Thomas A Smits1, Shareen Cox, Tsuyoshi Fukuda, Joseph R Sherbotie, Robert M Ward, Jens Goebel, Alexander A Vinks. 1. *Division of Clinical Pharmacology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; †Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio; ‡Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, Utah; and §Division of Nephrology and Hypertension, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.
Abstract
BACKGROUND: Mycophenolic acid (MPA) is a key immunosuppressive drug that acts through inhibition of inosine monophosphate dehydrogenase (IMPDH). MPA is commonly measured, as part of therapeutic drug monitoring, as the total concentration in plasma. However, it has been postulated that the free (unbound) fraction of MPA (fMPA) is responsible for the immunosuppressive effects. In this study, a sensitive low volume high-performance liquid chromatography (HPLC) assay was developed to measure fMPA concentrations to explore the relationship between fMPA and IMPDH activity. METHODS: To obtain fMPA concentrations, plasma samples were filtrated using Centrifree ultrafiltration devices. The ultrafiltrate was analyzed by HPLC using a Kinetex C18 column (2.6 μm, 3.0 × 75 mm). fMPA concentrations were compared with the total MPA concentrations available in 28 pediatric kidney transplant patients at 3 consecutive occasions after transplantation. The relationship between fMPA and IMPDH activity was analyzed using an Emax model. RESULTS: The HPLC assay, using 25 μL of the ultrafiltrates, was validated over a range from 2.5 to 1000 μL with good accuracy, precision, and reproducibility. Total and free MPA concentrations were well correlated (R = 0.85, P < 0.0001), although large intraindividual and interindividual variability in the bound MPA fractions was observed. The overall relationship between fMPA concentrations and IMPDH inhibition using the Emax model was comparable with that of total MPA, as previously reported. The model estimated EC50 value (164.5 μL) is in good agreement with reported in vitro EC50 values. CONCLUSIONS: This study provides a simple HPLC method for the measurement of fMPA and a pharmacologically reasonable EC50 estimate. The good correlation between the total and free MPA concentrations suggests that routine measurement of fMPA to characterize mycophenolate pharmacokinetic and pharmacodynamic does not seem warranted, although the large variability in the bound fractions of MPA warrants further study.
BACKGROUND:Mycophenolic acid (MPA) is a key immunosuppressive drug that acts through inhibition of inosine monophosphate dehydrogenase (IMPDH). MPA is commonly measured, as part of therapeutic drug monitoring, as the total concentration in plasma. However, it has been postulated that the free (unbound) fraction of MPA (fMPA) is responsible for the immunosuppressive effects. In this study, a sensitive low volume high-performance liquid chromatography (HPLC) assay was developed to measure fMPA concentrations to explore the relationship between fMPA and IMPDH activity. METHODS: To obtain fMPA concentrations, plasma samples were filtrated using Centrifree ultrafiltration devices. The ultrafiltrate was analyzed by HPLC using a Kinetex C18 column (2.6 μm, 3.0 × 75 mm). fMPA concentrations were compared with the total MPA concentrations available in 28 pediatric kidney transplant patients at 3 consecutive occasions after transplantation. The relationship between fMPA and IMPDH activity was analyzed using an Emax model. RESULTS: The HPLC assay, using 25 μL of the ultrafiltrates, was validated over a range from 2.5 to 1000 μL with good accuracy, precision, and reproducibility. Total and free MPA concentrations were well correlated (R = 0.85, P < 0.0001), although large intraindividual and interindividual variability in the bound MPA fractions was observed. The overall relationship between fMPA concentrations and IMPDH inhibition using the Emax model was comparable with that of total MPA, as previously reported. The model estimated EC50 value (164.5 μL) is in good agreement with reported in vitro EC50 values. CONCLUSIONS: This study provides a simple HPLC method for the measurement of fMPA and a pharmacologically reasonable EC50 estimate. The good correlation between the total and free MPA concentrations suggests that routine measurement of fMPA to characterize mycophenolate pharmacokinetic and pharmacodynamic does not seem warranted, although the large variability in the bound fractions of MPA warrants further study.
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