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International audience; BACKGROUND:We sought to evaluate, in patients on a liver transplantation waiting list, potential biomarkers of the base calcineurin pathway activity with use of a new model of nonstimulated peripheral blood mononuclear cells (PBMC) and ex vivo response to tacrolimus (TAC).METHODS:The calcineurin pathway activity was explored ex vivo in stimulated and nonstimulated PBMC from 19 patients. The inhibition of NFATC1+ translocation to PBMC, expression of intracellular IL-2, and membrane CD25 in different T-cell subsets were measured by multiparametric flow cytometry before and after exposure to TAC. We also studied the influence on the individual response of polymorphisms in 3 key genes of the calcineurin pathway: PPIA, PPP3CA, and IL2RA.RESULTS:All pharmacodynamics profiles closely fitted an I/Imax sigmoid model. Interindividual variability was higher in nonstimulated than in stimulated conditions, as well as in the presence of TAC. IL-2+CD8+ cells at TAC Imax showed the highest interindividual variability, suggesting its usefulness as a biomarker of individual TAC effects integrating many different sources of regulation and variability. Moreover, in the absence of TAC, patients with end-stage liver disease exhibited lower NFATC1 translocation and T-cell activation than healthy volunteers from a previous study under similar conditions. Multivariate statistical analysis showed strong and significant associations between TAC pharmacodynamic parameters and 2 polymorphisms in the gene-coding cyclophilin A (rs8177826 and rs6850).CONCLUSIONS:We show the feasibility of using nonstimulated PBMCs to explore the calcineurin pathway under more physiologic conditions and point toward potential biomarkers for TAC pharmacodynamic monitoring.

International audience

International audience; OBJECTIVES: Different associations between single nucleotide polymorphisms (SNPs) in cellular target, metabolism enzymes or transport proteins, and biopsy-proven acute rejection (BPAR) or adverse events have been reported in transplant patients receiving mycophenolate mofetil. This work aimed to study these in patients on enteric-coated mycophenolate sodium (EC-MPS). PATIENTS AND METHODS: The study included 189 renal transplant patients from the DOMINOS trial. Fifteen SNPs in IMPDH2, IMPDH1, ABCC2, SLCO1B3, UGT1A8, UGT1A9, UGT2B7, CYP2C8, HUS1, and IL12A were genotyped in all patients. Associations between SNPs and the first event of BPAR or diarrhea were investigated using multivariate logistic regressions. Associations between SNPs and leukopenia or anemia at nine different visits between days 0 and 190 after transplantation were studied using time-dependent Cox proportional hazards regression models. RESULTS: Multivariate analyses showed that the CYP2C8 rs11572076 wild-type genotype was associated significantly with a lower risk of leukopenia [GG vs. GA: hazard ratio (95% confidence interval) 0.14 (0.03, 0.59), P=0.00783]. Higher EC-MPS doses and the UGT2B7 c.-840 G>A variant allele were associated with an increased risk of anemia [EC-MPS per unit dose increase: 1.004 (1.003, 1.005), P

International audience; BACKGROUND: Recent studies have identified new candidate polymorphisms in the genes related to CYP3A activity or calcineurin inhibitor dose requirements in kidney transplant recipients. These genes and polymorphisms are CYP3A4 (cytochrome P450, family 3, subfamily A, polypeptide 4) (rs35599367-C>T; *22); POR [P450 (cytochrome) oxidoreductase] (rs1057868-C>T; *28); and PPARA (peroxisome proliferator-activated receptor alpha) (rs4253728-G>A). We investigated the impact of these polymorphisms on sirolimus (SRL) in vitro hepatic metabolism, SRL trough concentrations (C0), and SRL adverse events in kidney transplant recipients. METHODS: The clinical study included 113 stable kidney transplant patients switched from a calcineurin inhibitor to SRL (SRL C0 measured at 1, 3, and 6 months thereafter). We investigated SRL metabolism in vitro using human liver microsomes derived from individual donors (n = 31). Microsomes and patients were genotyped by use of Taqman® allelic discrimination assays. The effects of polymorphisms and covariates were studied using multilinear regression imbedded in linear mixed-effect models or logistic regressions. RESULTS: In vitro, the CYP3A4*22 allele resulted in approximately 20% lower metabolic rates of SRL (P = 0.0411). No significant association was found between CYP3A4, CYP3A5, or PPARA genotypes and SRL dose, C0, or C0/dose in kidney transplant patients. POR*28 was associated with a minor but significant decrease in SRL log-transformed C0 [CT/TT vs CC, β = -0.15 (0.05); P = 0.0197] but this did not have any impact on the dose administered, which limited the relevance of the finding. After adjustment for nongenetic covariates and correction for false discovery finding, none of the single-nucleotide polymorphisms tested showed significant association with SRL adverse events. CONCLUSIONS: These recently described polymorphisms do not seem to substantially influence the pharmacokinetics of SRL or the occurrence of SRL adverse events in kidney transplant recipients.

This is a non-final version of an article published in final form in Pharmacogenetics and Genomics; International audience; BACKGROUND: The mammalian target of rapamycin (m-TOR) inhibitor sirolimus is an immunosuppressive drug used in kidney transplantation. m-TOR binds with Raptor and phosphorylates p70S6 kinase, a protein involved in numerous cell signalling pathways. We examined the association of candidate polymorphisms in m-TOR, Raptor and p70S6K, sirolimus dose and exposure, and other time-independent as well as time-dependent covariates, with sirolimus-induced adverse events in kidney transplant recipients. METHODS: This study included a first group of 113 patients, switched from a calcineurin inhibitor to sirolimus, and a validation group of 66 patients from another clinical trial, with the same immunosuppressive regimen. The effects of gene polymorphisms and covariates on the total cholesterol, LDL cholesterol, triglycerides, haemoglobin, cutaneous adverse events, oedemas and infections were studied using multilinear regression, or logistic regression imbedded in linear mixed-effect models. RESULTS: An m-TOR variant haplotype was significantly associated with a decrease in haemoglobin levels in the two populations of patients (discovery group: β=-0.82 g/dl, P=0.0076; validation group: β=-1.58 g/dl, P=0.0308). Increased sirolimus trough levels were significantly associated with increased total cholesterol levels (discovery group: β=0.02 g/l, P

International audience; Cyclosporin A (CsA) is a substrate for cytochrome P450 3A and the efflux transporter P-glycoprotein (P-gp; ABCB1), both abundantly expressed in the kidney. In a long-term follow-up of a cohort of patients who had received kidney transplants between the years 1990 and 2005, we retrospectively investigated the effect of CYP3A4, CYP3A5, and ABCB1 polymorphisms in kidney graft donors on recipients' renal function and risk of subsequent graft loss. DNA samples from 227 donors and clinical data from the 259 respective recipients were analyzed. Graft loss was significantly associated with the presence of the ABCB1 variant haplotype 1236T/2677T/3435T in the donor (1236T/2677T/3435T vs. other haplotypes: hazard ratio = 9.346; 95% confidence interval (CI) (2.278-38.461); P = 0.0019) and with previous episodes of acute organ rejection (hazard ratio = 3.077; 95% CI (1.213-7.812); P = 0.0178). The variant haplotype was also associated with a greater decrease in renal function (homozygotes for TTT -3.047 mlxmin(-1)/year; heterozygotes for TTT -4.435 mlxmin(-1)/year; others -2.186 mlxmin(-1)/year; P = 0.0240). The study showed that the presence of ABCB1 polymorphisms in donors influences long-term graft outcome adversely with decrease in renal function and graft loss in transplant recipients receiving CsA.

International audience; AIM: In renal transplant patients given mycophenolate mofetil (MMF), we investigated the relationship between the digestive adverse events and polymorphisms in the UGT genes involved in mycophenolic acid (MPA) intestinal metabolism and biliary excretion of its phase II metabolites. METHODS: Clinical data and DNA from 256 patients transplanted between 1996 and 2006 and given MMF with cyclosporin (CsA, n = 185), tacrolimus (TAC, n = 49) or sirolimus (SIR, n = 22), were retrospectively analysed. The relationships between diarrhoea and polymorphisms in UGT1A8 (2; 518C>G, 3; 830G>A), UGT1A7 (622C>T), UGT1A9 (-275T>A), UGT2B7 (-840G>A) and ABCC2 (-24C>T, 3972C>T) or the co-administered immunosuppressant were investigated using the Cox proportional hazard model. RESULTS: Multivariate analysis showed that patients on TAC or SIR had a 2.8 higher risk of diarrhoea than patients on CsA (HR = 2.809; 95%CI (1.730, 4.545); P < 0.0001) and that non-carriers of the UGT1A8 2 allele (CC518 genotype) had a higher risk of diarrhoea than carriers (C518G and 518GG genotypes) (HR = 1.876; 95%CI (1.109, 3.175); P = 0.0192). When patients were divided according to the immunosuppressive co-treatment, a significant effect of UGT1A8 2 was found in those co-treated with CsA (HR = 2.414; 95%CI (1.089, 5.354); P = 0.0301) but not TAC or SIR (P = 0.4331). CONCLUSION: These results suggest that a possible inhibition of biliary excretion of MPA metabolites by CsA and a decreased intestinal production of these metabolites in UGT1A8 2 carriers may be protective factors against MMF-induced diarrhoea.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT Tacrolimus is an immunosuppressant agent, largely used in kidney transplantation, with a narrow therapeutic range. Therapeutic drug monitoring of tacrolimus exposure improves the efficacy and toxicity of this drug. Separated Bayesian estimators have been developed to estimate tacrolimus exposure following administration of Prograf® and the prolonged release formulation Advagraf®., WHAT THIS STUDY ADDS A population model was developed to compare the pharmacokinetics of 32 patients treated with Prograf® and 41 treated with Advagraf®. A mixture model and a model using formulation as covariates were developed to describe the bimodal distribution in the absorption rate following Advagraf® administration. Comparison of these two models showed that the nonmixture model was adequate. A single Bayesian estimator was developed to estimate the exposure for both formulations, which is more suitable for clinical practice., AIM To investigate the differences in the pharmacokinetics of Prograf® and the prolonged release formulation Advagraf® and to develop a Bayesian estimator to estimate tacrolimus inter-dose area under the curve (AUC) in renal transplant patients receiving either Prograf® or Advagraf®., METHODS Tacrolimus concentration-time profiles were collected, in adult renal transplant recipients, at weeks 1 and 2, and at months 1, 3 and 6 post-transplantation from 32 Prograf® treated patients, and one profile was collected from 41 Advagraf® patients more than 12 months post-transplantation. Population pharmacokinetic (popPK) parameters were estimated using nonmem®. In a second step, the popPK model was used to develop a single Bayesian estimator for the two tacrolimus formulations., RESULTS A two-compartment model with Erlang absorption (n= 3) and first-order elimination best described the data. In Advagraf® patients, a bimodal distribution was observed for the absorption rate constant (K(tr)): one group with a K(tr) similar to t.