XenoTech, LLC, Lenexa, Kansas 66219, USA.
Drug Metab Dispos. 2013 Apr;41(4):897-905. doi: 10.1124/dmd.112.050054. Epub 2013 Feb 1.
Lipophilic (logP > 1) and amphiphilic drugs (also known as cationic amphiphilic drugs) with ionizable amines (pKa > 6) can accumulate in lysosomes, a process known as lysosomal trapping. This process contributes to presystemic extraction by lysosome-rich organs (such as liver and lung), which, together with the binding of lipophilic amines to phospholipids, contributes to the large volume of distribution characteristic of numerous cardiovascular and central nervous system drugs. Accumulation of lipophilic amines in lysosomes has been implicated as a cause of phospholipidosis. Furthermore, elevated levels of lipophilic amines in lysosomes can lead to high organ-to-blood ratios of drugs that can be mistaken for active drug transport. In the present study, we describe an in vitro fluorescence-based method (using the lysosome-specific probe LysoTracker Red) to identify lysosomotropic agents in immortalized hepatocytes (Fa2N-4 cells). A diverse set of compounds with various physicochemical properties were tested, such as acids, bases, and zwitterions. In addition, the partitioning of the nonlysosomotropic atorvastatin (an anion) and the lysosomotropics propranolol and imipramine (cations) were quantified in Fa2N-4 cells in the presence or absence of various lysosomotropic or nonlysosomotropic agents and inhibitors of lysosomal sequestration (NH4Cl, nigericin, and monensin). Cellular partitioning of propranolol and imipramine was markedly reduced (by at least 40%) by NH4Cl, nigericin, or monensin. Lysosomotropic drugs also inhibited the partitioning of propranolol by at least 50%, with imipramine partitioning affected to a lesser degree. This study demonstrates the usefulness of immortalized hepatocytes (Fa2N-4 cells) for determining the lysosomal sequestration of lipophilic amines.
亲脂性（logP > 1）和亲水的药物（也称为阳离子亲脂性药物）具有可电离的胺（pKa > 6），可以在溶酶体中积累，这个过程被称为溶酶体捕获。这个过程有助于富含溶酶体的器官（如肝脏和肺）的预系统提取，再加上亲脂性胺与磷脂的结合，有助于许多心血管和中枢神经系统药物的大分布容积特征。亲脂性胺在溶酶体中的积累被认为是磷脂沉积的原因。此外，溶酶体中亲脂性胺水平的升高可导致药物在器官与血液中的比值升高，这可能被误认为是主动药物转运。在本研究中，我们描述了一种基于荧光的体外方法（使用溶酶体特异性探针 LysoTracker Red），用于鉴定永生化肝细胞（Fa2N-4 细胞）中的溶酶体趋向性药物。测试了具有各种物理化学性质的多种化合物，例如酸、碱和两性离子。此外，还在存在或不存在各种溶酶体趋向性或非溶酶体趋向性药物以及溶酶体隔离抑制剂（NH4Cl、 Nigericin 和 Monensin）的情况下，量化了非溶酶体趋向性阿托伐他汀（阴离子）和溶酶体趋向性普萘洛尔和丙咪嗪（阳离子）在 Fa2N-4 细胞中的分配。NH4Cl、 Nigericin 或 Monensin 可使普萘洛尔和丙咪嗪的细胞分配明显减少（至少减少 40%）。溶酶体趋向性药物也至少抑制了普萘洛尔的分配 50%，而丙咪嗪的分配受影响较小。这项研究表明，永生化肝细胞（Fa2N-4 细胞）可用于确定亲脂性胺的溶酶体隔离。
