Simultaneous quantification of cyclosporin, tacrolimus, sirolimus and everolimus in whole blood by UHPLC-MS/MS for therapeutic drug monitoring.

The aim of this study was to develop and validate a UHPLC-MS/MS assay to quantify cyclosporin (CYC), tacrolimus (TAC), sirolimus (SIR) and everolimus (EVE) in human whole blood for therapeutic drug monitoring. Analytes were extracted from 50 µL human whole blood by protein precipitation. The separation of the drugs was performed on an Acquity UPLC BEH C18 column. Analytes were eluted with a mobile phase consisting of 2 mM ammonium acetate with 0.1% formic acid (v/v) in deionised water and 2 mM ammonium acetate with 0.1% formic acid (v/v) in methanol at a flow rate of 300 µL/min in gradient elution. The method performance was evaluated by analysing patient blood samples and/or external quality control samples [proficiency testing (PT) scheme]. The method was linear from 23.75 to 1094.0, 1.3 to 42.4, 1.3 to 47.0 and 1.2-41.6 µg/mL for CYC, TAC, SIR and EVE, respectively. The within- and between-assay reproducibility results were ˂ 11%. Results from PT and patient sample quantification were comparable to those obtained previously by an in-house validated method using protein precipitation and liquid-liquid extraction. This method showed good analytical performance for quantifying CYC, TAC, SIR and EVE in whole blood over their respective calibration ranges.

Simultaneous Determination of Everolimus, Sirolimus, Tacrolimus, and Cyclosporine-A by Mass Spectrometry.

Measurement of immunosuppressive drug concentrations cyclosporine A (CyA), tacrolimus (TAC), sirolimus (SIR), and everolimus (EVE) in blood is an important application of therapeutic drug monitoring. These immunosuppressive agents are used in combined regimens and nowadays the liquid chromatography and tandem mass spectrometry is the best option for simultaneous analysis of these drugs in one short run. We developed an liquid chromatography and tandem mass spectrometry methodology in-house to measure the combination of immunosuppressants in a single blood sample from transplant patients in Brazil. We analyzed 235 combinations of 4 immunosuppressive drugs in patient blood to validate this study. The measuring ranges were 9 to 1000 ng/mL for CyA and 2 to 50 ng/mL for TAC, SIR, and EVE. Accuracy of the method was between 83.87% and 126.6% (coefficient of determination [r2] > 0.995). Validation of variation was ≤15% for lower limit of quantification. In our analysis 20% of patients treated with EVE showed concentration range of 6 to 6.9 ng/mL, 28% of patients treated with SIR showed a concentration range of 4 to 4.9 ng/mL to TAC, 22% of patients showed concentration range of 5 to 5.9 ng/mL, and finally 50% of patients treated with CyA showed concentration range of 20 to 30 ng/mL. Our routine laboratory was able to implement this new methodology in-house to measure simultaneous CyA, TAC, SIR, and EVE in a single blood sample from transplant patients with a sensibility and rapid quantification analysis.

Adequacy of Initial Everolimus Dose, With and Without Calcineurin Inhibitors, in Kidney Transplant Recipients.

BACKGROUND: This study investigates the adequacy of initial everolimus (EVR) dose, with and without calcineurin inhibitors (CNI), in kidney transplant recipients. METHODS: This retrospective cohort analysis involved data from 305 kidney transplant recipients participating in 3 randomized trials receiving reduced dose cyclosporin A (CsA) combined with EVR 0.75 mg BID (CSA/EVR0.75, N = 32) or 1.5 mg BID (CSA/EVR1.5, N = 31), reduced dose tacrolimus (TAC) combined with EVR 1.5 mg BID (TAC0.05/EVR1.5, N = 83), standard dose TAC combined with EVR 1.5 mg BID (TAC0.1/EVR1.5, N = 93), and EVR 1.5 mg BID (EVR1.5, N = 66) with TAC introduction after day 5. The adequacy of the initial EVR dose, based on EVR whole blood trough between 3 and 8 ng/mL, was compared using first EVR blood concentrations obtained at day 3 after transplantation. RESULTS: Recipient age, proportion of patients with diabetes mellitus, and proportion of grafts from living donors were different among the groups. Dose-corrected EVR concentrations were higher in patients receiving CsA than in those receiving TAC or no calcineurin inhibitors (6.7 ± 5.9 versus 5.4 ± 2.2 versus 2.4 ± 0.8 versus 2.5 ± 0.9 versus 2.2 ± 0.7, P = 0.000). No differences were observed comparing dose adjusted EVR concentrations combined with TAC or alone (P = 0.073). The proportion of patients with EVR concentration below <3 ng/mL was lower when EVR was combined with CsA (25 versus 3 versus 43 versus 33 versus 50%, P = 0.000). Later introduction of TAC did not influence EVR concentrations. There were no differences in mean CsA concentrations comparing patients receiving EVR 0.75 or 1.5 mg BID (240 ± 143 versus 213 ± 105 ng/mL). On the other hand, mean TAC concentrations were higher according to the initial TAC dose regimen (6.4 ± 3.9 versus 9.8 ± 5.9 ng/mL). CONCLUSIONS: In de novo kidney transplant recipients, the choice of the initial dose of EVR should consider the type of calcineurin inhibitor to reach target EVR concentration within the first week in a higher proportion of patients, maximizing the efficacy/toxicity profile.

Longitudinal Pharmacokinetics of Everolimus When Combined With Low-level of Tacrolimus in Elderly Renal Transplant Recipients.

BACKGROUND: Although the proportion of elderly patients among renal transplant recipients has increased, pharmacokinetic (PK) studies of immunosuppressants rarely include older patients. METHODS: We studied 12-hour everolimus (EVL) PK in 16 elderly renal transplant recipients (all whites; 10 men; mean age, 64 ± 2 years (61-71 years), in 4 separate timepoints (at 7, 30, 60, and 150 days) after EVL introduction, corresponding to a mean postrenal transplantation day: PK1 (43 ± 4 days), PK2 (65 ± 7 days), PK3 (106 ± 17 days), and PK4 (206 ± 40 days). Patients received EVL (target trough level (Ctrough, 3-8 ng/mL), prednisone, and tacrolimus (TCL) (target Ctrough, 2-5 ng/mL). RESULTS: Mean TCL-Ctrough was 7.2 ± 3.8, 4.9 ± 2.2, 4.9 ± 2.2, and 4.5 ± 1.2 ng/mL at PK1, PK2, PK3, and PK4, respectively. There were no differences among timepoints for mean EVL daily dose (data shown as PK3) (3.5 ± 1.3 mg/d), Ctrough (4.7 ± 2.5 ng/mL), AUC0-12h (106 ± 51 ng/h per mL), Caverage (8.8 ± 4.2 ng/mL), Cmax (19.2 ± 9.7 ng/mL), apparent Half-life (11.7 ± 4.2 hours), estimated total body clearance (0.39 ± 0.27 L/h), or fluctuation (166 ± 65%). Also, none of those PK parameters differed statistically when adjusted for body weight. EVL-Ctrough showed a very high correlation (r = 0.849) with AUC0-12h. CONCLUSIONS: Our data indicate that elderly renal transplant recipients starting EVL 1 month after transplantation along with a steady-state TCL level, present stable EVL-PK parameters without significant changes in dose or exposure during the first 6 months after renal transplantation.