Mycophenolic Acid Metabolites Acyl-Glucuronide and Glucoside Affect the Occurrence of Infectious Complications and Bone Marrow Dysfunction in Liver Transplant Recipients.

BACKGROUND Mycophenolic acid (MPA) prodrugs are anti-proliferative immunosuppressive agents commonly used after organ transplantation. Although they are generally well tolerated by patients, adverse effects may occur. It is postulated that MPA metabolites could also contribute to these adverse effects. MATERIAL AND METHODS The objective of this study was the assessment of concentrations of total MPA and its metabolites, phenyl glucuronide (MPAG), acyl glucuronide (AcMPAG) and glucoside (GluMPA), using liquid chromatography combined with mass spectrometry (LC/MS/MS) in two groups: kidney transplant recipients and liver transplant patients. Associations of MPA and its metabolites with adverse effects were analyzed. RESULTS The study group consisted of 211 recipients of liver or kidney transplants who received immunosuppressive therapy, including MPA prodrugs. Multivariant analysis showed a positive influence of MPA on gastroenterotoxicity in kidney transplant recipients. In liver patients, gastroenterotoxicity was associated with lower MPAG concentrations. A positive influence of AcMPAG on bacterial infections in liver transplant patients was observed. In liver transplant recipients, a positive influence of MPA and a negative influence of GluMPA levels on the PLT count were revealed. MPA and its metabolites did not influence the hemoglobin levels in both groups. There were no significant relationships among MPA, its metabolites and WBC counts. CONCLUSIONS In kidney transplant recipients, total MPA trough concentration is associated with gastroenterotoxicity and its monitoring could have important role in management of gastrointestinal complications. The quantification of AcMPAG in liver recipients receiving MPA may be helpful in avoiding bacterial infections. GluMPA seems to have a toxic effect on thrombopoiesis.

Two rapid ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) methods with common sample pretreatment for therapeutic drug monitoring of immunosuppressants compared to immunoassay.

Therapeutic drug monitoring of immunosuppressive agents is a critical and essential part of patient therapy after organ transplantation. We have developed high-throughput, robust, and rapid liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) methods with common pretreatment procedures for simultaneous quantification of four immunosuppressive agents (everolimus, sirolimus, tacrolimus, and cyclosporin A) in whole blood and one immunosuppressant (mycophenolic acid) in plasma. The new approach used in this work is based on improved sample preparation procedures allowing the analysis of five immunosuppressive drugs. Whole blood was prepared by transferring 100µL of blood into a 1.5-mL silanized conical test tube. Zinc sulfate solution (150µL), containing deuterated internal standards, was added to perform hemolysis. The samples were vortexing for 10s, followed by the addition of 250µL acetonitrile, containing internal standard for cyclosporin A, to precipitate proteins. The mixture was vortexed for 1min and centrifuged for 2min at 14,000rpm. The whole supernatant was transferred to a vial. To prepare blood plasma, the hemolysis step involving the addition of zinc sulfate was omitted and, instead of acetonitrile, methanol was used as the solvent for the internal standard (mycophenolic acid-d3). The volumes of chemicals used in this procedure were the same as those used in the procedure for immunosuppressants in whole blood. The basic validation parameters for the analytical methods were limits of detection (0.5ng/mL for everolimus, sirolimus and tacrolimus, 25ng/mL for cyclosporin A and 100ng/mL for mycophenolic acid), precision (<15%), recovery (>84%), repeatability and reproducibility. Possible mutual ion suppression was eliminated in the presence of internal standards. The method developed for the quantitation of immunosuppressants in whole blood was used to analyze 276 patient samples containing tacrolimus and 55 samples containing cyclosporin A. The results from LC/MS/MS were compared to those obtained from immunoassays of the same samples. Immunoassays significantly overestimated the concentrations of immunosuppressants.