Lysosomal Trapping (Lysosomotropism)

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 Vitro 25; 715-723

Protocol

Lysosomal trapping cell-based assay protocol

Test System	High content screening using lysosomal and nuclear dyes
Cell Lines	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
Quality Controls	Vehicle control Negative control = piroxicam Positive control = chloroquine
Test Article Requirements	50 µL of 30 mM DMSO solution or equivalent amount in solid compound
Data Delivery	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

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.

Vehicle Control

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.

Compound	cLogP	Basic pK_a	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)
Acetaminophen	0.51	9.46	>4000	>4000	>500	>4000	>4000	>500
Diclofenac	4.98	-2.1	>500	>500	>500	>500	>500	>500
Rosuvastatin	1.47	-2.8	>150	>150	>500	>150	>150	>500
Piroxicam	2.2	3.79	>300	>300	>150	>300	>300	>150
Amiodarone	7.24	8.47	6.34	7.14	3.8	144	75	12.6
Chloroquine	5.28	10.37	16.4	6.38	11.1	>150	>150	148.3
Chlorpromazine	5.18	9.2	9.5	16.5	5.8	59.2	27.5	20.9
Desipramine	4.02	10.02	35.6	59.2	4.6	>150	41.7	10.7
Fluoxetine	4.09	9.8	22.7	27.9	6.5	81.1	27.7	13.9
Paroxetine	3.1	9.77	19.2	20.6	5.3	67.7	18.2	10.7
Sertraline	5.06	9.85	6.43	14.2	6.6	42.3	11.3	12.2
Tamoxifen	5.93	8.76	15.2	22.2	3.8	70.4	24	6.6

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 pK_a of between 6.5 and 11 exhibit a higher propensity for lysosomotropism².

References

¹ Turk B and Turk V (2009) J Biol Chem 284; 21783-21787
² Nadanaciva S et al., (2011) Toxicology In Vitro 25; 715-723
³ Kazmi F et al., (2013) Drug Metab Dispos 41; 897-905