Screening for Mechanisms of Hepatotoxicity: Phospholipidosis, Steatosis, Apoptosis and Inflammatory Markers K.F. Marcoe, R. Keyser, P. TB. Nguyen, Yulia Ovechkina, and C. O’Day MDS Pharma Services – Bothell, WA, USA Multiparametric Hepatotoxicity Screening in HepG2 Cells with Comparison in Primary Hepatocytes K.F. Marcoe, Yulia Ovechkina, R. Keyser, P. TB. Nguyen, C. O’Day MDS Pharma Services – Bothell, WA, USA
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Screening for Mechanisms of Hepatotoxicity: Phospholipidosis, Steatosis,
Apoptosis and Inflammatory Markers
K.F. Marcoe, R. Keyser, P. TB. Nguyen, Yulia Ovechkina, and C. O’Day
MDS Pharma Services – Bothell, WA, USA
Multiparametric Hepatotoxicity Screening in HepG2 Cells with
Comparison in Primary Hepatocytes
K.F. Marcoe, Yulia Ovechkina, R. Keyser, P. TB. Nguyen, C. O’Day
MDS Pharma Services – Bothell, WA, USA
Liver major site of metabolism for most drugs
Based on safety, hepatotoxicity recognized as a leading cause for drug
withdrawal
Toxicity of new drug candidates routinely evaluated just prior to compounds
moving into clinical trial
Late stage In vivo toxicity studies have problems
− Costly (multiple animal species requirements)
− Large amounts of compounds
− Significant investment of resources tied to late findings
In vitro early stage toxicity studies afford
− Identification of hepatotoxic potential earlier (cost and time savings)
− Opportunities for ranking and prioritizing or development of alternatives
with lower toxicity
Multiparameter high content cell-based screening methods in drug discovery
contribute to better predictivity of human hepatotoxicity potential
Early safety screening current priority in drug development
Drug-Induce Hepatotoxicity
Early Safety Hepatotoxicity Screening Assays
Development of effective in vitro cell-based screening models to
assess human hepatotoxicity potential of drugs ideally requires:
Use of high content multiplexed technologies
Utilization of primary human cell and HepG2 cell line hepatocyte models
Measurement of parameters
− At the single cell level
− Morphological and biochemical
− Investigative of pre-lethal cytotoxic effects
− Representative of different mechanisms of toxicity
− Suitable for rapid throughput
− 384 well plate format
Minimal amount of compound for testing (1 - 2 mg)
Multiplexed High Content Screening Tools
IN Cell 1000 Analyzer automated fluorescent microscopy imaging of live or fixed cells allows
Subcellular localization AND quantitation of the cellular targets
Multiplexing capabilities: multiple data points from a single assay well
High sensitivity (nuclear staining allows for normalization of cellular signals against cell number)
Measurement of individual cell responses in the heterogeneous cell populations
Customized protocols for cell image quantitation (IN Cell Developer Software)
xMAP technology using Luminex
Flow based multiplexed microsphere assay system
Multi-analyte protein analysis in the same well
Nuclei staining with IN Cell imaging allows normalization of cellular signals against cell number
Evaluation of toxicity ‘window / safety margin’ and mechanism of death helps determine dosing and cost/benefit analysis of therapeutic agent based on prediction of in vivo toxicity potential
− Higher values predict higher in vivo safety margins
− In vitro cell-base safety margins use to rank compounds based on hepatotoxicity potential in humans
− 80% correlation between actual in vivo and in vitro cell-based toxicity results have been demonstrated (Shrivastava R, et al., O’Brien PJ, et al., Vivek C, et al.)
− Other factors contributing to toxicity profiles: drug properties, concentrations, protein binding and transport, pharmacokinetic characteristics
Provides information on the relative toxicities of candidate drugs within particular compound families to aid selection of lead candidates.
Offers insight into drug toxicity mechanism
Provides end-point-specific drug hepatotoxicities
Multiplexed In vitro Hepatotoxicity Assay
Multiplexed Hepatotoxicity Assay
HepG2 cells seeded in 384-well Collagen I coated optical plates, incubated
24 hrs
Cells incubated 72 hrs with test compounds serially diluted ½ log over 10
concentrations
Post 72 hrs incubation cells fixed and immunolabeled with:
− Anti-active Caspase-3 for detection of apoptosis
− Anti-phospho-Histone-3 for detection of cell cycle
− Stained with a nuclear dye for cell proliferation quantification
Automated fluorescence microscopy carried out using a GE Healthcare