Inhibition of PHA induced mononuclear cell proliferation by FK506 in combination with cyclosporine, methylprednisolone, 6-mercaptopurine and mycophenolic acid

Abstract

In vitro inhibition of human peripheral blood mononuclear cell (PBMC) proliferation by immunosuppressive drugs has been shown to correlate with clinical outcomes in kidney transplant patients. The aim of our study was to analyse the degree of variability of in vitro interactions of FK506 (FK) with combinations of cyclosporin A (CyA), methylprednisolone (MP), 6-mercaptopurine (6ME), and mycophenolic acid (MPA) using phytohaemagglutinin (PHA) stimulated PBMCs. Out hypotheses were: that a wide range of interindividual variation would be detected; and that certain combinations of drugs would have a synergistic inhibitory effect on PHA stimulated PBMCs. Cells were cultured in supplemented RPMI 1640 for 65 hours at 37°C in humidified 5% CO 2 - 95% air and proliferative responses measured by 3H-thymidien incorporation over a further 24 hours. Inhibition of PBMC proliferation was assessed over 10 FK concentrations ranging from 1 x 10 −8 to 10 μg/ml using both volunteer controls ( n = 51) and dialysis patients ( n = 23). A wide range of interindividual variability of FK inhibition occurred throughout the range of FK concentrations tested, becoming less variable at the higher concentrations. The interactions of FK with CyA, MP, 6ME and MPA were assessed using PBMCs from 12 volunteer controls. For FK at 1 x 10 −8 μg/ml; CyA, MP, 6ME and MPA were used at 0.01 μg/ml. For FK at 1 x 10 −7 μg/ml; CyA, MP, 6ME and MPA were used at 0.1 μg/ml. Both FK concentrations were tested with all possible combinations ( n = 7) of CyA, MP and 6ME; and these FK combinations were also tested with the inclusion of MPA to assess the effect of addition of MPA. Using FK at 1 x 10 −8 μg/ml and the other four drugs at 0.01 μg/ml there was a significant ( p < 0.05 Wilcoxon's test) increase in per cent inhibition in 5/15 FK combinations. Using FK at 1 x 10 −7 μg/ml and the other four drugs at 0.1 μg/ml there was a significant increase in per cent inhibition in 10/15 FK combinations. Significant reduction of FK inhibition did not occur in any combinations at either concentration level, implying that no drugs were directly antagonistic to each other. Significantly increased inhibition above the level achieved by FK or FK/MPA occurred in 15/30 FK containing combinations. The FK/CyA combination displayed the largest and most frequent inhibition increases compared with either FK or FK/MPA alone. FK combinations with CyA, MP and 6ME resulted in significantly increased inhibition in 11/14 (79%) combinations, compared to that achieved by FK alone. FK/MPA combinations with CyA, MP and 6ME resulted in significantly increased inhibition in 4/14 (29%) combinations, compared to that achieved by FK/MPA alone. This suggested that at these concentrations, the inclusion of MPA in FK containing combinations did not improve the inhibition levels achieved. using eight control PBMCs, FK and CyA were tested at equal concentrations of 0.01 and 0.1 μg/ml. The result for all eight individuals was the same. At these concentrations there were no significant improvements in percentage inhibition by using FK/CyA together compared with FK or CyA alone.