Data Sheet 1_Ex vivo perfusion model of mouse liver and its application to analyze the effects of OCT1 deficiency.pdf

IntroductionThe liver plays a critical role in drug pharmacokinetics. In in vivo experiments, it is difficult to isolate the liver’s contribution to drug systemic concentrations from that of the intestine and kidneys. Rat liver perfusion is well-established for studying liver-specific effects. However, rats are not easily genetically manipulated, complicating analyses of individual drug transporters and metabolizing enzymes. This study aimed to establish an ex vivo liver perfusion model in mice and to apply it to analyze the effects of mOct1 on drug metabolism.
MethodsAfter euthanizing, the liver of 6- to 28-week-old mice was perfused via an indwelling venous catheter in the portal vein as entry and into the caudal vena cava toward the heart as exit. Perfusion solutions were prewarmed to 42 °C and pumped at 2 mL/min. First, HBSS supplemented with 0.5 mM EDTA was used to exsanguinate the liver, followed by HBSS alone and then HBSS containing the drug of interest. Drug and metabolite concentrations in the perfusates were measured by LC-MS/MS.
Results and conclusionThe method enables reproducible and reliable perfusion of mouse livers. We applied it to study the effects of Oct1 knockout on drug metabolism. Oct1 knockout affected the first-pass metabolism of codeine, including the formation of the metabolites morphine and morphine-3-glucuronide, as well as the first-pass metabolism of proguanil and the formation of cycloguanil. The model is applicable to any mouse strain, genetic background, and substrate of interest and is thus applicable to a wide variety of research questions.

引言
肝脏在药物药代动力学中发挥关键作用。在体内实验中，难以将肝脏对药物全身浓度的贡献与肠道和肾脏的贡献区分开来。大鼠肝脏灌注模型已被广泛用于研究肝脏特异性效应，但大鼠不易进行基因操作，这使得针对单个药物转运蛋白及代谢酶的分析变得复杂。本研究旨在建立小鼠离体肝脏灌注模型，并利用该模型分析小鼠有机阳离子转运体1（mOct1）对药物代谢的影响。

方法
小鼠处死后，通过门静脉留置静脉导管作为灌注入口，经下腔静脉向心脏方向作为灌注出口，对6至28周龄小鼠的肝脏进行灌注。灌注液预先预热至42℃，并以2mL/min的流速泵入。首先使用添加了0.5mM乙二胺四乙酸（EDTA）的汉克平衡盐溶液（HBSS）对肝脏进行驱血，随后依次使用单纯HBSS以及含有目标药物的HBSS进行灌注。采用液相色谱-串联质谱法（LC-MS/MS）测定灌注液中的药物及代谢物浓度。

结果与结论
该方法可实现稳定且可重复的小鼠肝脏灌注。我们利用该模型研究了Oct1敲除对药物代谢的影响：Oct1敲除可影响可待因的首过代谢，包括其代谢产物吗啡及吗啡-3-葡糖苷酸的生成，同时还可影响氯胍的首过代谢及环氯胍的生成。该模型适用于任意小鼠品系、遗传背景以及目标底物，因此可应用于众多不同的研究课题。