Assay validation of phosphorylated S6 ribosomal protein for a pharmacodynamic monitoring of mTOR-inhibitors in peripheral human blood.
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.


CONCLUSIONS
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.
Department of Cardiac Surgery, University of Leipzig, Heart Center Leipzig, Leipzig, Germany.
Maja-Theresa
MT
Dieterlen
Hartmuth B
HB
Bittner
Sara
S
Klein
Sandy
S
von Salisch
Anja
A
Mittag
Attila
A
Tárnok
Stefan
S
Dhein
Friedrich W
FW
Mohr
Markus J
MJ
Barten
D016572:Cyclosporine / Q000097:blood / Q000494:pharmacology; D003907:Dexamethasone / Q000097:blood / Q000494:pharmacology; D016903:Drug Monitoring / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood / Q000494:pharmacology*; D009173:Mycophenolic Acid / Q000097:blood / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D038601:Ribosomal Protein S6 / Q000097:blood*; D020123:Sirolimus / Q000097:blood / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism*; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors*
D007166:Immunosuppressive Agents; D038601:Ribosomal Protein S6; D003907:Dexamethasone; D016572:Cyclosporine; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D009173:Mycophenolic Acid; D020123:Sirolimus