Intracellular sequestration of amiodarone: role of vacuolar ATPase and macroautophagic transition of the resulting vacuolar cytopathology

Abstract

Background and purpose:Tissue deposits of the anti-arrhythmic drug amiodarone are a major source of side effects (skin discoloration, etc.). We addressed the mechanism of the concentration of amiodarone in cells, and characterized the resulting vacuolar cytopathology and its evolution towards macroautophagy. Experimental approach: Sequestration of amiodarone in human cells (macrophages, smooth muscle cells, HEK 293a cells) was evaluated using its violet fluorescence and cytopathology using GFP-conjugated subcellular markers. Autophagic signalling was probed by immunoblotting for the effector protein LC3. A patient biopsy of amiodarone-induced blue-gray skin discoloration was investigated for the presence of macroautophagy (immunofluorescence for LC3). Key results: Most of the amiodarone (1-20 mM, 4-24 h) captured by cultured cells (macrophages were most avid) was present in enlarged vacuoles. The specific vacuolar ATPase (V-ATPase) inhibitors, bafilomycin A1 or FR167356, prevented vacuolization and drug uptake. Vacuoles in HEK 293a cells were positive for markers of late endosomes and lysosomes (GFP-Rab7, -CD63) and for an effector of macroautophagy, GFP-LC3. The vacuoles accumulated endogenous LC3 and filled with lipids (Nile red staining) following longer amiodarone treatments (Ն24 h). The electrophoretic mobility of both GFP-LC3 and endogenous LC3 changed, showing activation in response to amiodarone. Paraffin tissue sections of the pigmented skin exhibited granular LC3 accumulation in superficial dermis macrophages. Conclusion and implications: Vacuolar sequestration of amiodarone occurs at concentrations close to therapeutic levels, is mediated by V-ATPase and evolves towards persistent macroautophagy and phospholipidosis. This cytopathology is not cell type specific, but tissue macrophages appear to be particularly susceptible.

High affinity capture and concentration of quinacrine in polymorphonuclear neutrophils via vacuolar ATPase-mediated ion trapping: Comparison with other peripheral blood leukocytes and implications for the distribution of cationic drugs

“…Mature macrophages, e.g. those harvested from the lung alveolar space or differentiated in vitro from monocytes, were highly competent to concentrate a related drug, amiodarone, in previous studies (Baritussio et al, 2001;Morissette et al, 2009). Quinacrine uptake by cultured human vascular smooth muscle cells or in undifferentiated U937 monoblastoid cells, equally dependent on V-ATPase activity, was comparable in many respects to that of peripheral blood PMNLs, but of lower apparent affinity (K M 8.7 µM and 14.8 µM, respectively) and LC3 accumulation occurred at a somewhat higher drug concentration than in PMNLs (present study vs. Marceau et al, 2009;2013).…”

“…In examined cultured cells, the driving force of this pseudo-transport is provided by vacuolar (V)-ATPase, a proton pump expressed in the trans-Golgi and derived organelles (endosomes, lysosomes, secretory granules). Thus, specific vacuolar (V-)ATPase inhibitors such as bafilomycin A1 have been extensively used to document the cellular capture and retention of amines, such as triethylamine (Et 3 N; Morissette et al, 2005) and drugs from various therapeutic classes that can be considered to be substituted forms of this tertiary amine: in increasing order of lipophilicity, procainamide (Morissette et al, 2008), lidocaine (Bawolak et al, 2010), chloroquine (Zheng et al, 2011), quinacrine 2013) and amiodarone (Stadler et al, 2008;Morissette et al, 2009). The threshold concentration for inducing the retention of amines and the characteristic vacuolar morphology that derives from it decreases with increased lipophilicity because the first step of ion trapping is believed to be simple diffusion of the uncharged form though the plasma and vacuolar membranes.…”

Vacuolar ATPase-Mediated Cellular Concentration and Retention of Quinacrine: A Model for the Distribution of Lipophilic Cationic Drugs to Autophagic Vacuoles

“…The postulated first step of this form of sequestration is the crossing of plasma and vacuolar membranes by simple diffusion, a process that is inhibited for procainamide by acidifying the extracellular fluid (Morissette et al, 2008b) and which should be greater for drugs with increased lipophilicity. Consistent with this idea, the antiarrhythmic agent amiodarone (logP 7.2), which can be considered as a substituted triethylamine similar to the more hydrophilic agent procainamide (logP 1.13), was concentrated in cell vacuoles and activated LC3 processing (formation of LC3 II) at 5 to 20 M (Morissette et al, 2009). Accumulation of macroautophagic vacuoles has been observed in the skin of a patient with amiodarone-induced skin discoloration (Morissette et al, 2009), a side effect caused by drug deposition in tissues (Ammoury et al, 2008).…”

“…Consistent with this idea, the antiarrhythmic agent amiodarone (logP 7.2), which can be considered as a substituted triethylamine similar to the more hydrophilic agent procainamide (logP 1.13), was concentrated in cell vacuoles and activated LC3 processing (formation of LC3 II) at 5 to 20 M (Morissette et al, 2009). Accumulation of macroautophagic vacuoles has been observed in the skin of a patient with amiodarone-induced skin discoloration (Morissette et al, 2009), a side effect caused by drug deposition in tissues (Ammoury et al, 2008).…”

A Physiologically Based Pharmacokinetic Model of Amiodarone and its Metabolite Desethylamiodarone in Rats: Pooled Analysis of Published Data

“…Thus, a 'deep tissue' sub-compartment was incorporated into all the tissue compartments except fat and thyroid, to improve the model. AMD is a lipophilic cationic drug and could be sequestered in lysosomes [56,57]. The 'deep tissue' can be regarded as the lysosomes or other possible sites where AMD is very slowly released once bound.…”