Mycophenolic Acyl-glucuronide Research Articles

Development and validation of a novel UPLC‐MS/MS method for the simultaneous quantification of mycophenolic mofetil and its major metabolites in rat plasma and liver S9 hydrolysis assay in rats

A sensitive liquid chromatography‐tandem mass spectrometry method was developed and validated for the simultaneous quantification of mycophenolic mofetil (MMF) and its major metabolites, mycophenolic acid (MPA), mycophenolic acyl glucuronide (AcMPAG), and mycophenolic glucuronide (MPAG), in rat plasma. Upon administration, mycophenolic mofetil (MMF), a prodrug, undergoes extensive metabolism in the liver. Following hydrolyzation, the active metabolite MPA is produced. MPA can then be further metabolized to produce its glucuronide conjugated metabolites, AcMPAG and MPAG. This method can simultaneously determine both the parent compound and its major metabolites within biological samples. In addition, liver S9 fraction hydrolysis studies of MMF results in the production of MPA, demonstrating that the parent drug is first hydrolyzed within the liver. A novel UPLC method was developed to simultaneously quantify both compounds following S9 liver enzyme hydrolysis.A Shimadzu UHPLC system coupled to an AB Sciex QTrap 4000 mass spectrometer was used for the analysis. Separation was achieved using an Ultra Biphenyl 5µm column (100 × 2.1mm) with acetonitrile and 0.1% formic acid as the mobile phases. Analysis was performed under positive ionization mode using the multiple reaction monitoring (MRM) approach. Additionally, to determine the hydrolysis rate, different concentrations of MMF were incubated with liver S9 fraction (0.5µg/ml final concentration) for 30 mins, after which the reaction was terminated using 6% formic acid in acetonitrile. Samples were prepared and MPA concentrations were determined in the reaction system using UPLC for detection and quantification.The method was linear in the range of 9.77nM – 5000nM with correlation coefficient values > 0.99 for all components. The method has been shown to be reproducible, with intra‐ and inter‐day accuracy and precision ±12.3% of nominal values, for all analytes. The average extraction recovery rates for MMF, MPA, AcMPAG, and MPAG were 91.3%, 97.9%, 98.4%, and 90.5%, respectively. Matrix effect was in the acceptable range (<15%). The analytes in plasma were found to be stable under bench –top, freeze‐thaw, and storage (‐4°C) conditions. The metabolic studies showed that mycophenolic mofetil can be rapidly hydrolyzed by rat liver S9 fraction with Vmax and Km values of 0.66±0.78 nmol/min/µg and 80.06±123.1µM respectively.This novel UPLC‐MS/MS method can comprehensively evaluate and quantify the concentration of both mycophenolic mofetil and its major metabolites, mycophenolic acid, mycophenolic acyl glucuronide, and mycophenolic glucuronide in biological samples. This method can be used as a tool to further investigate and understand the extensive metabolism of mycophenolic mofetil in clinical practice.

Uridine Diphosphate Glucuronosyltransferase 2B7 Variant p.His268Tyr as a Predictor of Kidney Allograft Early Acute Rejection

BackgroundUridine diphosphate glucuronosyltransferase (UGT2B7) is responsible for conversion of mycophenolic acid to mycophenolic acyl-glucuronide (acylMPAG). Conflicting data exist regarding the role of UGT2B7 p.His268Tyr (802C>T, rs7439366) variant in the clinical course following organ transplantation. Study AimThe aim of this study was to reveal an association between UGT2B7 p.His268Tyr (802C>T, rs7439366) polymorphism and kidney transplantation outcome. Study Design, Patients, and MethodGenomic DNA of 235 kidney transplant recipients was genotyped for UGT2B7 802C>T using TagMan single nucleotide polymorphism (SNP) genotyping assay. Maintenance immunosuppression used mycophenolate mofetil (MMF) and cyclosporine A (n = 137) or tacrolimus (n = 98). Primary end-point was biopsy-confirmed acute rejection within 3 and 12 post-transplantation months. Secondary end-points included gastrointestinal side effects, leukopenia, lymphopenia, neutropenia, and infections. Statistical analysis was performed with the aid of SAS System using kernel-smoothed estimates of acute graft rejection hazard function. The log-rank test and hazard ratio were used to reflect association between UGT2B7 802C>T variant and risk of acute graft rejection. ResultsWithin 3 postimplantation months 38 (16.2%) patients experienced acute rejection; 33 were allele C carriers in UGT2B7 802C>T SNP and 5 were TT homozygotes (P < .0457). Allele C–associated risk of rejection was 2.50 and remained between 2.19 and 3.02 after adjustment for clinical confounders, ie, HLA mismatch, panel-reactive antibodies, donor age, repeated transplantation, induction therapy, donor type, delayed graft function, applied calcineurin inhibitor, or MMF dosing. We found no association between the polymorphism and gastrointestinal side effects, leukopenia, lymphopenia, neutropenia, and infections. ConclusionUGT2B7 802C>T genotyping may help identify patients with excessive early acute rejection risk.