Krogstad Veronica, Vethe Nils T, Robertsen Ida, Hasvold Grete, Ose Anne-Marthe D, Hermann Monica, Andersen Anders M, Chan Joe, Skauby Morten, Svensson My H S, Åsberg Anders, Christensen Hege
Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo.
Department of Pharmacology, Oslo University Hospital, Rikshospitalet, Oslo.
Ther Drug Monit. 2018 Jun;40(3):292-300. doi: 10.1097/FTD.0000000000000510.
Tacrolimus (TAC) is currently the cornerstone of immunosuppressive protocols for renal transplant recipients. Despite therapeutic whole blood monitoring, TAC is associated with nephrotoxicity, and it has been hypothesized that intrarenal accumulation of TAC and/or its metabolites are involved. As TAC is a substrate of P-glycoprotein (P-gp), the expression and activity of this efflux transporter could influence the levels of TAC in renal tissue. The primary aim of this study was to develop and validate a method for quantification of TAC in tissue homogenates from single human renal core biopsies. The secondary aim was to provide measures of P-gp expression and of the demethylated metabolites of TAC in the same renal biopsy.
Human renal tissue, with and without clinical TAC exposure, was used for method development and validation. Homogenates were prepared with bead-beating, and concentrations of TAC and its demethylated metabolites were analyzed with liquid chromatography tandem mass spectrometry after protein precipitation. A Western blot method was used for semiquantification of P-gp expression in the homogenates. The final methods were applied to renal core biopsies from 2 transplant patients.
The TAC assay showed within- and between-run mean accuracy between 99.7% and 107% and coefficients of variation ≤6.7%. Matrix effects were nonsignificant, and samples were stable for 3 months preanalytically when stored at -80°C. TAC concentrations in the renal core biopsies were 62.6 and 43.7 pg/mg tissue. The methods for measurement of desmethyl-TAC and P-gp expression were suitable for semiquantification in homogenates from renal core biopsies.
These methods may be valuable for the elucidation of the pharmacokinetic mechanisms behind TAC-induced nephrotoxicity in renal transplant recipients.
他克莫司（TAC）是目前肾移植受者免疫抑制方案的基石。尽管进行了治疗性全血监测，但TAC仍与肾毒性相关，据推测，TAC及其代谢产物在肾内的蓄积与之有关。由于TAC是P-糖蛋白（P-gp）的底物，这种外排转运蛋白的表达和活性可能会影响肾组织中TAC的水平。本研究的主要目的是开发并验证一种定量单个人类肾芯活检组织匀浆中TAC的方法。次要目的是在同一肾活检组织中提供P-gp表达及TAC去甲基化代谢产物的测量指标。
使用有或无临床TAC暴露的人类肾组织进行方法开发和验证。通过珠磨法制备匀浆，蛋白质沉淀后用液相色谱串联质谱法分析TAC及其去甲基化代谢产物的浓度。采用蛋白质印迹法对匀浆中P-gp表达进行半定量分析。最终方法应用于2例移植患者的肾芯活检组织。
TAC检测的批内和批间平均准确度在99.7%至107%之间，变异系数≤6.7%。基质效应不显著，样本在-80°C储存时在分析前3个月内稳定。肾芯活检组织中TAC浓度分别为62.6和43.7 pg/mg组织。去甲基-TAC测量方法和P-gp表达方法适用于肾芯活检组织匀浆的半定量分析。
这些方法对于阐明肾移植受者中TAC诱导的肾毒性背后的药代动力学机制可能具有重要价值。
