70
ICCS 2011
P34
Phospho-fow cytometry to assess biomarkers for
a therapeutic monitoring of immunsuppressive
drugs after organ transplantation
Sandy von Salisch, Maja-Theresa Dieterlen, Sara
Klein, Katja Eberhardt, Hartmuth Bittner, Stefan
Dhein, Friedrich Mohr, Markus Barten
Heart Center Leipzig, Leipzig, Germany
Backgrounds:
Therapeutic drug monitoring
(TDM) of immunosuppressive drugs after heart
transplantation like the mTOR-inhibitors sirolimus
(SRL) or everolimus (ERL) is based on measuring
serum levels alone, but often results in under- or
over-immunosuppression. Earlier studies have shown
the capability of measuring pharmacodynamic drug
effects for TDM. Since in combination therapies the
assessment of biomarkers for individual drugs is still
not clinical routine
,
we used phospho-fow cytometry
to assess the downstream effects of drug-induced
target inhibition.
Methods:
Different clinical relevant
concentrations of SRL (0.9-91.4µg/L), cyclosporine
A (CsA, 75.1-1202µg/L), mycophenolate acid
(MPA, 0.08-3.2mg/L) or Dexamethasone (DEX,
0.5-200ng/mL) were added to 200µl of whole-blood
from eight healthy volunteers, respectively. Whole-
blood was mitogen-stimulated to activate the mTOR
pathway, stained for phospho-S6 in T-cells and
analyzed by fow cytometry. For the validation we
determined coeffcient of inter-assay variability for 3
different samples and 15 measurements.
Results:
Phospho-fow analysis revealed that addition of SRL
suppressed phosphorylation of ribosomal protein
S6 in human T-cells, whereas CsA, MPA or DEX
did not inhibit mTOR-related S6-phosphorylation.
We determined the assay-specifc IC
50
for sirolimus
at 23.5nM. Between-run assay coeffcients of
variation ranged from 0.12 to 0.49 and showed the
robustness of the assay.
Conclusions:
In this study,
we established a phospho-fow cytometry assay for
biomarker assessment, which specifcally measures
the SRL effect on mTOR inhibition. Future studies
in organ transplanted human recipients will show
if such an assay has the potential to dose SRL in
combination with either CsA or MPA more safely
without loosing effcacy.
POSTER ABSTRACTS