Pharmacogenetic-Whole blood and intracellular pharmacokinetic-Pharmacodynamic (PG-PK2-PD) relationship of tacrolimus in liver transplant recipients.

Tacrolimus (TAC) is the cornerstone of immunosuppressive therapy in liver transplantation. This study aimed at elucidating the interplay between pharmacogenetic determinants of TAC whole blood and intracellular exposures as well as the pharmacokinetic-pharmacodynamic relationship of TAC in both compartments. Complete pharmacokinetic profiles (Predose, and 20 min, 40 min, 1h, 2h, 3h, 4h, 6h, 8h, 12h post drug intake) of twice daily TAC in whole blood and peripheral blood mononuclear cells (PBMC) were collected in 32 liver transplanted patients in the first ten days post transplantation. A non-parametric population pharmacokinetic model was applied to explore TAC pharmacokinetics in blood and PBMC. Concurrently, calcineurin activity was measured in PBMC. Influence of donor and recipient genetic polymorphisms of ABCB1, CYP3A4 and CYP3A5 on TAC exposure was assessed. Recipient ABCB1 polymorphisms 1199G>A could influence TAC whole blood and intracellular exposure (p<0.05). No association was found between CYP3A4 or CYP3A5 genotypes and TAC whole blood or intracellular concentrations. Finally, intra-PBMC calcineurin activity appeared incompletely inhibited by TAC and less than 50% of patients were expected to achieve intracellular IC50 concentration (100 pg/millions of cells) at therapeutic whole blood concentration (i.e.: 4-10 ng/mL). Together, these data suggest that personalized medicine regarding TAC therapy might be optimized by ABCB1 pharmacogenetic biomarkers and by monitoring intracellular concentration whereas the relationship between intracellular TAC exposure and pharmacodynamics biomarkers more specific than calcineurin activity should be further investigated.

Standardization of Flow Cytometric Immunophenotyping for Hematological Malignancies: The FranceFlow Group Experience.

Flow cytometry is broadly used for the identification, characterization, and monitoring of hematological malignancies. However, the use of clinical flow cytometry is restricted by its lack of reproducibility across multiple centers. Since 2006, the EuroFlow consortium has been developing a standardized procedure detailing the whole process from instrument settings to data analysis. The FranceFlow group was created in 2010 with the intention to educate participating centers in France about the standardized instrument setting protocol (SOP) developed by the EuroFlow consortium and to organise several rounds of quality controls (QCs) in order to evaluate the feasibility of its application and its results. Here, we report the 5 year experience of the FranceFlow group and the results of the seven QCs of 23 instruments, involving up to 19 centers, in France and in Belgium. The FranceFlow group demonstrates that both the distribution and applicability of the SOP have been successful. Intercenter reproducibility was evaluated using both normal and pathological blood samples. Coefficients of variation (CVs) across the centers were <7% for the percentages of cell subsets and <30% for the median fluorescence intensities (MFIs) of the markers tested. Intracenter reproducibility provided similar results with CVs of <3% for the percentages of the majority of cell subsets, and CVs of <20% for the MFI values for the majority of markers. Altogether, the FranceFlow group show that the 19 participating labs might be considered as one unique laboratory with 23 identical flow cytometers able to reproduce identical results. Therefore, SOP significantly improves reproducibility of clinical flow in hematology and opens new avenues by providing a robust companion diagnostic tool for clinical trials in hematology. © 2019 International Society for Advancement of Cytometry.

Tacrolimus diffusion across the peripheral mononuclear blood cell membrane: impact of drug transporters.

Measuring tacrolimus (TAC) concentration in peripheral blood mononuclear cells (PBMCs) could better reflect the drug effect on its target (calcineurin (CaN) in lymphocytes) than whole blood concentrations. Mechanisms influencing TAC diffusion into PBMC are not well characterized. This work aimed at describing, ex vivo, TAC diffusion kinetics into PBMC and investigating the contribution of membrane transporters to regulate TAC intracellular concentration as well as the impact on CaN activity. PBMCs were incubated with TAC for 5 min to 4 h and under several experimental conditions: 37 °C (physiological conditions), 4 °C (inhibition of influx and efflux active transport), 37 °C + transporter inhibitors (verapamil, carvedilol, and probenecid and bromosulfophthalein, respectively, inhibitors of P-gp, OAT, and OATP). TAC concentration and CaN activity were measured in PBMC using liquid chromatography coupled with mass spectrometry. TAC intra-PBMC concentration was maximal after 1 h of incubation. Mean TAC PMBC concentrations were significantly lower in samples incubated at 4 °C compared to the 37 °C groups. Addition of verapamil slightly increased TAC accumulation in PBMC while other inhibitors had no effect. A significant correlation was found between TAC intra-PBMC concentration and the level of inhibition of CaN. Using an ex vivo cellular model, these results suggest that P-gp is involved in the drug efflux from PBMC while influx active transporters likely to regulate TAC intra-PBMC disposition remain to be identified. TAC concentration in PBMC is correlated with its pharmacodynamic effect. Then, TAC intra-PBMC concentration appears to be a promising biomarker to refine TAC therapeutic drug monitoring.