Smits Thomas A, Cox Shareen, Fukuda Tsuyoshi, Sherbotie Joseph R, Ward Robert M, Goebel Jens, Vinks Alexander A
*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.
Ther Drug Monit. 2014 Dec;36(6):716-23. doi: 10.1097/FTD.0000000000000081.
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.
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.
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.
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.
霉酚酸（MPA）是一种关键的免疫抑制药物，通过抑制肌苷单磷酸脱氢酶（IMPDH）发挥作用。作为治疗药物监测的一部分，MPA通常作为血浆中的总浓度进行测定。然而，据推测，MPA的游离（未结合）部分（fMPA）才是产生免疫抑制作用的原因。在本研究中，开发了一种灵敏的小体积高效液相色谱（HPLC）测定法来测量fMPA浓度，以探讨fMPA与IMPDH活性之间的关系。
为了获得fMPA浓度，使用Centrifree超滤装置对血浆样本进行过滤。超滤液通过使用Kinetex C18柱（2.6μm，3.0×75mm）的HPLC进行分析。将fMPA浓度与28例儿科肾移植患者移植后连续3次的总MPA浓度进行比较。使用Emax模型分析fMPA与IMPDH活性之间的关系。
使用25μL超滤液的HPLC测定法在2.5至1000μL的范围内进行了验证，具有良好的准确性、精密度和重现性。尽管观察到结合MPA部分存在较大的个体内和个体间变异性，但总MPA浓度和游离MPA浓度之间具有良好的相关性（R = 0.85，P < 0.0001）。使用Emax模型，fMPA浓度与IMPDH抑制之间的总体关系与先前报道的总MPA的关系相当。模型估计的EC50值（164.5μL）与报道的体外EC50值高度一致。
本研究提供了一种简单的HPLC方法来测量fMPA，并给出了药理学上合理的EC50估计值。总MPA浓度和游离MPA浓度之间的良好相关性表明，尽管MPA结合部分的巨大变异性值得进一步研究，但常规测量fMPA以表征霉酚酸的药代动力学和药效学似乎没有必要。
