C-440T/T-331C polymorphisms in the UGT1A9 gene affect the pharmacokinetics of mycophenolic acid in kidney transplantation.

INTRODUCTION: The immunosuppressive agent mycophenolic acid (MPA) is metabolized by uridine diphosphate glucuronosyltransferase 1A9 (UGT1A9) to 7-O-glucuronide (MPAG) and excreted by multidrug resistance-associated protein 2 in the bile. By contrast, the production of the acyl MPAG, a minor MPA metabolite, was ascribed to UGT2B7 and UGT1A8. Several polymorphisms in the genes encoding for UGT1A9, UGT2B7 and MRP2 proteins have been described. However, their functional role in vivo on MPA metabolism remains poorly defined. METHODS: A total of 40 Caucasian kidney transplant patients, given induction therapies (with Campath-(1)H or the combination basiliximab/rabbit antithymocyte globulin) and on maintenance immunosuppression with cyclosporine in combination with mycophenolate mofetil (MMF) in a steroid-free regimen, were enrolled in the pharmacogenetic study. Patients had clinical and hematochemical evaluations at month 6 after transplantation, as well as complete MPA pharmacokinetic assessment. They were genotyped for SNPs in UGT1A9 C-2152T, T-1887G, C-665T, C-440T, T-331C, T-275A, T98C, for the nonsynonymous C802T SNP in UGT2B7, and for ABCC2 SNPs C-24T and G1249A. The association of these polymorphisms with MPA pharmacokinetic parameters was investigated. RESULTS: Differences in the MPA pharmacokinetic profiles confirmed large interpatient variability of MPA exposure, with AUC(0-12) values ranging from 7.9 to 50.1 mg*h/ml. MPA AUC(0-12) was significantly associated with the presence of UGT1A9 -440/-331 genotypes (TT/CC: 61.5 +/- 2.7 mg*h/ml/g MMF; TC/CT: 45.4 +/- 14.0 mg*h/ml/g MMF; CC/TT: 40.8 +/- 10.8 mg*h/ml/g MMF; p = 0.005), whereas MPAG exposure was mainly influenced by renal function. The positive association between MPA AUC and SNPs in position -440/-331 found in kidney transplant patients confirmed previous in vitro findings showing that the abovementioned SNPs had a significant impact on UGT1A9 protein content in the liver. The presence of ABCC2 promoter C-24T and exon 10 G1249A SNPs did not cause any significant variation in MPA and MPAG pharmacokinetic parameters. CONCLUSION: The study demonstrated a significant impact of C-440T/T-331C SNPs in the promoter region of the UGT1A9 gene on MPA pharmacokinetics in renal allograft recipients.

Development of a CE method for the determination of mycophenolic acid in human plasma: a comparison with HPLC.

Mycophenolate mofetil (MMF) is a widely used drug for the maintenance of immunosuppressive therapy in renal-transplant recipients. MMF is rapidly metabolized in vivo to mycophenolic acid (MPA), a reversible, noncompetitive inhibitor of inosine monophosphate dehydrogenase, which represents a limiting enzyme in lymphocyte proliferation. MPA shows large interindividual pharmacokinetic variability: its monitoring is therefore of primary importance to achieve adequate immunosuppression with minimized risk of graft rejection or toxicity. We developed a CE method for the determination of total MPA (tMPA) in plasma, based on easy sample preparation; CE evaluation of tMPA was performed in 30 mmol/L sodium-borate with 10 mmol/L SDS (pH 10.00) at 25 degrees C using a 60 cm (54.5 to window) uncoated capillary with UV detection at 254 nm wavelength. MPA was readily detectable in plasma; the CE method was linear in the range of 0.7-120 microg/mL (r >0.992). Intra- and interassay imprecision was <7% except for the lowest spiked MPA concentration, which had an intra-assay RSD% of 14.7 compared to 18.3 interassay. Data by CE were compared with results obtained by a validated HPLC method. CE assay of tMPA exhibited a very good correlation (r(2) >0.988) with respect to HPLC; Bland-Altman difference versus average showed a mean of -0.18 microg/mL +/- 1.14 SD. CE determination of tMPA is a robust, sensitive and reproducible method with the advantage over HPLC of being fast, simple and unexpensive, also enabling quick assessment of MPA for pharmacokinetic studies.

Comparison of the Innofluor certican assay with HPLC-UV for the determination of everolimus concentrations in heart transplantation.

OBJECTIVES: Therapeutic monitoring of everolimus with chromatographic methods (HPLC) enabled effective immunosuppression while limiting the incidence of drug-related adverse events. A fluorescence polarization immunoassay (FPIA) has been recently developed for the assessment of everolimus levels. The present study was designed to evaluate FPIA performance and to compare it to HPLC. DESIGN AND METHODS: The performance of HPLC and FPIA was initially tested using drug-free whole blood spiked with different amount of everolimus concentrations and, subsequently, by analyzing 113 trough blood samples from heart transplant recipients chronically given everolimus as part of their immunosuppressive regimen. RESULTS: Inaccuracy and imprecision of both methods were below 15%. The correlation between everolimus concentrations and measured FPIA and HPLC was good, with a Pearson coefficient of 0.9118. The FPIA gave a mean overestimation of 24.3% as compared with HPLC. As additional analysis, cross-reactivity ranging from 85.4% to 138.0% was found with sirolimus, an immunosuppressant with a chemical structure close to everolimus. CONCLUSION: The FPIA demonstrated acceptable performance for therapeutic drug monitoring of everolimus, and is a viable alternative to HPLC-based methods.

Mycophenolic acid formulation affects cyclosporine pharmacokinetics in stable kidney transplant recipients.

A novel monitoring strategy based on the blood concentration at two hours post-dose (C2) has been recently proposed for the assessment of cyclosporine (CsA) absorption and daily exposure, and therapeutic windows for C2 levels have been identified. These guidelines have been derived from patients given mycophenolate mofetil (MMF) or azathioprine, and never tested in those treated with the enteric-coated formulation of mycophenolic acid (EC-MPS). The authors have compared full CsA pharmacokinetic evaluations in 12 kidney transplant recipients given EC-MPS with those from 20 patients on MMF at months 6, 12, 18 and 24 postsurgery. At month 6 postsurgery, patients on EC-MPS had a shift to the right in the CsA peak concentration as compared to that in patients given MMF, an effect associated with significant differences in CsA Tmax (1.9 +/- 0.3 h vs. 1.5 +/- 0.6 h, P < 0.05), C2 (988 +/- 259 vs. 720 +/- 214 ng/mL, P < 0.01), and C3 levels (539 +/- 119 vs. 435 +/- 119 ng/mL, P < 0.05). Interestingly, the authors found that the majority of patients on EC-MPS had CsA peaking at 2-h postdosing, whereas most of patients on MMF had CsA Cmax at 1 h. Similar results were observed also at months 12, 18, and 24 postsurgery. These findings indicate that the pharmacokinetics of CsA is significantly affected by the concomitant administration of different MPA formulations. This would imply the need of specific algorithms to adequately estimate CsA dose adjustment in patients given, in addition to CsA, EC-MPS or MMF.

Simultaneous determination of everolimus and cyclosporine concentrations by HPLC with ultraviolet detection.

In the clinical practice of organ transplantation everolimus (RAD) is used in combination with cyclosporine (CsA), the most common antirejection agent. Both drugs show a narrow therapeutic window, which requires strict monitoring of their blood concentration. Simple methods for simultaneous measurement of RAD and CsA concentration are needed. As we have recently developed an HPLC-UV assay for RAD determination, we decided to implement it to allow concomitant measurement of CsA. The within- and between-day coefficients of variation of the measurement were less than 12.1% for RAD and 9.8% for CsA. The within- and between-day inaccuracy of quality control samples were less than 9.7% for RAD and less than 4.9% for CsA. The method was found accurate and precise and useful for simultaneous therapeutic monitoring of the two drugs.

Comparison of different cyclosporine immunoassays to monitor C0 and C2 blood levels from kidney transplant recipients: not simply overestimation.

BACKGROUND: Immunoassays used for the measurement of cyclosporine (CsA) usually show cross-reactivity for CsA metabolites, usually resulting in unacceptable bias. METHODS: To assess the performance of different immunoassays, CsA concentrations were analyzed in 132 samples using ACMIA, EMIT-VIVA, CEDIA-PLUS, and HPLC. Samples were collected from kidney transplant patients monitored with the traditional blood CsA trough level (C0, n=73) and the new sampling at 2-h post CsA dosing (C2, n=59). RESULTS: Overall, the correlations between HPLC and other methods were good (r values ranging from 0.85 to 0.97). The use of C2 concentrations to monitor CsA exposure were associated with an overall better performance of all the immunoassays as compared with C0 values. However, none of the immunoassays agreed with the guidelines proposed in the Lake Louis Consensus Conference. Of note, the CEDIA-PLUS was the only that provided a linear relationship with HPLC for both sampling times. A false positive case associated with ACMIA was also documented in blood samples from a patient withdrawn from CsA for 1 month. CONCLUSION: These data suggest that the performance of some of the most used immunoassays is not satisfactory, eventually leading to incorrect therapeutic decision guided by erroneous CsA monitoring.

High-performance liquid chromatography with ultraviolet detection for therapeutic drug monitoring of everolimus.

We developed and validated a high-performance liquid chromatography-ultraviolet (HPLC-UV) method for determining everolimus concentrations in human whole blood. Sample preparation involved a solid-phase extraction after protein precipitation. The separation of everolimus from internal standard (IS) and endogenous components was achieved using an isocratic elution on an octyl column. The method showed a linear relationship between peak height ratios and blood concentrations in the range of 1-200 ng/mL (r(2)=0.9997). The observed intra- and inter-day assay imprecision had a coefficient of variation (CV)=12.8%, and inaccuracy was 11.4%. The method was found to be precise, accurate, and sensible making it useful for routine therapeutic monitoring of everolimus.

Therapeutic drug monitoring of sirolimus: effect of concomitant immunosuppressive therapy and optimization of drug dosing.

Sirolimus (SRL) is a new immunosuppressant which shares a common metabolic pathway with several other immunosuppressive agents. This leads to potential pharmacokinetic interactions that might affect SRL blood levels with relevant clinical consequences. As a validated laboratory, 2658 SRL trough samples (corresponding to 495 kidney transplant recipients treated with different immunosuppressive regimens) from more than 40 Italian Transplant Units were analyzed. We found that dose-normalized SRL trough levels were significantly higher in patients treated with cyclosporine (CsA) and SRL (4.15 +/- 2.23 ng/mL/mg SRL), compared with patients treated with mycophenolate mofetil (MMF) and SRL (3.26 +/- 1.86 ng/mL/mg SRL; p < 0.01) or with MMF, steroids and SRL (2.52 +/- 1.73 ng/mL/mg SRL; p < 0.01). Mean intra- and interpatient variabilities were 19% and 47%, respectively. Both parameters are significantly affected by the time postsurgery, with the first week post transplantation being associated with the greatest variability. As additional analysis, a simple dose-adjustment formula has been proposed as a useful tool to guide SRL dose changes. The proposed equation has been able to predict SRL concentration after a dose change in 73% of the tested samples. These findings suggest that different immunosuppressants significantly interfere with SRL bioavailability. Strategies aimed at reducing variability in SRL exposure may have a positive clinical impact.

Pharmacogenetics and pharmacogenomics of immunosuppressive agents: perspective for individualized therapy.

Immunosuppressive therapy has markedly improved over the past years with the advent of highly potent and rationally targeted immunosuppressive agents. Since these drugs are characterized by a narrow therapeutic index, major efforts have been carried out to define therapeutic windows based on the blood levels of each immunosuppressant, and relating those concentrations to clinical events. Although pharmacokinetic-based approaches are currently used as useful tools to guide drug dosing, they present several limitations. Pharmacogenomics - a science that studies the inherited basis of differences between individual responses to drugs in order to identify the best dose and therapy for each patient - might represent a complementary support. Preliminary studies that have focused on polymorphisms of genes encoding enzymes involved in drug metabolism, drug distribution, and pharmacological target, have shown promising results. Indeed, pharmacogenomics holds promise for improvement in the ability to individualize pharmacological therapy based on the patient's genetic profile.