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Assay validation of phosphorylated S6 ribosomal protein for a pharmacodynamic monitoring of mTOR‐inhibitors in peripheral human blood
Author(s) -
Dieterlen MajaTheresa,
Bittner Hartmuth B.,
Klein Sara,
von Salisch Sandy,
Mittag Anja,
Tárnok Attila,
Dhein Stefan,
Mohr Friedrich W.,
Barten Markus J.
Publication year - 2012
Publication title -
cytometry part b: clinical cytometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.646
H-Index - 61
eISSN - 1552-4957
pISSN - 1552-4949
DOI - 10.1002/cyto.b.21005
Subject(s) - whole blood , pharmacodynamics , pharmacology , flow cytometry , medicine , sirolimus , everolimus , pi3k/akt/mtor pathway , ribosomal protein s6 , pharmacokinetics , therapeutic drug monitoring , phosphorylation , chemistry , immunology , biochemistry , p70 s6 kinase 1 , signal transduction
Background: Therapeutic drug monitoring (TDM) of immunosuppressive drugs after organ transplantation is based on measuring blood levels alone, which often results in under‐ or over‐immunosuppression. Previous studies have shown the potential of measuring pharmacodynamic drug effects for TDM, but assessment of biomarkers for individual drugs is still not clinical routine. Therefore, we validated a specific assay to measure the pharmacodynamic effects of mammalian target of rapamycin (mTOR)‐inhibitors on phosphorylated S6 ribosomal protein (p‐S6RP), a downstream target of mTOR. Methods: Clinical relevant concentrations of sirolimus (SRL, 0.9–91.4 μg/L), cyclosporine A (CsA, 75.1–1202 μg/L), mycophenolate acid (MPA, 0.08–3.2 mg/L), or dexamethasone (DEX, 0.5–200 ng/mL) were added to whole‐blood from healthy volunteers. Activated whole‐blood was analyzed by phospho‐flow cytometry to measure p‐S6RP in T cells. Results: Phospho‐flow analysis revealed that SRL suppressed p‐S6RP in human T cells in a dose‐dependent manner with a half‐maximal inhibitory concentration (IC 50 ) at 19.8 nM and a maximal inhibitory effect ( I max %) at 91.9%. Neither CsA, MPA, nor DEX inhibited mTOR‐related S6RP‐phosphorylation. Coefficient of variations from 0.03 to 0.05, 0.12 to 0.25, and 0.14 to 0.38 for intra‐, interassay, and interindividual variability respectively, showed robustness of our assay. Furthermore, samples can be stored at RT or 4°C up to 2 h after withdrawal. Conclusion: We validated a robust whole‐blood assay that allows the specific measurement of SRL‐ and everolimus‐induced inhibition of T cells' function through detection of p‐S6RP. Future studies in organ transplanted recipients will show if this assay has the potential to enhance a TDM for mTOR‐inhibitor drugs in combination therapies. © 2011 International Clinical Cytometry Society