Étude des relations pharmacodynamiques, pharmacogénétiques et pharmacocinétiques des immunosuppresseurs anticalcineurines chez les transplantés hépatiques

Abstract

Calcineurin inhibitors (CNI) are the immunosuppressants most employed in solid organ transplantation and despite their toxicity and suboptimal efficacy they will remain the first therapeutic option for time ahead. Their effects show huge intra- and inter-individual variability, not explained by differences in drug doses, concentrations or areas under the concentration-time curve, limiting the benefits of therapeutic drug monitoring and suggesting that other factors contribute to response variability. As CNI target T cell function, CNI concentration in lymphocytes was studied as a surrogate marker of the immune function. Actually, intra-lymphocyte TAC levels in liver transplant patients were better associated with acute rejection than whole blood levels, probably because they integrate potential inter-individual variations of drug entry into the lymphocytes. In the same line, previous studies showed that intrahepatic TAC levels displayed excellent correlation with the Banff rejection scores, whereas trough blood levels did not. Both may represent interesting, although difficult to assess, pharmacokinetic biomarkers. To qualify as an index of CNI immunosuppressive activity, a pharmacodynamic biomarker should be directly affected by drug activity, i.e. not too far from the drug target and at the same time close to its clinical effects. As a large amount of evidence supports the concept that T cell suppression is the key mechanism by which CNI achieve immunosuppression to prevent cellular rejection, T cell function and activation are attractive candidates for pharmacodynamic monitoring strategies. However, so far no single pharmacodynamic (PD) biomarker has met all the ideal requirements, i.e. non-invasiveness, reliability, sensitivity, specificity, reproducibility and short analytical time. Objectives and populations To search for suitable PD biomarkers, i.e., with high specificity for calcineurin inhibition and most affected by inter-individual variability, our works aimed at exploring the pharmacodynamics of CNI, the strength and variability of signal translation along the calcineurin pathway, as well as the steps where sources of internal (genetic) or external variability are the most influential. In order to achieve this, we assessed simultaneously NFAT1 translocation into the nucleus of peripheral blood mononuclear cells (PBMC) (NFTA1 being the main NAFT isoform in resting and activated lymphocytes), the intracellular expression of IL-2 in CD3+, CD4+ and CD8+ T cell subsets and the membrane expression of CD25 (IL-2Rα), a surface marker of T cells activation, in T cells at large. A non-interventional clinical trial was set up in healthy volunteers, patients registered on a liver transplantation waiting list (WLP) and liver transplant recipients (LTR). A different question was addressed in each group: The healthy volunteer study (n=35): explored TAC PD along the calcineurin pathway by exposing PBMC ex-vivo; modelled signal translation along this cascade; examined the interindividual variability of TAC PD parameters; and investigated the sources of the variability observed and their contribution at each step of the calcineurin pathway. Furthermore, it allowed us to evaluate the analytical variability of our techniques as well as the intra-individual variability of TAC PD parameters. WLP (n=19) were enrolled to confirm in patients with liver diseases the results obtained in healthy volunteers, as well as to test the potential influence of their initial disease on the ex-vivo pharmacodynamics of TAC. The aims of the transversal study of LTR on CNI (n=80) were to further explore the interindividual variability in the PD of CNI in realistic clinical conditions, i.e. in situations of residual PD activities under tacrolimus or cyclosporine exposure, and the potential pharmacogenetic (PG) sources of such variability. The (still small) group of liver transplant patients (n=6) enrolled immediately before transplantation and followed-up with serial monitoring along the first year post-transplantation was intended to explore the relationships between CNI PD and clinical responses. In summary, IL-2 and CD25 in CD8+ T cells and CD25 in CD4+ T cells may be reliable biomarkers of CNI activity, with the largest inter-individual variability. Moreover, a few clinical cases suggest that NFAT1 levels in PBMC nuclei might help to anticipate infection episodes, while Tregs diminution and high levels of IL-2 expression in CD8+ T cells might predict acute cellular rejection.