Pharmacokinetics of Mycophenolic Acid in Patients post Hematopoietic Stem Cell Transplantation

[Purpose] Mycophenolate mofetil (MMF), an ester prodrug of mycophenolic acid (MPA), is widely used as a maintenance immunosuppressive regimen in solid organ transplant patients. It is increasingly used for the prophylaxis and treatment of graft-versus-host disease (GVHD) in hematopoietic stem cell transplantation (HSCT) patients. MPA is metabolized primarily to phenolic MPA glucuronide (MPAG) and minorly to acyl MPA glucuronide (AcMPAG). In patients with normal renal and liver function, MPA and MPAG are highly bound to serum albumin, 97-99% for MPA and 82% for MPAG. The optimal MMF dosing and preferred targets are still under investigation in HSCT patients due to the substantial intra- and inter-individual pharmacokinetic variability of MPA and broad range of transplants. Therefore, the overall goal of this thesis is to elucidate the clinical pharmacokinetics of MPA in HSCT patients. To approach this goal, three specific aims are 1) To develop a reliable UPLC-MS/MS assay for the simultaneous monitoring of MPA, MPAG and AcMPAG in human plasma samples; 2) To evaluate the pharmacokinetic variations of MPA, from plasma protein binding and metabolism perspectives, in both pediatric and adult patients following HSCT; 3) To investigate population pharmacokinetics of unbound MPA in pediatric and adolescent HSCT patients. [Methods] Plasma proteins were precipitated with acetonitrile and the chromatographic separation was achieved on a C18 column with a gradient elution. The detection was performed by a triple quadrupole mass spectrometer in the positive electrospray ionization (ESI) and multiple reaction monitoring (MRM) mode. Stability of MPA and its glucuronide metabolites was also thoroughly evaluated in human blood and plasma samples under short- and long- term storage conditions. Twenty pediatric patients with a median age of 3 years (range, 0.2-12 years) and thirteen adult patients with a media age of 54 years (range, 18-63 years) were enrolled. Sparse sampling design was used in this study. Blood samples were collected on days 0, 7, 14, 21 and 30 after allogeneic HSCT. Total and free MPA, as well as MPAG were quantified using the validated UPLC-MS/MS assay. Nonlinear mixed-effects modelling (NONMEM) was employed to analyze MPA pharmacokinetic data. A total of 89 unbound MPA plasma concentration-time datum points from 23 patients with a median age of 3 years (range, 0.2-20 years) were available for model development. [Results] Linearity of the assay was demonstrated over the range of 20-10,000 ng/ml for MPA and MPAG, and 2-1,000 ng/ml for AcMPAG in human plasma. The assay was precise and accurate with coefficient of variation and bias less than 15%. The plasma protein binding of MPA and MPAG did not change significantly in pediatric patients over the one month sampling period post HSCT. However, it increased in adult patients from day 7 to day 30 post HSCT, from 97.3±0.8% to 98.3±0.6% for MPA, and 74.6±9.4% to 82.9±8.1% for MPAG (P <0.05). The plasma protein binding of MPA was significantly higher in males compared to females in both pediatric (98.3±1.1 vs 97.4±1.1%) and adult (98.1±0.7 vs 97.4±1.2%) patients (P <0.05). The MPAG/MPA ratios on an mg/kg dose basis in adult patients were significantly higher than those in pediatric patients (4.3±3.4 vs 2.4±2.6; P <0.05). Pharmacokinetics of unbound MPA was described by a two-compartment model with first-order elimination. Given the range of body sizes, clearance and volume of distribution were scaled using standard weight-based allometric exponents. Final estimates in a standard 70 kg individual for clearance, inter-compartmental clearance, volumes of distribution in the central and peripheral compartments were 1720 L/h, 1180 L/h, 3260 L and 4120 L, respectively. No significant differences were observed in weight-adjusted clearance between males and females. Because of age-dependent differences in weight-adjusted clearance, the calculated unbound MPA AUC was higher in younger patients compared with those in older patients receiving 15 mg/kg MMF. [Conclusions] The UPLC-MS/MS assay for simultaneous quantification of MPA and its glucuronide metabolites was developed and fully validated in human plasma samples per US FDA Guideline. Extended stabilization procedures were suggested to improve the accuracy of the analysis before routine application. Time-dependent plasma protein binding, sex and age-related differences in MPA metabolism, at least in part, impact the reported large inter- and intra-individual variability in MPA pharmacokinetics. The final population pharmacokinetic model successfully described unbound MPA population pharmacokinetics, which could be used to explore dosing guidelines for safe and effective immunotherapy in pediatric HSCT patients. Future research should be multi-institutional and focus on developing clinical decisions with adequate statistical power to improve clinical care of HSCT recipients.