Monitoring Calcineurin Inhibitor Therapy: Localizing the Moving Target (2010)

The introduction of cyclosporine 30 years ago created a revolution in transplantation medicine, and its use made transplantation the preferable therapeutic intervention for end-stage kidney, liver, heart, and lung disease. In the late 1980s, another immunosuppressant, tacrolimus, was added to the armamentarium of immunosuppressive drugs in transplantation medicine. The clinical success of these drugs is evident, as cyclosporine or tacrolimus are now present in the immunosuppressive protocol of more than 94% of kidney transplant recipients. Cyclosporine and tacrolimus are calcineurin inhibitors (CNIs) and act by inhibiting the phosphatase activity of calcineurin through binding of cyclosporine-cyclophilin and tacrolimus-FKBP12 complexes to calcineurin, which is an essential enzyme in the nuclear factor of activated T-cell (NFAT) pathway involved in T-cell activation. However, calcineurin and NFAT are not T-cell specific, and the use of cyclosporine and tacrolimus is associated with toxicity beyond immunosuppression, such as metabolic alterations (hyperlipidemia and diabetes mellitus), hypertension, nephrotoxicity, neurotoxicity, and cosmetic changes (gingival hypertrophy and hypertrichosis). To minimize CNI exposure and avoid these side effects, CNIs are mostly used in combination with other drugs (corticosteroids, mycophenolate, sirolimus, everolimus, and so forth) (1). The main goal after transplantation is to maintain a reasonable balance between efficacy (avoid rejection), side effects of general immunosuppression (malignancies and infections), and specific toxicity of the immunosuppressive agents used. This is an important issue for cyclosporine and tacrolimus, which are characterized by a poor dose-level correlation, an unpredictable level-effect association, and an unclear level-toxicity relation (1). The correlation between very high peripheral blood levels and toxicity was evident in the early clinical studies with cyclosporine and was confirmed by the first dose-finding studies with tacrolimus. After it became clear that cyclosporine and tacrolimus are drugs with a narrow therapeutic window, great care was given to keep their blood levels within the preset target ranges. Nevertheless, therapeutic drug monitoring of CNIs is not the entire solution. Side effects are still observed with drug levels below the standard therapeutic range (2), while high peripheral blood levels of cyclosporine or tacrolimus are not fully protective against rejection. The levels necessary to provide sufficient immunosuppression overlap with the levels that can cause side effects. The therapeutic window of the CNI levels is, therefore, even nonexisting for many patients. In addition, there has been much debate on the best strategy for CNI level monitoring. With these problems, it could seem surprising that the CNIs are still used in clinical practice. However, there is a lack of good alternative treatment strategies, and recent data show a benefit of low level CNI use over other drugs that are less efficient at decreasing the risk for rejection and are associated with worse graft survival (2). Until we get access to newer agents or strategies for better treatment of transplant recipients, we need to further improve our use of the currently available immunosuppressive protocols, which often include CNIs. Apart from the issues with CNI pharmacokinetics, much of the problems with the clinical use of cyclosporine and tacrolimus are caused by their unpredictable pharmacodynamics and the variable inhibitory effects of the CNIs on T-cell responses. Direct pharmacodynamic monitoring of CNI-based therapy could, therefore, be the answer, with several levels of information, including clinical and histologic background analysis, T-cell response assays, and specific monitoring tools based on the action mechanism of CNIs. The most obvious and currently used clinical marker of CNI efficacy is the routine measurement of graft function and graft histology. Rapid deterioration of graft function is often a clear indication of ongoing problems and failure of the immunosuppressive regimen, and the individualized treatment targets can be based on the patient's and his graft's medical history. This is the way most centers nowadays tailor the immunosuppressive therapy target levels to the individual patient, with reasonable clinical success and acceptable graft and patient outcome. Within this same concept, some centers use systematic “protocol” biopsies to guide their immunosuppressive regimens and base the clinical decisions partly on the results of the subclinical histologic appearance of the transplanted organs. Given the broad range of attacks to the transplanted organs (immune-mediated-like rejection, or nonimmune phenomena-like infection and drug toxicity), histologic reading of biopsies is probably one of the most reliable tools to get a detailed view on both efficacy and toxicity of the immunosuppression used. The implementation of a protocol biopsy program is, however, associated with invasive procedures and considerable organizational problems, and the studies that demonstrate a clinical benefit are currently lacking (3). Noninvasive indirect assays that measure immune responses represent a second level of pharmacodynamic monitoring of immunosuppressive therapy. The most straightforward matrix would be peripheral blood or lymphocytes, although recent studies also show promising results with urine samples. As most of the currently used immunosuppressive drugs inhibit T-lymphocyte proliferation, several groups have studied T-cell proliferation assays and activation markers. Both in the laboratory as in a clinical setting, a broad range of techniques are being used to monitor the degree of immunosuppression at the individual level, going from flow cytometry over enzyme-linked immunosorbent spot assays to single-gene expression studies and whole-genome transcriptomics with microarray technology. A clear strength of these approaches is that not one single drug is evaluated, but the combination of the immunosuppressive drugs used. Therefore, these techniques could not only be relevant for the currently used immunosuppressive protocols but also be suitable for future protocols with newer therapeutic agents. Nevertheless, large prospective interventional studies have not yet been performed, and the optimal strategy for T-cell monitoring is still under debate (4). Finally, several groups have developed more specific assays for pharmacodynamic monitoring of CNIs. These assays are based on the well-known action mechanisms of cyclosporine and tacrolimus, through measurement of calcineurin phosphatase activity in peripheral blood or through measurements of the levels of interleukin-2 production or the levels of cytokines such as interferon-γ or tumor necrosis factor-α. Many of these tools have been evaluated in small clinical studies and support the concept of pharmacodynamic monitoring. However, larger and preferably also interventional studies are needed to confirm their clinical usefulness. A major drawback with these tools is that only the immunosuppressive actions of the CNIs are measured, and much less the toxic characteristics of these drugs. Also, the monitoring of only one drug will never be sufficient to fully determine a patient's risk, and the other drugs included in the immunosuppressive regimen will always remain a major interfering factor. The study by Sommerer et al. (5) in the current issue of Transplantation reports on such a specific pharmacodynamic monitoring tool for CNIs in a clinical setting. The use of an NFAT-regulated gene expression assay in a large patient cohort allowed the investigators to demonstrate the correlation between this assay and both efficacy (biopsy-proven acute rejection) and side effects related to the level of immunosuppression (recurrent infections). The same group previously reported on the potential clinical benefits of this tool in patients treated with cyclosporine, although they point to important differences in the results between the two currently used CNIs. As it is not excluded that many of the side effects of the CNIs are also mediated through the NFAT-calcineurin pathway, it is possible that in larger studies also an association will be found between the NFAT-regulated gene expression assay and the specific CNI toxicity. These studies by the Heidelberg group are very valuable, and the NFAT-regulated gene expression assay, with further validation, may provide clinical utility for pharmacodynamic monitoring of both cyclosporine and tacrolimus. Nevertheless, these studies leave many important questions unanswered. Although multivariate analyses showed that the association of the NFAT-regulated gene expression with rejection and infection is significant, independent of time, the study was only performed in a cross-sectional group of transplanted patients. The best timing to use the assay is, therefore, not elucidated. Furthermore, it is not known whether different results need to be adapted for differences in recipient age. The differences observed between a cyclosporine-based regimen and a tacrolimus-based regimen also demonstrate the dependence of this tool on different immunosuppressive regimens. Therefore, the results of this study cannot be simply generalized to different immunosuppressive protocols. Is this tool only applicable in stable patients, or is it also relevant immediately after transplantation, and what is its value in patients with difficult clinical problems, such as interval viral infection, rejection, posttransplant lymphoproliferative disorder, that is, the groups of patients most in need of well targeted therapy? How stable is the NFAT-regulated gene expression over time in the same patient and how often do we need to use this tool in clinical practice? Could this tool help us to decide which patients to switch to other immunosuppressive regimens and which patients are best treated with a CNI-based protocol? To answer these questions and to confirm or discard the clinical usefulness of pharmacodynamic monitoring in routine clinical transplantation, we need larger and more systematic studies. Pharmacodynamic tools should routinely be implemented in all clinical studies assessing new or comparing different immunosuppressive regimens in transplantation. Perhaps, it is also time to consider larger interventional trials that implement reasonable pharmacodynamic tools for guidance of therapeutic decisions. This could be based on aforementioned tools such as NFAT-regulated gene expression, T-cell monitoring or gene expression assays, or on the subclinical histologic evolution in protocol biopsies. Without the proper tools to assess the efficacy and toxicity of the immunosuppressive agents we use, we will not be able to redefine any target, especially not when our targets are moving over time.