding sites, which are abundant in WB and in vivo, but absent in culture medium.

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