Follow the Area Under the Curve Not the Trough Concentration: A Case Study of Tacrolimus Monitoring in a Kidney Transplant Recipient Cotreated With Phenobarbital.

ABSTRACT: The authors described tacrolimus dosing in a kidney transplant patient concurrently treated with phenobarbital, where measuring the tacrolimus area under the curve was necessary to achieve adequate drug exposure and improve kidney function.

Volumetric absorptive microsampling for the quantification of tacrolimus in capillary blood by high performance liquid chromatography-tandem mass spectrometry.

Volumetric absorptive microsampling (VAMS) is an innovative alternative strategy to venipuncture for monitoring tacrolimus levels in transplant recipients. In this study, we aimed to validate a new high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for quantifying tacrolimus in blood collected by VAMS. Tacrolimus was extracted from dried blood tips in an original process involving sonication, protein precipitation and salting out. The assay was validated in accordance with EMA and IATDMCT guidelines. For clinical validation, the tacrolimus concentrations measured in liquid venous whole blood (with the reference method) were compared with those measured in capillary whole blood collected simultaneously with VAMS by a nurse. The assay was then used to monitor tacrolimus exposure in transplant recipients. The method was linear, sensitive and fast. Within-day and between-day precisions and overall bias were within ±15%. No significant hematocrit effect was observed. The matrix effect was negligible and recovery exceeded 80% for every concentration and hematocrit levels. Tacrolimus was stable in blood collected by VAMS for 1 week at room temperature, 48 h at 60 °C and 4 °C and 1 month at -80 °C. Clinical validation (n = 42 paired samples) demonstrated a strong correlation between the two methods (r = 0.97 Pearson correlation). Bland-Altman analysis revealed that more than 90% of the differences between VAMS and liquid blood paired concentrations were within the ±20% acceptable range. The method had a satisfactory analytical performance and fulfilled clinical requirements. This minimally invasive VAMS-based assay appears reliable for the determination of tacrolimus levels in blood from transplanted patients.

A simple and fast liquid chromatography tandem mass spectrometry method to determine cyclosporine A concentrations in endomyocardial biopsies.

Measuring cyclosporine A (CsA), an immunosuppressive drug used to prevent heart transplant rejection, concentrations in myocardial biopsies might be more informative than its measurement in whole blood. Therefore, a fast, accurate and reproductive method to determine CsA concentration in this complex matrix is needed. We report the validation of a liquid chromatography tandem mass spectrometry method to measure CsA concentration in heart parenchyma, applicable to everyday practice. The method was found to be precise, accurate, reproducible, specific of CsA, and without any matrix effect or carry-over. The lower limit of quantification was 50 pg of CsA in myocardium. The method was linear up to 2000 pg of CsA in myocardium. Samples were found stable for one year at - 80 °C. At last, 40 drugs which could be prescribed to heart transplant recipients were tested with the method and showed no interference with CsA signal. The method was suitable to quantify CsA in endomyocardial biopsies from heart transplanted patients. This method allows designing clinical studies aiming at exploring the relationship between CsA intra-graft concentrations and outcome.

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.