HPLC is currently the preferred method for accurate measurement of mycophenolic acid (MPA). This study was designed to validate the Emit compared with HPLC in relation to clinical outcome measurements.Pediatric renal-transplant recipients (n = 50) on an immunosuppressive triple regimen consisting of cyclosporin A, prednisone, and mycophenolate mofetil (600 mg/m(2) twice per day) were investigated in an open-label prospective study. Pharmacokinetic profiles over 12 h were obtained at 1 week, 3 weeks, 3 months, and 6 months posttransplant. Plasma MPA was measured by both reversed-phase HPLC and the Emit immunoassay.There was an association between the risk of acute rejection episodes and low area under the curve values from t(0) to t(12h) (AUC(0-12)) for MPA (MPA-AUC(0-12)) or predose concentrations of MPA derived from both HPLC and Emit measurements. According to ROC analysis, an AUC value of 33.8 mg x h/L for MPA from t(0) to t(12h) (MPA-AUC(0-12)) determined by HPLC had a diagnostic sensitivity of 80% and a diagnostic specificity of 57%. The corresponding value of the Emit was 36.1 mg x h/L. For the predose concentration (MPA-c(12)), a concentration of 1.2 mg/L determined by HPLC and 1.4 mg/L determined by Emit gave a sensitivity of 80% and a specificity of 60%, respectively. There was no association of any pharmacokinetic variables derived from total MPA measurements with an increased risk of side effects related to mycophenolate mofetil.The Emit assay appears to have a comparable diagnostic efficacy to HPLC for assessing the risk of acute rejection in pediatric renal-transplant recipients. However, because of the cross-reactivity of the antibody used in the Emit assay with the active MPA acyl glucuronide metabolite, the decision thresholds for the Emit were higher than those calculated from HPLC measurements.
Gilbert syndrome is a clinically inconsequential entity of mild unconjugated hyperbilirubinemia caused by an A(TA)(n)TAA insertion polymorphism (UGT1A1*28) in the promoter region of the gene coding for the enzyme UDP-glucuronosyltransferase 1 (EC 2.4.1. 17; UGT1A1). Present methods for genotyping this polymorphism are laborious.Hybridization probes were designed complementary to the wild type (TA)(6) and to alleles with (TA)(7) and (TA)(8) repeats in the promoter region. Melting points were measured in samples representing all currently known alleles with (TA)(5) to (TA)(8) repeats. Probe melting points were predicted with a thermodynamic nearest-neighbor model for Watson-Crick paired probes. The dominant secondary structures resulting from probe hybridization were predicted by thermodynamic free energy calculations. Alternatively samples were genotyped based on amplicon size resolved by high-resolution polyacrylamide gel electrophoresis.Only short probes (22-24 bases) could be successfully used for genotyping this locus because of the very low stability of this TA repeat. Assays based on (TA)(7) or (TA)(8) genotype-compatible hybridization probes effectively discriminated five to eight TA repeats. The consecutive use of two different detection probes was necessary for better discrimination of some heterozygous genotypes. All results were in concordance with the alternative genotyping method. Of 100 investigated Caucasians (50 males, 50 females), 9 (9%) were homozygous for the (TA)(7) allele.The presented method for genotyping the (TA)(n) promoter polymorphism of the UGT1A1 gene with the LightCycler has the potential to genotype all currently known (TA)(n) repeats in a single assay and is sensitive toward possible new genotypes. Our findings also show that thermodynamic calculations are of practical value for the design of hybridization probe assays for the genotyping of insertion/deletion polymorphisms.
Dosage guidelines for mycophenolate mofetil (MMF), an ester prodrug of the immunosuppressant mycophenolic acid (MPA), are still preliminary in children. This study compares the pharmacokinetics of MPA and its major metabolite MPA glucuronide (MPAG) in pediatric renal transplant recipients receiving 600 mg MMF/m2 body surface area twice a day to those of adults on the currently recommended oral dose of 1 g of MMF twice a day. Concentration-time profiles of 18 children (age, 10.7+/-0.72 yr; range, 5.9 to 15.3 yr) and 10 adults were investigated 1 and 3 wk after transplantation. Plasma concentrations of MPA and MPAG were measured by reverse-phase HPLC. Because MPA is extensively bound to serum albumin and only the free fraction is presumed to be pharmacologically active, the MPA free fraction was also analyzed by HPLC after separation through ultrafiltration. The areas under the concentration-time curves (AUC0-12) of total and free MPA throughout the 12-h dosing interval in children were, in general, comparable to the corresponding data in adult patients. The mean AUC0-12 of MPA and free MPA did not change significantly over the first 3 wk after transplantation, but there was substantial intra- and interindividual variation. MPAG-AUC0-12 values in children with primary renal transplant dysfunction were threefold higher than in those with functioning transplants. Renal impairment had no consistent effect on total MPA-AUC0-12 values, but the MPA free fraction in children (median, 1.65%; range, 0.40 to 13.8%) was significantly (r2=0.46) modulated by renal transplant function and serum albumin levels. In conclusion, concentration-time profiles of pediatric renal transplant recipients administered 600 mg MMF/m2 body surface area twice a day are comparable to those in adults on 1 g MMF twice a day in the first 3 wk after transplantation. Renal impairment and decreased serum albumin levels led to an increase in the free fraction of MPA and the free MPA-AUC0-12 values. Because the pharmacologic activity of MPA is a function of unbound drug concentration, these findings might be relevant for the pharmacodynamic effects of MPA.
Donor-derived cell-free DNA (dd-cfDNA) is a noninvasive biomarker for comprehensive monitoring of allograft injury and rejection in kidney transplantation (KTx). dd-cfDNA quantification of copies/mL plasma (dd-cfDNA[cp/mL]) was compared to dd-cfDNA fraction (dd-cfDNA[%]) at prespecified visits in 189 patients over 1 year post KTx. In patients (N = 15, n = 22 samples) with biopsy-proven rejection (BPR), median dd-cfDNA(cp/mL) was 3.3-fold and median dd-cfDNA(%) 2.0-fold higher (82 cp/mL; 0.57%, respectively) than medians in Stable Phase patients (N = 83, n = 408) without rejection (25 cp/mL; 0.29%). Results for acute tubular necrosis (ATN) were not significantly different from those with biopsy-proven rejection (BPR). dd-cfDNA identified unnecessary biopsies triggered by a rise in plasma creatinine. Receiver operating characteristic (ROC) analysis showed superior performance (P = .02) of measuring dd-cfDNA(cp/mL) (AUC = 0.83) compared to dd-cfDNA(%) (area under the curve [AUC] = 0.73). Diagnostic odds ratios were 7.31 for dd-cfDNA(cp/mL), and 6.02 for dd-cfDNA(%) at thresholds of 52 cp/mL and 0.43%, respectively. Plasma creatinine showed a low correlation (r = 0.37) with dd-cfDNA(cp/mL). In a patient subset (N = 24) there was a significantly higher rate of patients with elevated dd-cfDNA(cp/mL) with lower tacrolimus levels (<8 μg/L) compared to the group with higher tacrolimus concentrations (P = .0036) suggesting that dd-cfDNA may detect inadequate immunosuppression resulting in subclinical graft damage. Absolute dd-cfDNA(cp/mL) allowed for better discrimination than dd-cfDNA(%) of KTx patients with BPR and is useful to avoid unnecessary biopsies.
