Whole-Blood Calcineurin Activity Is Not Predicted by Cyclosporine Blood Concentration in Renal Transplant Recipients

Abstract

Background: In transplant patients, current cyclosporine (CsA) dose monitoring with classic pharmacokinetics has demonstrated limitations. Evaluation of the activity of calcineurin (CN), the serine-threonine phosphatase enzyme target of CsA, has been proposed as a reliable way to optimize CsA dosing. Methods: CN activity was measured in whole blood in an attempt to overcome the high variability of results obtained previously with peripheral blood mononuclear cells (PBMCs). We also explored, in vitro, a possible relationship between the CsA concentration and CN inhibition in whole blood. Finally, we assessed whether the CsA blood trough concentration correlates with whole-blood CN activity in kidney transplant recipients (n = 15) on maintenance immunosuppression with CsA. Results: In 14 healthy individuals, less scattered CN activity values were documented in whole blood than in the PBMC fraction. Whole-blood CN activity was higher than the sum of the enzyme activity in each cell blood fraction. After ex vivo incubation of whole blood from healthy subjects (n = 5) with increasing concentrations of CsA (50–1000 μg/L for 1 h), a concentration-dependent inhibition of CN activity was found comparable to that in the PBMC fraction. Moreover, in 15 kidney transplant recipients, no relationship was found between CsA pharmacokinetic parameters and CN activity at time 0. However, a highly significant correlation was found between CN area under the CN activity-time curve, which represents the extent of the CN daily inhibition, and CN activity at time 0 (r = 0.79; P <0.01) and at 12 h postdosing (r = 0.96; P <0.01). Conclusions: Measuring CN activity in whole-blood samples is a reproducible method. In kidney transplant recipients, CsA trough concentrations do not predict baseline CN activity. Moreover, a single CN activity monitoring at baseline or at time 12 h post-CsA dosing may be a useful surrogate for the inhibition of this enzyme by CsA during 12 h.

Distribution of calcineurin activity in blood cell fractions and impact of tacrolimus inhibition

“…in whole blood, or after isolation. In agreement with a previous report [19], we found that 62.3% of the total PP2B activity is recovered from the fractionated blood. This difference may be related to the analytical variability of HPLC for each PP2B measurement and to a potential loss of PP2B activity occurred within different extraction steps of each cellular fraction.…”

“…However, PP2B activity in PBMC was the highest when it was expressed per million of cells. Indeed, Caruso et al [19] demonstrated the highest expression of PP2B a catalytic subunit in PBMC among the other blood cell types. As the cell target of tacrolimus is the lymphocyte in order to prevent acute rejection, we measured PP2B activity in isolated lymphocytes and monocytes, both cells included in PBMC.…”

“…Finally, to limit the variability in the measurement of PP2B activity, Caruso et al [19] advised the use of whole blood as biological matrix rather than PBMC. These authors reported a greater variability in the measurement of PP2B activity in PBMC, which could be explained by PBMC isolation, frozen storage or the use of a radioactive method.…”

The calcineurin activity profiles of cyclosporin and tacrolimus are different in stable renal transplant patients

“…Pharmacokinetic monitoring has been the method of choice in the transplantation units to the present, but pharmacodynamic monitoring of the calcineurin inhibitors as a sole monitoring tool or as a supplement could be helpful in TDM [8], hopefully resulting in prolonged graft survival. The pharmacodynamic assay used here is based on whole blood measurements, which have been shown to be superior to human peripheral blood mononuclear cells in a study by Caruso et al [9].…”

24‐h Monitoring of Calcineurin Phosphatase Activity in Healthy Subjects

“…Although some attention has been paid to the measuring of CaN activity in patients treated with CaN inhibitors [10][11][12][13], little is known about the natural behaviour of the enzyme CaN. Caruso et al [14] investigated three control individuals and found no indications of variation in CaN activity during a 12-h period.…”