Lysosomes are essential for the degradation of old organelles and engulfed microbes and also play a role in programmed cell death1.
Lysosomotropic agents e.g., chloroquine, accumulate preferentially in the lysosomes of cells in the body.
These agents tend to have both lipophilic or amphiphilic compounds with basic moieties. Once inside the acidic environment of the lysosome, the drug becomes protonated and trapped in the organelle2.
Trapping of drugs in the lysosome may be one mechanism leading to drug-induced phospholipidosis for cationic amphiphilic drugs (CAD).
Cyprotex’s lysosomal trapping service is a cell based assay which uses high content screening to identify both lysosomotropism and cytotoxicity according to a recently published method2.
Although there is a limited understanding of how lysosomotropism contributes to toxicity, an association between the two has been observed and therefore assays that identify lysosomal impairment compounds are desired.
2Nadanaciva S, Lu S, Gebhard DF, Jessen BA, Pennie WD, Will Y (2011) Toxicology In Vitro25; 715-723
Lysosomal trapping cell-based assay protocol
High content screening using lysosomal and nuclear dyes
HepG2, human liver carcinoma cell line. (Other cell lines available on request, e.g., rat cardiomyocyte derived H9c2 cells)
Test Article Concentrations
8 point dose curve with top concentration based on cell loss or solubility limit
Vehicle control Negative control = piroxicam Positive control = chloroquine
Test Article Requirements
50 µL of 30 mM DMSO solution or equivalent amount in solid compound
Summary report Minimum effective concentration (MEC) and AC50 value for each cell health parameter (cell loss, nuclear morphology, DNA fragmentation and lysosomotropism) Graphical representation of data
Data for Lysosomal trapping
Known lysosomotropic and non-lysosomotropic agents were screened in the lysosomal trapping assay. Data generated were compared to those published in the literature.
Chloroquine (60 µM)
Piroxicam (150 µM)
Figure 1 Representative high content screening images for cells treated with (A) vehicle control (B) 60 µM chloroquine (a lysosomotropic agent) and (C) 150 µM piroxicam (a non-lysosomotropic agent) over a 4 hr period.
Drugs which are lysosomotropic such as chloroquine competitively inhibit uptake of the lysosomal dye into the lysosomes.
Figure 2 Graphical representation of lysosomal trapping data for chloroquine (a lysosomotropic agent) and piroxicam (a non-lysosomotropic agent).
Chloroquine causes a concentration dependent decrease in lysosomal staining compared to vehicle control treated cells. No effect was observed for piroxicam. No cytotoxic effects were observed for either compound at the concentration range tested. Data represents mean of triplicate incubations ± standard deviation.
Cyprotex decrease in lysosomal staining IC50 (µM) in HepG2
Cyprotex decrease in lysosomal staining IC50 (μM) in H9c2
Literature decrease in lysosomal staining IC50 (µM)
Cyprotex cytotoxicity IC50 (µM) in HepG2
Cyprotex cytotoxicity IC50 (µM) in H9c2
Literature cytotoxicity IC50 (µM)
Table 1 Lysosomal trapping and cytotoxicity data for a set of 12 compounds with comparison against literature data2.
Cyprotex data correlates well with data generated in the literature. It has been suggested that compounds with a cLogP > 2 and a basic pKa of between 6.5 and 11 exhibit a higher propensity for lysosomotropism2.
1 Turk B and Turk V (2009) J Biol Chem284; 21783-21787 2 Nadanaciva S et al., (2011) Toxicology In Vitro25; 715-723 3 Kazmi F et al., (2013) Drug Metab Dispos41; 897-905
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