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In Vitro Lysosomal Trapping (Sequestration) Studies

Lysosomal Trapping (or sequestration) is a physicochemical (non-enzymatic and non-transporter mediated) process by which membrane-bound organelles within hepatocytes can sequester lipophilic amine drugs, potentially limiting clearance and reducing therapeutic effect.

We offer an in vitro definitive study designed to evaluate inhibition and uptake (mechanistic determination) to predict lysosomotropic behavior to better assess risk impacted by pharmacokinetics, drug-drug interactions (DDIs), and other safety parameters.

Lysosomal Trapping Diagram

Our Approach to Lysosomal Trapping Studies in Support of Preclinical Drug Development

If your drug candidate is lipophilic or amphiphilic with ionizable amines, you should consider investigating lysosomal trapping potential because lysosomal trapping (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.

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 lysosomotropics 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.

Assays to Determine Lysosomal Activity, Inhibition IC50 and Mechanistic Action

Test System

As the liver is a lysosome rich organ, we use immortalized hepatocytes (Fa2N-4) 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 metabolism or transporter activity, but do propagate in culture and readily attach to collagen plates.

LysoTracker Inhibition Assay

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. 

  • Fa2N-4 cells plated in a 96-well plate as a test system
  • LysoTracker Red used as the fluorescent probe
  • Seven 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

Mechanistic Determination Assay

Our scientists 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 choride or a lysosomotropic compound is used to determine partitioning into lysosomes 
  • Studies customized to fit your needs

The table below 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.

Molecular Species: Acid

DrugDrug ClassLogPlogDph7.4PSA(A2)Acidic or anionic pKaBasic or cationic pKaLysoTracker Red IC50(μM)Cytotxicity (LDH release)EVIDENCE FOR LYSOSOMAL TRAPPING
DiclofenacNSAID4.261.1049.334.00>500Little or noneNone
IbuprofenNSAID3.841.3437.304.85>500Little or noneNone
KetaprofenNSAID3.610.3954.373.88>500Little or noneNone
KeterolacNSAID2.28-0.9659.303.84>500Little or noneNone
TenoxicamNSAID0.34-0.7199.603.63, 7.214.78>500Little or noneNone
Atorvastatin Statin5.392.44111.794.33, 11.82>500Little or noneNone
FluvastatinStatin3.831.0682.694.56>500Little or noneNone
PravastatinStatin1.651.38124.294.21>500Little or noneNone
RosuvastatinStatin1.921.24140.924.00>500Little or noneNone

Molecular Species: Base

DrugDrug ClassLogPlogDph7.4PSA(A2)Acidic or anionic pKaBasic or cationic pKaLysoTracker Red IC50(μM)Cytotxicity (LDH release)EVIDENCE FOR LYSOSOMAL TRAPPING
FluoxetineAnitdepressant (SSRI)4.171.8321.269.80150Little or noneYes
ParoxetineAnitdepressant (SSRI)3.150.8339.729.77130Little or noneYes
DesipramineAntidepressant (Tricyclic)3.901.3715.2710.02220Little or noneYes
ImipramineAntidepressant (Tricyclic)4.282.486.489.20260Little or noneYes
AstemizoleAntihistamine5.393.9742.326.54, 8.75110YesPossible
AmodiaquineAntimalarial4.532.2448.399.126.46, 10.23>500Little or nonePossible
ChloroquineAntimalarial3.930.8828.167.29, 10.32220Little or noneYes
DextromethorphineAntitussive3.491.0812.479.85>500Little or nonePossible
LabetalolBeta Blocker2.971.2695.588.059.80>500Little or nonePossible
PropranololBeta Blocker2.580.3641.499.6792Little or noneYes

Molecular Species: Neutral

DrugDrug ClassLogPlogDph7.4PSA(A2)Acidic or anionic pKaBasic or cationic pKaLysoTracker Red IC50(μM)Cytotxicity (LDH release)EVIDENCE FOR LYSOSOMAL TRAPPING
AcetaminophenAnalgesic0.910.9049.339.46>500Little or noneNone
ErlotinibAnticancer3.203.2074.734.59>500Little or noneNone
GefitinibAnticancer3.753.6468.744.51, 6.8577Little or noneYes
LapatinibAnticancer4.644.43106.353.80, 7.20380YesPossible
FluconazaoleAntifungal antibiotic0.560.5681.6512.711.96. 2.56>500Little or noneNone
NifedipineCalcium channel blocker1.821.81110.453.89>500Little or noneNone

Molecular Species: Zwitterion

DrugDrug ClassLogPlogDph7.4PSA(A2)Acidic or anionic pKaBasic or cationic pKaLysoTracker Red IC50(μM)Cytotxicity (LDH release)EVIDENCE FOR LYSOSOMAL TRAPPING
CetirizineAntihistamine3.580.7453.013.602.12, 7.79>500Little or noneNone
RacloprideAntipsychotic3.292.0361.806.268.47>500Little or noneNone

See the science

Read our scientists’ study demonstrating the utility of immortalized hepatocytes (Fa2N-4 cells) for determining the lysosomal sequestration of lipophilic amines

Study director analyst in vitro ADME studies

Performing your own assays?

We provide immortalized hepatocytes (Fa2N-4 cells) and accompanying media available for use as a test system in your in-house lysosomal trapping assays

Immortalized hepatocytes in lysosomal trapping assay in drug development