Figure 1: ABCB1 expression analysis by fluorescence, microscopy. (A) Untransfected (CTL) HEK293, (B) transfected with empty plasmid (CTL pcDNA3.1) HEK pcDNA3.1 (C) HEK1199G and (D) HEK1199A cells were stained with anti-ABCB1 antibodies (green fluorescence). DAPI was used to stain nuclei (blue).
Our in vitro research activities have allowed unraveling the mechanistic reasons of decreased cellular transport of tacrolimus, an immunosuppressant, in the presence of a particular genetic variant when a transport protein (ABCB1) is overexpressed in a recombinant cell line model. This observation is in agreement with our previously published clinical data. Our study emphasizes thus the importance of ABCB1 polymorphisms to explain differences in drug response.
In 2013, our group has shown that two SNPs in complete linkage disequilibrium located in the gene of PPARα was associated with a higher risk of developing new onset diabetes after transplantation (NODAT), a common toxic effect observed with tacrolimus. In parallele, Patrice D. Cani (MNUT, LDRI) has shown in his lab that knock-out mice for hepatic Myd88 (Myd88 HKO) under high fat diet were characterized by a decreased production of biliary acids production.
Previous studies also observed that PPARα deficient mice showed enhanced fatty acid oxidation, decreased glucose degradation and increased insulin resistance. Therefore our hypothesis is that the insulin resistance observed in Myd88 HKO is due to the decreased in PPARα activity and might predispose to diabetes development with Tacrolimus (figure2).
To test this hypothesis in collaboration with MNUT (Patrice D. Cani) and LTAP (IREC, Vincent Haufroid), we will study the development of insulin resistance in Myd88 HKO after treatment with tacrolimus compared to wild-type controls (figure 3). In addition, as PPARα has been also associated with a decreased CYP3A4 activity, we will performed a complete PK analysis in these mice to see the loss of Myd88 activity in the liver and the consequent decreased in PPARα activity has an impact on tacrolimus exposure that might in fine explain differences in phenotypes and predisposition to diabetes development.
Patient survival and graft outcome after kidney transplantation have drastically improved in recent decades, mainly because of major improvements in immunosuppressive therapy. However, optimal immunosuppression is difficult to achieve in an individual patient, as the majority of immunosuppressive agents are characterized by highly variable PK and a narrow therapeutic window. The use of immunosuppressive drugs is further complicated by their high toxicity profile. The susceptibility to develop adverse events or experiencing therapeutic failure varies strongly between individuals. An important part of this variability in drug response is thought to be the consequence of substantial inter-individual differences in drug metabolism. Some patients have relatively fast drug clearance, while others exhibit a slower drug elimination rate. This variation in drug clearance is of importance, since it might be related to an increased risk of under- or overexposure, which can ultimately lead to a higher frequency of acute graft rejection or adverse events. A collaboration with the Erasmus MC (Rotterdam, The Nethelands) and the KUL (Louvain, Belgium) has permitted to highlight that carriership of genetic variants in the Cytochrome P450-mediated drug metabolism is associated with a rough 30% reduction in in vivo metabolic activity. In addition, it was demonstrated that immunosuppressant steady-state drug clearance was equally decreased with the presence of this genetic variant, leading to 50% lower dose requirements. In addition to genetic differences, drug interaction can also impact on therapy outcome. In a hepatic pediatric transplant cohort, we have set up a popPK model to estimate the drug clearance and to decipher how individual characteristics explain the inter-patient clearance variability. Not only had we confirmed that patient genotypes correlate with drug metabolism but also that concomitant use of CYP3A4 inhibitors impact on drug exposure. All those important discoveries have led our group to propose new dosage guidelines (see figure 4 above) that can be useful in the frame of pre-emptive genotyping and dosage adjustment prior to transplantation, before the initiation of immunosuppressive therapy. In theory, this would lead to a reduced risk of under or over-exposure to the drug in every patient and in fine, to decreased risk of undesirable therapy outcome. In a more recent study, we also found that the presence of this Cytochrome P450 genetic variant was associated with the susceptibility of developing cancer with long term immunosuppressive drug treatment, emphasizing the importance of our discovery and that it appears as really imperative to take genetic predisposition into account regards to therapy outcome.
In 2016, our new hypothesis with the implication of PPARα in the clinical response with tacrolimus (see above, animal studies and figure 2), we will work in collaboration with Erasmus MC (Rotterdam) to see if Myd88 genotypes have an impact on the risk of developing NODAT. To that purpose, a total of 101 patients from the IMPACT study have been followed after transplantationand the need of antidiabetic medication after start of therapy has been recorded and is assumed to reflect the development of NODAT. To date, we have selected 5 SNPs in MYD88 that we will screen in the entire population. They all have a minor allelic frequency of >5% and at least one citation in Pubmed with a potential clinical impact.