Lysosomal Trapping

Overview

Lysosomes are acidic organelles (pH 4-5) that play a key role in various metabolic processes, however they can also sequester lipophilic amine drugs through a physiochemical (non-enzymatic and non-transporter mediated) process known as lysosomal trapping. This “trapping” by lysosomes (in conjunction with phospholipid binding in tissues) can cause many cationic drugs to exhibit large volumes of distribution and high liver-to-blood ratios. While lipophilic amine drugs readily diffuse across cell membranes at physiological pH by passive diffusion, upon diffusion into lysosomes, they become protonated (positively charged), which restricts their diffusion back across the lysosomal membrane into the cytosolic space.

Lysosomal Trapping Diagram
The diagram above (click to expand) represents the basis of pH partitioning of lipophilic amines into lysosomes (adapted from Kazmi et al., 2013)

Competition for lysosomal trapping has been the subject of some speculation as a potential mechanism of DDIs. As many CNS and cardiovascular drugs are lysosomotropics (drugs that undergo lysosomal sequestration) there is the possibility that concomitant administration of lysomotropics could lead to elevated drug exposure levels as competition for lysosomal sequestration increases or lysosomal pH is elevated by virtue of amine accumulation. Furthermore, compounds that accumulate in lysosomes have a strong association with the development of drug induced phospholipidosis, characterized by the excess accumulation of phospholipids.

As the liver is a lysosome rich organ, Sekisui XenoTech uses immortalized hepatocytes or Fa2N-4 cells as the in vitro test system to evaluate lysosomal trapping of lipophilic amine drugs. These cells make an ideal test system because they are transformed human hepatocytes that do not retain significant transporter activity, but do propagate in culture and readily attach to collagen plates.

Assays

Sekisui XenoTech offers two distinct in vitro assays to determine whether your compound is sequestered in lysosomes:

  • LysoTracker Inhibition Assay (Screen) – The fluorescent probe Red DND-99 (also known as LysoTracker Red), has been shown to be highly specific for lysosomal accumulation and has been used in numerous non-hepatic cell lines. Co-incubation with lysosomotropic compounds will block the accumulation of LysoTracker Red. Sekisui XenoTech offers this approach for the rapid screening of drugs for their potential to be trapped in lysosomes.
    • Fa2N-4 cells plated in a 96-well plate as a test system
    • LysoTracker Red used as the fluorescent probe
    • Six test article concentrations
    • Test article and LysoTracker Red are incubated for 30 min concurrently
    • LDH release assay is used for cytotoxicity testing
    • Samples are analyzed on a fluorescent plate reader
    • Data are presented as LysoTracker Red inhibition IC50 curves

Lysosomal Trapping Physicochemical Properties
This table (click to expand) represents predicted physicochemical properties and experimentally-determined LysoTracker Red IC50 values for 27 compounds (adapted from Kazmi et al., 2013)
*Note: All physicochemical properties were calculated with MarvinSketch 5.9.0 as described in the Materials and Methods; compounds that showed >25% LysoTracker Red inhibition at the highest tested concentration but did yield an IC50 value or exhibited concentration-dependent cytotoxicity were classified as ‘Possible’ for undergoing lysosomal sequestration; LDH is lactate dehydrogenase; SSRI is selective serotonin reuptake inhibitor; NSAID is non-steroidal anti-inflammatory drug; PSA is polar surface area.*

Lysosomal Trapping Photomicrograph
This photomicrograph represents epifluorescence microscopy of cryopreserved human hepatocytes and immortalized hepatocytes (Fa2N-4 cells) treated with LysoTracker Red (adapted from Kazmi et al., 2013)

  • Mechanistic Determination – Sekisui XenoTech can also perform mechanistic experiments to provide more detailed information on the propensity of a compound to be trapped in lysosomes.
    • Plated Fa2N-4 cells are incubated with the test article 
    • Test article quantified by LC/MS 
    • Test article partitioning (uptake) into cells determined
    • Treatment with ammonium chloride or other lysosomotropic is used to determine partitioning into lysosomes 
    • Studies customized to fit your needs

Lysosomal Trapping Mechanistic Determination
The graphs above (click to expand) represent time-course of the partitioning of propranolol and imipramine in Fa2N-4 cells with and without ammonium chloride (adapted from Kazmi et al., 2013).

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