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Background: The type 1 plasminogen activator inhibitor (PAI-1) is involved in the development of fibrosis, and its intrarenal expression is increased in interstitial fibrosis and tubular atrophy (IFTA). Moreover, a 4G/5G polymorphism of the PAI-1 gene has been described associating 4G haplotype with higher PAI-1 plasma activity. We investigated the relationship between the donor and recipient PAI-1 polymorphism and kidney graft survival., Methods: The PAI-1 genotype was determined for both the 304 donors and the 337 corresponding recipients. In recipients, PAI-1 antigen levels were also determined. We compared 4G/4G donors versus donors with other genotypes., Results: Donor or recipient genotype did not influence the PAI-1 plasma level in recipients. Actuarial kidney graft survival was significantly reduced in the 4G/4G donor group (107 months versus 147.5 months, P = 0.013), while recipient PAI-1 genotype did not show any influence on graft survival. Moreover, graft loss due to IFTA proved significantly higher in the 4G/4G donor group (13% versus 6%, P = 0.03). Multivariate analysis showed that the significant independent variables associated with graft loss were the donor 4G/4G genotype, acute clinical rejection and donor age., Conclusion: Our study suggests that donor PAI-1 polymorphism influences kidney graft survival and that the donor 4G/4G genotype is an independent risk factor for graft loss. Prospective studies are needed to confirm these results.

Mycophenolic acid (MPA) is metabolized primarily by glucuronidation to form the biologically inactive 7-O-glucuronide conjugate (MPAG), which is the major urinary excretion product. MPA is also converted to acyl-glucuronide metabolite (AcylMPAG), which has been suggested to be involved in the generation of MPA-related adverse events such as diarrhea or leucopenia. This conversion of MPA to AcylMPAG is catalyzed by UDP-glucuronosyltransferase 2B7 (UGT2B7). We studied the impact of the -840G>A polymorphisms in the UGT2B7 gene on the pharmacokinetics of AcylMPAG. We also investigated whether the plasma concentrations of AcylMPAG are correlated with MPA-related toxicity to further evaluate its potential clinical significance. In a randomized, controlled trial, comparing fixed-dose mycophenolate mofetil (MMF) with concentration-controlled MMF therapy, patients undergoing renal transplantation were treated with a calcineurin inhibitor, MMF, and corticosteroids. Informed consent was obtained from 332 patients for genotyping. In all patients, blood samples were drawn (three samples within the first 2 hours after administration) on Day 3, Day 10, Week 4, and Months 3, 6, and 12 to measure MPA and AcylMPAG plasma concentrations. The pharmacokinetics of AcylMPAG were correlated with the -840G>A single nucleotide polymorphism (SNP) in the UGT2B7 gene. Heterozygosity for the -840G>A SNP in the UGT2B7 gene was found in 145 patients (145 of 332 [44%]) and 93 (93 of 332 [28%]) patients were homozygous for the -840G>A allele. No difference was found in the dose-normalized AcylMPAG trough (C0) levels and dose-normalized AcylMPAG areas under the concentration-time curve (AUCs) at each visit between carriers and noncarriers of the -840G>A SNP. Also, metabolic ratios, expressed as AcylMPAG/MPA and AcylMPAG/MPAG, were not related to UGT2B7 genotype. The dose-normalized AcylMPAG-C0 and AcylMPAG AUC were higher in the cyclosporine-treated group compared with the tacrolimus-treated patients at each visit. There was no difference in AcylMPAG concentrations (trough or AUC) or AcylMPAG/MPAG ratio between patients with compared with patients without diarrhea. None of the -840G>A UGT2B7 SNPs was disproportionately present among the patients with diarrhea. There was a higher incidence of diarrhea in tacrolimus-treated patients [26 of 163 (16.0%)] compared with cyclosporine-treated individuals [five of 51 (9.8%)], although AcylMPAG concentrations were lower in tacrolimus-treated patients. In this study, we have found no influence of the -840G>A UGT2B7 SNP on AcylMPAG exposure in patients undergoing renal transplantation. There also was no association between this variant genotype and the incidence of diarrhea or leucopenia, two adverse events for which a role for AcylMPAG has been suggested.

Objective: Patients expressing the tacrolimus-metabolizing enzyme, cytochrome P450 (CYP) 3A5, require more tacrolimus to reach target concentrations. We studied the influence of the CYP3A5(*)3 allele, which results in the absence of CYP3A5 protein, on tacrolimus dose and exposure, as well as the incidence of biopsy-proven acute rejection (BPAR) after renal transplantation., Methods: A total of 136 patients participating in a prospective, randomized-controlled clinical trial with the primary aim of comparing the efficacy of a fixed-dose versus a concentration-controlled mycophenolate mofetil immunosuppressive regimen, were genotyped for CYP3A5(*)3. The patients described herein, participated in a pharmacogenetic substudy and were all treated with mycophenolate mofetil, corticosteroids and tacrolimus. Tacrolimus predose concentrations (C(0)) were measured on day 3 and 10, and month 1, 3, 6 and 12., Results: Compared with CYP3A5(*)3/(*)3 individuals (n=110), patients carrying at least one CYP3A5(*)1 (wild-type) allele (CYP3A5 expressers; n=26) had a lower tacrolimus C(0) on day 3 only (16.6 versus 12.3 ng/ml, respectively), whereas dose-corrected tacrolimus C(0) were significantly lower in the latter group at all time points. After day 3, the overall daily tacrolimus dose was 68% higher in CYP3A5 expressers (P<0.001). The incidence of BPAR was comparable between CYP3A5 expressers and nonexpressers (8 versus 16%, respectively; P=0.36)., Conclusion: We conclude that patients expressing CYP3A5 need more tacrolimus to reach target concentrations and have a lower tacrolimus exposure shortly after transplantation. This delay in reaching target concentrations, however, did not result in an increased incidence of early BPAR and therefore, genotyping for CYP3A5 is unlikely to improve short-term transplantation outcome.

Introduction: The polymorphic enzyme UGT2B7 metabolizes mycophenolic acid into acyl-mycophenolic acid-glucuronide (AcMPAG), a presumably toxic metabolite. This study aimed at investigating in vitro and in vivo the impact on AcMPAG production of: (i) the UGT2B7 gene G-842A single nucleotide polymorphism, in complete linkage disequilibrium with most other known single nucleotide polymorphisms in the promoter region of this gene and with the C802T single nucleotide polymorphism in exon 2 (UGT2B*2); and (ii) of the other immunosuppressants given to renal transplant patients in association with mycophenolate mofetil., Methods: We compared the production of AcMPAG by human liver microsomes genotyped for the UGT2B7 G-842A and C802T single nucleotide polymorphisms, and plasma AcMPAG concentrations in genotyped renal transplant patients administered mycophenolate mofetil associated with sirolimus (n=40), tacrolimus (n=24) or cyclosporin (n=28) and decreasing doses of corticosteroids, over the first 3 months after transplant. The effect of corticosteroids was also investigated in vitro using rats' liver microsomes., Results: The two polymorphisms studied were in complete reverse linkage disequilibrium. AcMPAG production was 1.25 and 1.56-fold higher in G-842A and -842AA human liver microsomes, respectively, compared with GG-842 human liver microsomes (P=0.01). Enzyme kinetics showed 1.4 and 3.7-fold higher Vmax in the respective pools of human liver microsomes. Km values were 0.20, 0.25 and 0.44 mmol/l for the GG-842, G-842A and -842AA genotypes, respectively. This clear increase in Vmax is in favor of the implication of the promoter region polymorphisms, whereas the slighter increase in Km might be due to the UGT2B7*2 single nucleotide polymorphism. Consistently, the UGT2B7 genotype significantly influenced AcMPAG area under the curve (AUC0-9 h)/dose in patients on sirolimus at months 1 and 3 after transplant (P=0.04 for both). No effect was observed in patients on tacrolimus and possibly also on cyclosporin, maybe owing to pharmacokinetic interaction with mycophenolate. AcMPAG production was increased in corticosteroid-induced rat liver microsomes, consistent with the observed in-vivo decrease of mycophenolic acid metabolites AUC0-9 h/dose with time after transplant., Conclusion: Both UGT2B7 polymorphisms and co-medications significantly influenced AcMPAG production, but cyclosporin and tacrolimus hindered the phenotypic impact of this trait.

Background: In the multicenter, open-label Myriade study, renal transplant patients were randomized to early cyclosporine microemulsion (CsA-ME, day 0) or delayed CsA-ME (day 6) with enteric-coated mycophenolate sodium (EC-MPS), steroids and interleukin-2 receptor induction. One-yr results have been published previously. We now report the results of an extension study in which patients were followed up for a period of three yr post-transplant., Methods: All patients completing the one-yr core study on-treatment were eligible to enter the extension study., Results: Of the 203 patients, 153 completed the core trial on-treatment; 144 (94%) entered the extension study with a minimum follow-up of one yr (73 early CsA-ME, 71 delayed CsA-ME). In 75% of patients receiving EC-MPS during the extension, the recommended dose was administered (1440 mg/d). Median creatinine clearance remained constant (57 mL/min) at 12, 24 and 30 months post-transplant and was similar in the early and delayed CsA-ME groups as well as in subpopulations with or without delayed graft function. One patient in the early CsA-ME group died. No grafts were lost. The incidence of BPAR from time of transplant to the end of the extension study was 17% (24/139). Seven patients (5%) discontinued the extension study prematurely because of adverse events., Conclusion: These results suggest that a regimen of CsA-ME, EC-MPS and steroids results in excellent survival rates with stable renal function over a mean follow-up of 30 months. Immediate introduction of CsA-ME has no deleterious effect on long-term renal function, even among patients with delayed graft function.

Monitoring of human herpesvirus-6 (HHV-6) reactivation is important, especially in immunocompromised patients such as transplant recipients. Reverse transcription PCR (RT-PCR) is a useful method to distinguish between latent and active infection. Here, a RT-nested PCR coupled with a colorimetric plate hybridization assay was established to detect HHV-6 types A and B U79/80 mRNAs. After confirming the reliability of the assay on HHV-6 cultures, it was applied to the detection of HHV-6 reactivation after renal (27 patients), bone marrow (14 patients) or heart (7 patients) transplantation. A total of 206 blood samples were tested from renal (137), bone marrow (58) and heart (11) transplant recipients. U79/80 mRNAs were found in 32 samples that were considered as indicative of HHV-6 reactivation: 15, 13 and 5 from kidney, bone marrow and heart transplant recipients, respectively. Finally, U79/80 mRNA detection was correlated with clinical manifestations including leucopenia, skin rash, graft rejection or dysfunction and diarrhoea.

A roundtable meeting to discuss the use of therapeutic drug monitoring (TDM) to guide immunosuppression with mycophenolate mofetil was held in New York in December 2004. Existing recommendations for the initial months after transplantation were updated. After ensuring adequate levels of mycophenolic acid (MPA, the active metabolite of mycophenolate mofetil) immediately after transplantation, optimal efficacy may require only a few dose adjustments, because intrapatient variability in exposure seems low. Recommendations based on current knowledge were made for posttransplantation sampling time points and for target MPA concentrations. Algorithms for estimating MPA exposure using limited sampling strategies were presented, and a new assay for MPA discussed. It was agreed that because of interpatient variability and the influence of concomitant immunosuppressants, TDM might help optimize outcomes, especially in patients at higher risk of rejection. The value of TDM in the general transplant population will be assessed from large, ongoing, randomized studies.

Human cytomegalovirus (HCMV) and human herpesvirus-6 (HHV-6) are two closely related viruses, which belong to the Herpesviridae family. Following primary infection, they are thought to persist for life as latent forms in mononuclear cells. HCMV and HHV-6 can cause considerable morbidity in immunocompromised individuals, such as transplant patients. A sensitive and specific LightCycler multiplex real-time PCR assay based on fluorescence energy transfer (known as FRET) was developed. This assay, by using two sets of hybridization probes specific for HHV-6 (A and B) and HCMV, can differentiate reliably and quantify simultaneously both viruses in order to diagnose reactivation processes. The assay was optimized and the lower limit of detection for both viruses was determined to be 10 viral genome copies per reaction. Both viruses were quantified in 83 peripheral blood mononuclear cells (PBMCs) and 87 polymorphonuclear leukocytes (PMNLs) collected from 32 transplant recipients. This multiplex real-time quantitative PCR was finally compared with two other quantitation and detection assays used daily in laboratory (PCR DIG detection and antigenemia for HCMV, TaqMan Assay for HHV-6). This technique can be useful for the differentiation and quantitation of HCMV and HHV-6 for monitoring transplant patients.

For ten years, an increase in the number of elderly patients on renal transplant waiting lists has occured. In an attempt to close the widening gap between supply and demand and because the demand for kidneys for younger patients already surpasses the supply, transplant physicians nowadays accept organs from older donors that might have been deemed inappropriate in the past. Programs of age matching between donors and recipients and of dual-kidneys transplantation have emerged. The initial results of these programs are encouraging with excellent patient and graft survival at one and three years.

Objectives: The objectives of the present study were: (i) to analyse the population pharmacokinetics of sirolimus in renal transplant recipients co-administered mycophenolate mofetil, but no calcineurin inhibitor over the first 3 months post-transplantation and study the influence of different potential covariates, including genetic polymorphisms of cytochrome P450 (CYP) metabolic enzymes and active transporters, on pharmacokinetic parameters; and (ii) to develop a Bayesian estimator able to reliably estimate the individual pharmacokinetic parameters and exposure indices in this population., Methods: Twenty-two adult renal transplant patients treated with sirolimus participated in this study. Ninety concentration-time profiles (938 sirolimus whole blood samples) were collected at days 7 and 14, and months 1 and 3 post-transplantation. The population pharmacokinetic study was conducted using the nonlinear mixed effects model software, NONMEM, and validated using both the bootstrap and the cross-validation approaches. Finally, a Bayesian estimator based on a limited sampling strategy was built using the post hoc option., Results: A two-compartment open model with first-order elimination and Erlang's distribution (to describe the absorption phase) best fitted the data. The mean pharmacokinetic parameter estimates were 5.25 h(-1), 218L and 292L for the transfer rate constant, the apparent volume of the central and peripheral compartments, respectively. The CYP3A5*1/*3 polymorphism significantly influenced the apparent oral clearance: mean oral clearance = 14.1 L/h for CYP3A5 non expressers (CYP3A5*3/*3 genotype) versus 28.3 L/h for CYP3A5 expressers (CYP3A5*1/*3 and *1/*1 genotypes). The standard errors of all the parameter estimates were <15%. Maximum a posteriori Bayesian forecasting allowed accurate prediction of sirolimus area under the concentration-time curve from 0 to 24 hours using a combination of only three sampling times (0, 1 and 3 hours post-dose), with a non-significant bias of -2.1% (range -22.2% to +25.9%), and a good precision (root mean square error = 10.3%). This combination is also easy to implement in clinical practice., Conclusion: This study presents an accurate population pharmacokinetic model showing the significant influence of the CYP3A5*1/*3 polymorphism on sirolimus apparent oral clearance, and a Bayesian estimator accurately predicting sirolimus pharmacokinetics in patients co-administered mycophenolate mofetil, but no calcineurin inhibitor.

Background and Objectives: Population pharmacokinetic studies of ciclosporin microemulsion are needed to identify the individual factors influencing ciclosporin pharmacokinetic variability in transplant patients and to design efficient tools for the accurate estimation of ciclosporin overall exposure (area under the plasma concentration-time curve from 0 to 12 hours [AUC12]). In the present retrospective study, a large database of heart, lung (with or without cystic fibrosis) and kidney (both adult and paediatric) transplant patients receiving ciclosporin microemulsion was analysed with the aims of (i) building a population pharmacokinetic model and finding the main covariates linked with ciclosporin microemulsion pharmacokinetic parameters; and (ii) developing a maximum a posteriori probability Bayesian estimator (MAP-BE) to estimate ciclosporin microemulsion pharmacokinetic parameters using a limited-sampling strategy., Methods: 3,072 concentration data from 147 patients (i.e. 309 full pharmacokinetic profiles) were analysed using the nonlinear mixed-effects model program NONMEM. The influence of numerous covariates was tested, and the final model was validated by data splitting. For Bayesian estimation, the best limited-sampling strategy was determined based on the D-optimality criterion, and validation performed in an independent group of 60 patients., Results: The pharmacokinetics of ciclosporin microemulsion were accurately described by a two-compartment model with Erlang distribution for the absorption process. The type of graft and post-transplantation period were identified as significant sources of variability of the absorption parameter. Both apparent volume of the central compartment after oral administration (V1/F) and apparent oral clearance (CL/F) increased with bodyweight. The best limited-sampling strategy for Bayesian estimation was 0 hour, 1 hour and 3 hour post-dose, providing accurate estimation of ciclosporin microemulsion AUC12 in all patients of the test group, with a mean bias of 2.0 +/- 10.5% (range: -19.1% to -21.4% and 95% CI -0.6, +4.7)., Conclusion: Population pharmacokinetic analysis of ciclosporin microemulsion in allograft transplants resulted in the design of a new pharmacokinetic model for ciclosporin microemulsion, identification of significant covariates and the design of an accurate MAP-BE based on three blood concentrations and these covariates.

Introduction: Therapeutic drug monitoring of mycophenolate mofetil is recommended because of the interindividual variability in the exposition to its active moiety, mycophenolic acid. However, most of the pharmacokinetic studies involved patients cotreated with cyclosporine (INN, ciclosporin)., Methods: We analyzed the pharmacokinetics of mycophenolic acid in 13 renal graft recipients treated with sirolimus in an anticalcineurin-free regimen and compared it with that of 17 patients cotreated with cyclosporine. The area under the concentration versus time curve over a 12-hour period (AUC 0-12 ) of mycophenolic acid was estimated at 2 weeks, 1 month, 2 months, and 3 months after transplantation., Results: At the first 3 time points, patients cotreated with sirolimus had significantly higher mycophenolic acid AUC 0-12 values compared with patients cotreated with cyclosporine, as follows: 81 mg . h/L (SD, 39 mg . h/L) versus 43 mg . h/L (SD, 11 mg . h/L) (P < .001), 72 mg . h/L (SD, 17 mg . h/L) versus 48 mg . h/L (SD, 13 mg . h/L) (P < .001), and 70 mg . h/L (SD, 25 mg . h/L) versus 47 mg . h/L (SD, 17 mg . h/L) (P < .01) at week 2, month 1, and month 2, respectively. At all time points, patients cotreated with sirolimus had significantly higher dose-normalized mycophenolic acid AUC 0-12 values. At months 1 and 2, white blood cell counts were lower in the sirolimus group than in the cyclosporine group, as follows: 4.8 x 10(3)/mL (SD, 1.1 x 10(3)/mL) versus 6.5 x 10(3)/mL (SD, 2.2 x 10(3)/mL) (P < .01) at month 1 and 4.6 x 10(3)/mL (SD, 1.1 x 10(3)/mL) versus 5.9 x 10(3)/mL (SD, 2.0 x 10(3)/mL) (P < .05) at month 2., Conclusion: These data show that exposure to mycophenolic acid is higher in patients cotreated with sirolimus than in those cotreated with cyclosporine.

Post-transplant lymphoproliferative disease (PTLD) is a rare but severe complication of renal transplantation. Reduction of immunosuppression is essential for controlling PTLD but may induce graft loss. Retransplantation after PTLD is considered dangerous, because immunosuppressive treatment resumption may trigger hematological relapse. We retrospectively report six patients (five adults, one child) who underwent a second renal transplantation after successfully treated PTLD. Epstein-Barr virus (EBV) serology was positive before the first transplantation in all patients except the child. Post-transplant lymphoproliferative disease was detected 6.6 months (range 4.5-9.4) after transplantation. Lymphoproliferation was always monomorphic, B-cell, and EBV-related. Post-transplant lymphoproliferative disease was confined to the renal allograft (n = 4), multilocular (n = 1) or cerebral (n = 1). Immunosuppression tapering (6/6) and transplantectomy (5/6) led to hematological remission in all patients. Retransplantation was performed 77 months (range 50-128) after PTLD diagnosis. Immunosuppression included steroids (n = 6), ATG (n = 2), anti-CD25 (n = 2), cyclosporine (n = 4), tacrolimus (n = 2), mycophenolate mofetil (n = 4) and azathioprine (n = 1). After a median follow up of 30 months (range 24-47) patient survival was 100%, with no recurrence of PTLD. In conclusion, renal retransplantation can be considered in patients with monomorphic PTLD history, without major risk of hematological recurrence.

Background: Therapeutic drug monitoring of mycophenolic acid (MPA) may minimise the risk of acute rejection after transplantation. Area under the curve (AUC) rather than trough concentration-based monitoring is recommended and models for AUC estimation are needed., Objectives: To develop a population pharmacokinetic model suitable for Bayesian estimation of individual AUC in stable renal transplant patients., Patients and Methods: The population pharmacokinetics of MPA were studied using nonlinear mixed effects modelling (NONMEM) in 60 patients (index group) receiving MPA on a twice-daily basis. Ten blood samples were collected at fixed timepoints from ten patients and four blood samples were collected at sparse timepoints from 50 patients. Bayesian estimation of individual AUC was made on the basis of three blood concentration measurements and covariates. The predictive performances of the Bayesian procedure were evaluated in an independent group of patients (test group) comprising ten subjects in whom ten blood samples were collected at fixed timepoints., Results: A two-compartment model with zero-order absorption best fitted the data. Covariate analysis showed that bodyweight was positively correlated with oral clearance. However, the weak magnitude of the reduction in variability (from 34.8 to 28.2%) indicates that administration on a per kilogram basis would be of limited value in decreasing interindividual variability in MPA exposure. Bayesian estimation of pharmacokinetic parameters using samples drawn at 20 minutes and 1 and 3 hours enabled estimation of individual AUC with satisfactory accuracy (bias 7.7%, range of prediction errors 0.43-15.1%) and precision (root mean squared error 12.4%) as compared with the reference value obtained using the trapezoidal method., Conclusion: This paper reports for the first time population pharmacokinetic data for MPA in stable renal transplant patients, and shows that Bayesian estimation can allow accurate prediction of AUC with only three samples. This method provides a tool for therapeutic drug monitoring of MPA or for concentration-effect studies. Its application to MPA monitoring in the early period post-transplantation needs to be evaluated.

Objective: To develop a maximum a posteriori probability (MAP) Bayesian estimator for the pharmacokinetics of oral cyclosporin, based on only three timepoints, and evaluate its performance with respect to a full-profile nonlinear regression approach., Patients: 20 adult patients with stable renal transplants given orally administered microemulsified cyclosporin and mycophenolate., Methods: Cyclosporin was assayed by liquid chromatography-mass spectrometry. Nonlinear regression and MAP Bayesian estimation were performed using a home-made program and a previously designed pharmacokinetic model including an S-shaped absorption profile described by a gamma distribution., Outcome Measures and Results: MAP Bayesian estimation using the best limited sampling strategy (before administration, and 1 and 3 hours after administration) was compared with nonlinear regression (taken as the reference method) for the prediction of the different pharmacokinetic parameters and exposure indices. Median relative prediction error was -0.49 and -3.42% for area under the concentration-time curve over the administration interval of 12 hours (AUC12) and estimated peak drug concentration (Cmax), respectively (nonsignificant). Relative precision was 2.00 and 4.32%, and correlation coefficient (r) was 0.985 and 0.955, for AUC12 and Cmax, respectively., Conclusion: This paper reports preliminary results in a stable renal transplant patient population, showing that MAP Bayesian estimation can allow accurate prediction of AUC12 and Cmax with only three samples (0, 1 and 3 hours). Although these results require confirmation by further studies in other clinical settings, using other drug combinations, other analytical methods and commercially available pharmacokinetic software, the method seems promising as a tool for the therapeutic drug monitoring of cyclosporin in clinical practice or for exposure-controlled studies.