Monitoring of Ex Vivo Cyclosporin a Activity in Healthy Volunteers Using T Cell Function Assays in Relation to Whole Blood and Cellular Pharmacokinetics

Abstract

Therapeutic drug monitoring (TDM) of calcineurin inhibitors (i.e., tacrolimus and cyclosporin A) is standard of care after solid organ transplantation. Although the incidence of acute rejection has strongly decreased, there are still many patients who experience severe side effects or rejection after long-term treatment. In this healthy volunteer study we therefore aimed to identify biomarkers to move from a pharmacokinetic-based towards a pharmacodynamic-based monitoring approach for calcineurin inhibitor treatment. Healthy volunteers received a single dose of cyclosporine A (CsA) or placebo, after which whole blood samples were stimulated to measure ex vivo T cell functionality, including proliferation, cytokine production, and activation marker expression. The highest whole blood concentration of CsA was found at 2 h post-dose, which resulted in a strong inhibition of interferon gamma (IFNy) and interleukin-2 (IL-2) production and expression of CD154 and CD71 on T cells. Moreover, the in vitro effect of CsA was studied by incubation of pre-dose whole blood samples with a concentration range of CsA. The average in vitro and ex vivo CsA activity overlapped, making the in vitro dose–effect relationship an interesting method for prediction of post-dose drug effect. The clinical relevance of the results is to be explored in transplantation patients on calcineurin inhibitor treatment.

Immune Monitoring of Mycophenolate Mofetil Activity in Healthy Volunteers Using Ex Vivo T Cell Function Assays

“…Concentrations of MPA, the active form of the pro-drug MMF, were measured in plasma, peripheral blood mononuclear cells (PBMCs), and T cells. Samples were processed as described previously [ 17 ], and the isolated cells were frozen in PBS until analysis. The quantification of MPA in plasma, PBMCs, and T cell samples was performed by the ISO15189-accredited Clinical Pharmaceutical Laboratory of the Department of Clinical Pharmacy and Toxicology, Leiden University Medical Center, the Netherlands.…”

“…MPA concentration in plasma was quantified using a previously validated LC-MS/MS assay [ 18 ]. A new method was developed for the quantification of intracellular concentrations in PBMCs and T cells, which was performed in a similar way as for the measurement of intracellular cyclosporine A concentrations [ 17 ]. In short, the calibration standards and quality controls used were mycophenolic acid (Alsachim, Illkirch-Graffenstaden, France) and mycophenolic acid-D3-C13 (Alsachim, France) prepared in acetonitrile (10 mg/L).…”

“…For measurement of cytokine production, whole blood was stimulated for 24 h with 10 μg/mL phytohemagglutinin (PHA) (Sigma Aldrich, St. Louis, MO, USA), as described previously [ 17 ]. At the pre-dose time point, the in vitro MPA concentration–effect relationship for each individual subject was studied by incubating whole-blood samples with concentrations of 50, 10, 2, 0.4, and 0.08 μg/L MPA (Sigma Aldrich).…”

“…T cell proliferation was measured in the same way as in our previous study [ 17 ] using an EdU kit (Thermo Fisher Scientific, (Waltham, MA, USA). A MACSQuant 16 analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany) was used for flow cytometry analysis of EdU incorporation.…”

“…In these studies, we showed that production of IFN-γ and IL-2 in ex vivo -stimulated whole blood presented good biomarkers for the immunosuppressive effect of CNIs. Moreover, T cell proliferation and expression of CD154 and CD71 in T cells were also suitable to demonstrate CNI effects [ 16 , 17 ]. In our search for biomarkers that can reflect the overall immune status of the transplantation patient, we aimed to identify the effect of MMF treatment on these previously tested biomarkers.…”