A novel flow cytometry based assay for therapeutic monitoring of mTOR-inhibitor induced immunosuppression after heart transplantation

Objective: Therapeutic drug monitoring (TDM) of immunosuppressive drugs after transplantation (Tx) like the mTOR-inhibitors sirolimus (SRL) or everolimus (ERL) is based on measuring blood levels alone. But this often results in under- or over-immunosuppression leading to rejection or infection, respectively. Earlier studies have shown the potential value of measuring pharmacodynamic drug effects for TDM. Therefore we developed an assay to measure drug effects on the mTOR-pathway.

Methods: Blood from five volunteers was obtained. Different clinical relevant concentrations of SRL (0.9–91.4g/L), cyclosporine A (CsA, 75.1–1202g/L), mycophenolate acid (MPA, 0.08–3.2mg/L) or Dexamethasone (DEX, 0.5–200ng/mL) were added to only 200l of whole-blood. Activated whole-blood was analyzed by flow cytometry to measure phospho-S6 in T-cells.

Results: Phospho-flow analysis revealed that addition of SRL suppressed phosphorylation of ribosomal-protein S6 in human T-cells, whereas CsA, MPA or DEX as known from their mechanism of action did not inhibit mTOR-related S6-phosphorylation. We determined the assay-specific IC₅₀ for SRL at 23.5nM. The maximum inhibitory effect (I_max%) of SRL on S6 phosphorylation in T-cells was obtained at 89%. Reproducibility of this assay was verified with slide-based cytometry. Inter-assay and intra-assay coefficients of variation ranged from 0.12 to 0.25 and 0.03 to 0.05 respectively.

Conclusions: We established a specific whole-blood assay to assess drug effects on the mTOR-pathway through measuring phosphorylated ribosomal protein S6. Future studies in HTx recipients will show if this assay has the potential to dose mTOR-inhibitors SRL or ERL in combination with either CsA or MPA more safely without loosing the efficacy.