Progress in tacrolimus monitoring in organ transplantation : a multidisciplinary approach

Tacrolimus (Tac), a macrolide lactone produced by the fungus Streptomyces tsukubaensis, is nowadays the primary immunosuppressive drug used in organ transplantation. As cyclosporine, Tac is a calcineurin inhibitor preventing cellular rejection through the selective inhibition of interleukin-2 production by T-cells. Immunosuppressive agents are critical dose drugs; this means they have a narrow therapeutic index, exhibiting the desired therapeutic effect with acceptable tolerability only within a narrow range of blood concentrations. Furthermore, they exhibit a high degree of both inter- and intra-individual pharmacokinetic and -dynamic variability, increasing the risk of therapeutic failure if these agents are used at uniform doses in all patients. Hence, therapeutic drug monitoring (TDM) and dosage individualisation are highly recommended to reduce the occurrence of adverse events and to optimise patients outcomes. Practically, trough blood concentrations have been widely used as guidance for Tac dose individualisation. If monitoring Tac trough blood concentrations has contributed to improve efficacy and reduce the toxicity of Tac, the relationship between these concentrations and graft rejection is still unclear. Indeed, incidence of graft rejection remains hardly predictable based on single trough concentrations, underlying the need to find and validate additional markers of efficacy. The first aims of this work were to identify new biomarkers more closely linked to the occurrence and the severity of rejection, than blood concentrations. We therefore focused our research on tacrolimus concentrations in transplanted tissue and in peripheral blood mononuclear cells (PBMCs), a blood compartment enriched in lymphocytes. The first step was to develop and validate analytical methods for the detection and the quantification of tacrolimus in these unconventional matrices. These methods had to be sensitive enough to allow low tacrolimus concentrations detection and quantification. Subsequently, we have investigated the influence of genetic polymorphisms of biotransformation enzymes (CYP3A5 and CYP3A7), or their regulatory proteins as well as several transport proteins (P-gp, MRP2, OATP-C) on the tacrolimus pharmacokinetics and, more specifically on tissue and PBMCs concentrations. The final goals of this thesis are to propose new approaches to improve tacrolimus therapeutic drug monitoring. The first step of this work was to validate analytical methods to measure tacrolimus in the hepatic tissue and in PBMCs. These methods have been developed on LC-MS/MS after liquid-liquid extraction. After the optimisation of these methods, they were validated according to the FDA guidelines. In agreement with the ethic committee of our university, and after obtaining informed consent from patients, three major clinical trials were initiated: two in liver transplantation (LT-1, LT-2) and one in kidney transplantation (KT-1). The first study, LT1, in liver transplantation involved 146 adult recipients under tacrolimus monotherapy. Each of these patients underwent a protocol biopsy of their graft 7 days post transplantation for both histological (Banff scoring) and analytical (tacrolimus dosage) purposes. Tacrolimus trough blood levels were daily monitored. Based on the Banff score, blood and intra-hepatic tacrolimus concentrations have been compared as marker of efficacy. Donors have been genotyped for 14 genetic polymorphisms for genes coding for CYP3A5, CYP3A7, P-gp, MRP2, OATP-C and PXR. Then, the influence of these polymorphisms on blood and tissue tacrolimus concentrations was investigated. This study demonstrated that biopsy proven rejections (Banff scores > 6) were characterised by a significant lower intra-graft tacrolimus concentrations when compared to those observed in absence of rejection (Banff scores A and 2677G>T/A single nucleotide polymorphisms (SNPs) appeared to reduce the activity of P-gp on Tac. In the second study, LT-2, performed on 30 liver recipients, we investigated the relationship between PBMCc trough concentrations at day 1, 3, 5, and 7 post liver transplantation and the incidence and the severity of rejection episodes. As in LT-1, patients underwent a protocol biopsy 7 days post-transplantation, for both analytical and histological purposes, and tacrolimus trough blood levels were daily monitored. All of these parameters (intra-hepatic, PBMCs, and trough blood tacrolimus concentrations) were compared to the Banff score as marker of efficacy. Tissue and PBMCs levels determined by liquid-chromatography tandem mass-spectrometry, at days 3, 5 and 7 displayed good correlation with day 7 liver Banff rejection score, whereas mean blood levels and day-1 PBMCs levels did not. Clinically significant rejection (Banff > 6) was characterised by significant lower mean TAC PBMCs concentrations at day 3, 5 and 7 post-transplantation and by lower tissue concentrations. TAC tissue levels are significantly correlated with TAC PBMCs levels from day 5 post transplantation. This study suggests that TAC PBMCs levels could be a marker of immunosuppression efficacy in the early phase after LT. The last clinical trial involved 96 adult kidney recipients. All patients were under tri-therapy (tacrolimus, mycophenolate, and steroids). In this study we investigated the influence of recipient genetic polymorphisms of genes coding for CYP3A5 and P-gp on tacrolimus PBMCs and trough blood concentrations in the early phase after transplantation and at steady-state (one month after transplantation). Tacrolimus dose requirement (based on blood therapeutic drug monitoring) was higher among patients expressing CYP3A5 compared with patients who did not. Furthermore, expression of CYP3A5 significantly influenced Tac dose-adjusted PBMCs concentrations. In contrast, ABCB1 polymorphisms significantly influenced Tac PBMCs concentrations, whereas their impact on blood concentrations seemed negligible. Among these ABCB1 polymorphisms, the 1199G>A, 3435C>T and 2677G>T/A SNPs appeared to reduce the activity of P-gp, increasing Tac PBMCs concentrations. Increased Tac intracellular concentrations should contribute to the enhancement of the immunosuppressive status and to the prevention from rejection. The recipient’s genotyping for ABCB1 polymorphisms would be therefore useful to better individualise Tac therapy in renal transplantation. In conclusion, this work identified and emphasised the interest of two new matrices for tacrolimus determination: intra-graft and PBMCs tacrolimus levels. These compartments appear to be more significantly related to the occurrence and the severity of rejection episodes than trough blood concentrations. Nevertheless, it should be stressed that the association found between liver-tissue Tac concentration and the incidence or severity of liver rejections may not anymore be relevant after kidney transplantation. Indeed, since tacrolimus is characterised by a well established nephrotoxicity, its renal-tissue concentration might be more closely associated to side effects than to immunological efficacy. Regarding this consideration, and the fact that PBMCs tacrolimus concentrations could be considered as the active concentrations (at the pharmacological site of action), we believe that PBMCs tacrolimus could be a more relevant matrix for tacrolimus efficacy management. We confirmed that CYP3A5 intron 3 polymorphism is significantly associated with Tac dose requirement in the early phase post-transplantation. Furthermore, we also reported a higher influence of ABCB1 polymorphisms on Tac intra-hepatic and PBMCs concentrations, than on Tac blood concentrations. Both ABCB1 1199A and ABCB1 3435T allele led to an increase in Tac PBMCs concentrations, most likely due to a lower P-gp activity. Hence, it appears likely that these polymorphisms are directly involved in the Tac immunosuppressive activity. Taking these results into account and given the importance to rapidly achieve a well balanced immunosuppressive status, we could postulate that a pre-transplantation recipient’s CYP3A5 and ABCB1 pharmacogenetic testing, could contribute to a better individualisation of immunosuppressive therapy. The measurement of Tac within the PBMCs is long and tedious, and its short-term clinical implementation appears unlikely. However the findings reported here may shed new light in the optimisation of Tac therapy, leading either to the analytical improvement of such determinations (through industrial partnerships for instance), or to the development of a multivariate modelling able to predict PBMCs concentrations based on different co-variables (e.g. blood concentrations, genetic polymorphisms, type of graft, age, etc...).(FARM 3) -- UCL, 201