Quantitation of Lysosomal Trapping of Basic Lipophilic Compounds Using In Vitro Assays and In Silico Predictions Based on the Determination of the Full pH Profile of the Endo-/Lysosomal System in Rat Hepatocytes

Abstract

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 50 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 pK a values as input, allowing early predictions even prior to chemical synthesis. The concentration dependency-i.e., the saturability of the trappingcan then be determined by the IC 50 values generated in vitro. Thereby, a more quantitative assessment of the susceptibility of basic lipophilic compounds for lysosomal trapping is possible. This work was financed by Bayer AG, Germany. https://doi.org/10.1124/dmd.118.084541. s This article has supplemental material available at dmd.aspetjournals.org. ABBREVIATIONS: DAPI -49,6-diamidino-2-phenylindole; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LTR, LysoTracker Red DND-99; PBS, phosphate-buffered saline. 49

Existing highly accumulating lysosomotropic drugs with potential for repurposing to target COVID-19

“…Consequently, as the drug moves into more acidic compartment, the equilibrium between protonated and neutral forms is shifted towards the protonated form and a higher fraction of drug molecules become trapped inside the cell [ 8 ]. The highest concentration of lysosomotropic drugs is reached in lysosomes with pH of 4–5, and in late endosomes with pH of 5–6 [ 9 ]. For strong lysosomotropic drugs it was estimated that 50–70 % of intracellularly accumulated compound is stored in lysosomes and endosomes [ 10 ], leading to extreme concentrations in these compartments.…”

“…The impact of lysosomotropic drug accumulation on cells is obvious: lysosomes increase in volume [ 14 ], lysosomal function is impaired leading to downregulation of autophagy [ 15 , 16 ], endocytosis and the entire membrane trafficking in the cell is reduced [ 17 ]. Lysosomal and endosomal pH increases as a consequence of the overload of basic compounds [ 9 , 18 ]. Due to the cationic drug binding to phospholipid bilayer and change of the surface bilayer charge, the degradation of phospholipids is slowed [ 12 ] resulting in the accumulation of excess phospholipid membrane and vesicles inside the cell, which may lead to an adverse effect known as phospholipidosis [ 19 ].…”

Lysosomal Biogenesis and Implications for Hydroxychloroquine Disposition

“…This results in concentrations significantly higher than other compartments within the cell (Duvvuri and Krise, 2005). Key mechanisms contributing to pharmacokinetics have been considered and consolidated into in vitro mathematical models to describe cellular uptake of compounds with similar physicochemical properties to HCQ, namely weakly basic and lipophilic (Ishizaki et al, 2000;Trapp et al, 2008;Kornhuber et al, 2010;Schmitt et al, 2019). These models assume the cell behaves as a static system of compartments, but recent evidence suggests that lysosomotropic compounds induce changes in the lysosome itself.…”

Improved predictions of time-dependent drug-drug interactions by determination of cytosolic drug concentrations

“…Hence, the F ic of such compounds becomes inflated, resulting in an overestimation of the cytosolic bioavailability 7 . Determination of the intracellular bioavailability in the presence of chloroquine or other compounds that increase the lysosomal pH and eliminate the trapping of basic drugs inside endo-lysosomal compartments 12,18,19 could give a better estimate for the relevant concentration at the CYP3A4 enzyme metabolic site, since it should better reflect the cytosolic bioavailability (F cyto ).…”