Bayer AG, Pharmaceuticals R&D, Translational Sciences, Research Pharmacokinetics, Berlin, Germany (M.V.S., P.L., A.R.); and Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany (M.V.S., G.F.).
Bayer AG, Pharmaceuticals R&D, Translational Sciences, Research Pharmacokinetics, Berlin, Germany (M.V.S., P.L., A.R.); and Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany (M.V.S., G.F.)
Drug Metab Dispos. 2019 Jan;47(1):49-57. doi: 10.1124/dmd.118.084541. Epub 2018 Nov 8.
Lysosomal sequestration may affect the pharmacokinetics, efficacy, and safety of new basic lipophilic drug candidates potentially impacting their intracellular concentrations and tissue distribution. It may also be involved in drug-drug interactions, drug resistance, and phospholipidosis. However, currently there are no assays to evaluate the lysosomotropic behavior of compounds in a setting fully meeting the needs of drug discovery. We have, therefore, integrated a set of methods to reliably rank order, quantify, and calculate the extent of lysosomal sequestration in rat hepatocytes. An indirect fluorescence-based assay monitors the displacement of the fluorescence probe LysoTracker Red by test compounds. Using a lysosomal-specific evaluation algorithm allows one to generate IC values at lower than previously reported concentrations. The concentration range directly agrees with the concentration dependency of the lysosomal drug content itself directly quantified by liquid chromatography-tandem mass spectrometry and thus permits a quantitative link between the indirect and the direct trapping assay. Furthermore, we have determined the full pH profile and corresponding volume fractions of the endo-/lysosomal system in plated rat hepatocytes, enabling a more accurate in silico prediction of the extent of lysosomal trapping based only on p values as input, allowing early predictions even prior to chemical synthesis. The concentration dependency-i.e., the saturability of the trapping-can then be determined by the IC values generated in vitro. Thereby, a more quantitative assessment of the susceptibility of basic lipophilic compounds for lysosomal trapping is possible.
溶酶体隔离可能会影响新的亲脂性基础候选药物的药代动力学、疗效和安全性，从而潜在地影响它们的细胞内浓度和组织分布。它也可能涉及药物相互作用、药物耐药性和磷脂沉积症。然而，目前还没有在完全满足药物发现需求的环境中评估化合物溶酶体向性行为的检测方法。因此，我们整合了一组方法，可用于可靠地对大鼠肝细胞中的溶酶体隔离进行排序、定量和计算。一种基于间接荧光的测定方法可监测荧光探针 LysoTracker Red 被测试化合物取代的情况。使用溶酶体特异性评估算法可以在低于先前报道的浓度下生成 IC 值。浓度范围与通过液相色谱-串联质谱法直接定量的溶酶体药物含量的浓度依赖性直接一致，从而可以在间接和直接捕获测定之间建立定量联系。此外，我们已经确定了在贴壁大鼠肝细胞中内体/溶酶体系统的完整 pH 曲线和相应的体积分数，这使得仅基于 p 值作为输入就可以更准确地进行体内溶酶体捕获程度的计算机预测，甚至可以在化学合成之前进行早期预测。然后可以通过体外生成的 IC 值来确定捕获的浓度依赖性，即饱和度。从而可以对亲脂性碱性化合物对溶酶体捕获的易感性进行更定量的评估。
