Heike Bittersohl, Juliane Herbinger, Werner Steimer, Peter B. Luppa
Institute of Clinical Chemistry and Pathobiochemistry, Klinikum rechts der Isar der TU München, Ismaninger Str. 22, 81675 Munich, Germany
Therapeutic monitoring of protein unbound (free) immunosuppressants has the potential to better predict the clinical outcome compared to conventional monitoring of drug levels in whole blood or plasma. Only a small proportion of drugs are free in the patient blood, hence, requiring a sensitive analytical method for their determination. We established an LC-MS/MS method able to simultaneously measure levels of cyclosporine A and mycophenolic acid in the picomolar range. This procedure is currently applied in a study on samples from kidney transplant recipients taking both drugs as a combination therapy.
Therapeutic drug monitoring (TDM) of most commonly used immunosuppressants is essential for achieving optimal patient care after solid organ transplantation due to their narrow therapeutic window. While overdosing can cause severe toxicity and long term morbidity, underdosing can lead to transplant rejection. Presently, regular drug level determinations in lysed whole blood samples or, less commonly, in plasma are standard of care. But, despite this intensive monitoring, rates of acute rejection still remain high, varying from 8 % up to 30 % in the first two years post-transplantation [1, 2].
In whole blood, most immunosuppressants are located inside blood cells or are bound to plasma proteins. However, only the protein unbound (free) drug molecules can cross membranes and bind to receptors to produce the desired pharmacological effect. Only free drug molecules are supposed to be pharmacologically active and some early investigations indicate that monitoring of protein unbound immunosuppressants could better reflect the clinical outcome [3, 4].
We have established a sensitive liquid chromatography-tandem mass spectrometry method for simultaneous determinations of cyclosporine A (CsA) and mycophenolic acid (MPA) free fractions, which is currently applied to samples from renal transplant patients. The research study has been approved by the institutional ethics committee and informed consent was obtained from all patients prior to their participation. Ultracentrifugation and microdialysis techniques are used to separate free from protein bound drug molecules. After this first step, known amounts of deuterated internal standards are added to the samples, following purification of the target compounds by use of liquid-liquid extraction. The liquid chromatography procedure features a run time of 6 minutes, using a binary gradient of aqueous and methanolic mobile phases and a column oven temperature of 60°C. The tandem mass spectrometer operates in electrospray positive mode and multiple reaction monitoring transitions have been chosen for drug quantification. First study results of 12 samples from transplant patients taking cyclosporine A and mycophenolic acid show free drug concentrations of 1.3±0.3 ng/mL (MW±SD) for cyclosporine A and 11.4±6.2 ng/mL (MW±SD) for mycophenolic acid. The corresponding drug concentrations in microdialysate samples display mean concentrations of 0.4 ng/mL (CsA) and 93.0 ng/mL (MPA).
Our new LC-MS/MS method overcomes analytical challenges such as small drug amounts, high lipophilicity and high protein binding. Thus, the method can serve as a suitable tool to conduct larger studies on free immunosuppressant drug concentrations, which have the potential to personalize immunosuppressive drug therapy and might improve clinical outcomes of organ recipients.
This work is supported by the European Union in the seventh framework program project NANODEM ("NANOphotonic DEvice for Multiple therapeutic drug monitoring” - Grant agreement no: 318372).
1. Wallemacq P, Armstrong VW, Brunet M et al. (2009) Opportunities to optimize tacrolimus therapy in solid organ transplantation: report of the European consensus conference. Ther Drug Monit 31:139–152.
2. Boudjema K, Camus C, Saliba F, et al. (2011) Reduced-dose tacrolimus with mycophenolate mofetil vs. standard-dose tacrolimus in liver transplantation: a randomized study. Am J Transplant 11:965–76.
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4. Zahir H, McCaughan G, Gleeson M, et al. (2003) Factors affecting variability in distribution of tacrolimus in liver transplant recipients. Br J Clin Pharmacol 57:298–309.