Utilising magnetically isolated lysosomes for direct quantification of intralysosomal drug concentrations by LC MS/MS analysis: An investigatory study with imipramine

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Abstract

Lysosomes are acidic intracellular organelles that can extensively sequester basic lipophilic drugs due to pH and membrane partitioning, and therefore may significantly influence subcellular drug concentrations. Current in vitro methods for lysosomal drug sequestration evaluation typically lack the ability to accurately and sensitively quantify drug concentrations directly within the lysosome. In the current study, magnetic lysosomal isolation was used in the lysosome rich rat NR8383 cell line and combined with LC-MS/MS analysis to quantify intralysosomal concentrations and lysosomal partitioning (KpLysosome) values of imipramine. The purity of the isolated lysosomes was validated by enzymatic and electron microscopy analysis. Lysosomal imipramine accumulation was explored using two methods: addition of imipramine to cells followed by lysosomal isolation (Method 1), and direct addition of imipramine to isolated lysosomes (Method 2). This work highlighted that both experimental buffers and ATP influence intralysosomal drug concentrations, and non-specific drug binding and/or re-distribution limits the use of Method 1. Method 2 may benefit future lysosomal drug accumulation studies, as imipramine demonstrated high KpLysosome values (3,500), comparable to in silico predictions. This study reports a novel method for the direct quantification of intralysosomal drug concentrations that has the ability to be adapted to other cell types.

Bibliographical metadata

Original languageEnglish
Pages (from-to)2891-2901
JournalJournal of Pharmaceutical Sciences
Volume109
Issue number9
Early online date3 Jun 2020
DOIs
Publication statusPublished - 1 Sep 2020