Elsevier

Clinica Chimica Acta

Volume 413, Issues 17–18, 8 September 2012, Pages 1379-1386
Clinica Chimica Acta

Invited critical review
Calcineurin inhibitors and NFAT-regulated gene expression

https://doi.org/10.1016/j.cca.2011.09.041Get rights and content

Abstract

Calcineurin inhibitors (CNIs) have a narrow therapeutic window; therefore, regular monitoring of the drug is necessary to balance sufficient efficacy with minimal toxicity. Until now, monitoring of immunosuppressive drugs is performed by pharmacokinetic assessments, mainly by trough concentrations (C0) of the drug. All these methods rely on pharmacokinetic data, which does not reflect the biological effects of CNI on the immune system. Several approaches have been undertaken to measure the biologic effects of CNI-based immunosuppression. Recently, a new quantitative analysis of gene expression has been employed to calculate the inhibition of the transcription of NFAT-regulated genes in peripheral blood. Methodological aspects and clinical data on the potential benefit of this specific CNI monitoring assay are discussed.

Introduction

Calcineurin-inhibitors (CNI) are widely used in organ transplantation and more than 80% of de novo renal and heart transplant recipients are on CNI therapy [1]. Both CNIs, ciclosporin A (CsA) and tacrolimus (Tac), have confirmed an excellent efficacy profile with low acute rejection rates. The current 1-year survival rate of renal allografts reaches 90% [2]. Unexpectedly, the overall renal allograft survival remained stable during the last decades indicating a shift of graft failure due to rejection to consequences of long-term immunosuppression [3]. CNIs have a narrow therapeutic window; therefore, regular monitoring of the drug is necessary to balance sufficient efficacy with minimal toxicity. Common CNI induced side effects include nephrotoxicity, hypertrichosis, gingival overgrowth and hypertension in CsA therapy, and hyperglycemia as well as neurotoxicity in Tac immunosuppression [4], [5], [6], [7]. Most of these adverse reactions are dose dependent. On the other side, sub-therapeutic drug levels significantly increase the risk of allograft rejection. Until now, monitoring of immunosuppressive drugs is performed by pharmacokinetic assessments, mainly by trough concentrations (C0) of the drug. Detailed information about total drug exposure is provided by 12-h pharmacokinetic profiles. The area under the concentration–time curve (AUC0-12) predicts efficacy and safety [8], [9]. However, with 12 blood samples taken in 12 h these pharmacokinetic profiles are very inconvenient, costly and almost not feasible in regular routine practice. Data concerning the relationship between C0 and AUC0-12 are conflicting, whereas C2 levels are reported to correlate most closely with AUC0-12 [9], [10], [11], [12]. There are only a few prospective clinical studies comparing single time point concentrations or limited sampling strategies with the corresponding clinical outcome [9], [12], [13]. Long-term graft loss, acute rejection and nephrotoxicity were associated with deviations in the CsA C2 but not the C0 levels [14], [15]. Therefore C2 monitoring might provide a convenience of a single time point measurement with a good correlation to clinical events. All these methods rely on pharmacokinetic data, which does not reflect the biological effects of CNI on the immune system. In addition, similar drug concentrations cause highly variable pharmacodynamic effects in individual patients, reflecting the heterogeneity of their immunotype.

Altogether, there is a great need for a more informative monitoring beyond the traditional therapeutic drug monitoring by blood concentration. Several approaches have been undertaken to measure the biologic effects of CNI-based immunosuppression including calcineurin phosphatase activity, release of cytokines and gene expression [16], [17], [18], [19], [20], [21].

Section snippets

Mode of action and possible specific pharmacodynamic monitoring assays

Detailed knowledge about the drug-specific mode of action is required to establish and introduce a pharmacodynamic (PD) monitoring assay. The proposed immunosuppressive mode of CsA and Tac is the inhibition of the phosphatase activity of calcineurin after binding of the corresponding CNI–immunophilin complexes [22], [23], Fig. 1. The main substrate of calcineurin in T-cells is the phosphorylated transcription factor NFAT. NFAT dephosphorylation by calcineurin is required for the nuclear

Assessment of CNI therapy by NFAT-regulated gene expression

A PD monitoring assay is only useful if it provides reliable and valid results and if it is easy to perform. Today, gene expression assays are established and used in daily routine. Analysis of gene expression provides reliable and valid results and is used widely in medical diagnostics [32]. Even in PD monitoring assays assessment of gene expression has been used in several studies and concerning several targets [19], [20]. The available technique of real-time PCR provides a fast, highly

NFAT-regulated gene expression: PK/PD studies

An inverse correlation between drug blood concentration and expression of NFAT-regulated genes has been observed in CsA and in Tac treated patients [31], [40]. The highest inhibition of gene expression occurred at the time of the maximum blood concentration 2-h after CsA intake or about 1.5-h after Tac intake. Since the maximal inhibition of NFAT-regulated genes correlates with the peak drug concentration, one might suggest that CsA C2 or Tac C1.5 level reflects immunosuppressive activity of

Clinical studies — efficacy and safety in CsA treated renal allograft recipients

It is obvious that a PD monitoring assay is only useful if a clinical benefit is confirmed. Despite the various methods described for the PD monitoring of CNIs in the literature, until now none of these assays has been implemented in clinical practice. Several clinical studies affirmed the approach of residual NFAT-regulated gene expression as a useful tool with potential to individualize CNI therapy, Table 1.

One of the first prospective observational trials in stable renal allograft recipients

Clinical studies — efficacy and safety in Tac treated renal allograft recipients

The utility and clinical benefit of pharmacodynamic monitoring of Tac therapy by residual NFAT-regulated gene expression was evaluated in stable renal allograft recipients [40]. The median minimal residual gene expression varied widely from 0% to 138% 1.5 h after oral Tac intake. More than half of the 262 stable renal transplant patients exhibited a residual gene expression below 30% and 25 patients exhibited gene expression above 80%. Tac concentration at 1.5 h, but not Tac trough concentration

NFAT-regulated gene expression in other solid organ transplantation

In addition, PD monitoring by residual NFAT-regulated gene expression has been evaluated in other solid organ transplantation like heart and liver allograft recipients with CsA therapy [41], [53]. In the pilot study a strong inhibition of all three NFAT-regulated genes, IL-2, IFNγ and GM-CSF could be shown 2 h after drug intake in kidney, heart and liver transplant patients [31]. Corresponding to the daily CsA dosage heart transplant patients (2.9 (1.4–6.1) mg/kg CsA per day) demonstrated the

NFAT-regulated gene expression in special patient cohorts

In addition, assessment of NFAT-regulated gene expression has been evaluated in pediatrics and senior renal allograft recipients.

In an observational study it could be confirmed that pediatric renal allograft recipients with recurrent infections presented a significantly stronger inhibition of NFAT-regulated gene expression (18.2%) than patients without recurrent infections (31.7%; p = 0.012) [38]. Multivariate regression analysis showed that besides patient age residual NFAT-regulated gene

Conclusions

Several cross-sectional and prospective observational clinical studies in different patient cohorts indicate NFAT-regulated gene expression as promising tool to individualize CsA- and Tac-based immunosuppression. Quantitative analysis of NFAT-regulated gene expression provides a reliable quantification of functional immunosuppression at the target cell levels in solid organ recipients. Especially, beneficial effects concerning adverse events like recurrent infections or malignancies have been

Conflict of interest

None of the authors has any conflicts of interest.

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