Screening for Mechanisms of Hepatotoxicity: Phospholipidosis, Steatosis, Apoptosis and Inflammatory Markers

1. 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

2. Drug-Induce Hepatotoxicity
 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

3. 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)

4. 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

5. Multiplexed High Content Screening
Hepatotoxicity Early Safety Platform
HCS Hepatotoxicity Early Safety Platform
Hepato-toxicity
(cell proliferation, apoptosis, mitosis)
Hepato-Lipid Accumulation
(cell proliferation, phospholipidosis, neutral lipids)
Hepato-Cytokine Secretion
(cell proliferation, inflammatory markers)

6. Multiplexed In vitro Hepatotoxicity Assay
In vitro hepatotoxicity assessment
 Cultured HepG2 cells (human hepatocellular carcinoma cell line) useful
screening reagent
 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
− In vitro cell-based safety margin = cytotoxic concentration – on-target potency
concentration (cell-based efficacy)
− 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

7. 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
IN Cell Analyzer 1000
 Images collected with a 4X objective

8. Multiplexed In vitro Hepatotoxicity Assay
Vehicle Vinblastine
Labels: Nuclei - green; Apoptotic cells - blue; Mitotic cells - red
Cell Proliferation Apoptosis Induction Cell Cycle Block
Percent of Control
over Background
over Background
Fold Induction
160 100 6
Fold Induction
140
80
120
100 60 4
80
60 40
2
40 20
20
0 0
-13 -12 -11 -10 -9 -8 -7 -6 -13 -12 -11 -10 -9 -8 -7 -6 0
-13 -12 -11 -10 -9 -8 -7 -6
[Vinblastine], M [Vinblastine], M
[Vinblastine], M
HepG2, 72 hr assay

9. Primary human hepatotoxicity assay:
MTS and HCS comparison
Amiodarone Valproic acid Amitriplyline
100 160
120
140
100 80 120
MTS 80 100
POC
POC
POC
60
80
60
40 60
40
40
20
20 20
0 0 0
0.01 1 100 0.01 1 100 0.0 1 100
Concentration microM Concentration microM 1 Concentration microM
HCS
24 hour compound treatment of human primary hepatocytes; High Content Screening approach (HCS)

10. Primary human hepatotoxicity assay:
MTS and HCS comparison
HCS % of attached
MTS Viability IC50
Compound live cells IC50
(microM)
(microM)
Tamoxifen 36.7 ± 5.6 19.8 ± 0.7
Chlorpramazine 28.0 ± 6.9 26.4 ± 1.8
Amitriplyline 49.39 ± 4.06 62.3 ± 4.2
Amiodarone 125 ± 10 146 ± 22
Valproic acid >500 >500
Astemizole 6.39 ± 2.38 12.2 ± 2.1
Rosiglitazone 354 ± 133 413 ± 80
Troglitzqone 157 ± 6 122 ± 42
24 hour compound treatment of human primary hepatocytes; High Content Screening approach (HCS)

11. Multiplexed In vitro Hepato-Lipid
Accumulation Assay
 In vitro hepato-lipid accumulation assessment
− Cultured HepG2 cells (human hepatocellular carcinoma cell line)
 Phospholipidosis accumulation of excess phospholipids in cells
− Cationic amphiphilic drugs often induce phospholipidosis in vivo
− Toxic effect due to drug or metabolite accumulation in affected tissue, leads
to acute and chronic disease
− Liver and lung common targets
 Neutral lipid accumulation
− Steatosis accumulation of fatty acids
− Other mechanisms of lipid accumulation
− Can cause enlargement of the liver and irreversible cell damage
 Flags drug candidate hepatotoxicity potential in the lead optimization stage of
drug discovery
 End-point-specific drug-induced mechanism of hepatotoxicity

12. Multiplexed In vitro Hepato-Lipid
Accumulation Assay
Multiplexed Hepato-Lipid Accumulation Assay
 HepG2 cells seeded in 384-well Collagen I coated optical plates, incubated
24 hrs
 Cells incubated for 48 hrs with
− Fluorescently-labeled phospholipid (Invitrogen, H34350) for phospholipid
accumulation detection
− Test compounds serially diluted ½ log over 10 concentrations
 Post 48 hrs incubation cells fixed and stained with
− Neutral lipid dye (Invitrogen, H34476) for neutral lipid detection
− Nuclear dye for cell proliferation quantification
 Automated fluorescence microscopy carried out using a GE Healthcare
INCell Analyzer 1000
 Images were collected with a 4X objective.

13. Multiplexed In vitro Hepato-Lipid
Accumulation Assay (HepG2)
HepG2, 48 hr assay

14. Multiplexed In vitro Hepato-Lipid
Accumulation Assay (HepG2)
Hepato-Neutral Lipid Accumulation Assay
Labels: Nuclei - green; Neutral lipids - red
HepG2, 48 hr assay

15. Multiplexed In vitro Hepato-Lipid
Accumulation Assay (HepG2)
HepG2, 48 hr assay

16. In vitro Hepato-Lipid Accumulation Assay
using primary human hepatocytes in 384 WP

17. In vitro Hepato-Lipid Accumulation Assay
using primary human hepatocytes in 384 WP
Amiodarone Amitriplyline

18. Multiplexed In vitro Hepato-Cytokine
Secretion Assay
Multiplexed Hepato-Cytokine Secretion Assay
IN Cell
xMAP™
Automated
technology
fluorescent
using
microscopy
Luminex 
imaging 
Markers of
cell count
inflammation
normalization

19. xMAP technology-Multiple Analytes /Well
 Multiplexing: Up to 100 analytes/well
 Analytes cytokines or other inflammatory markers
 Flow based assay system. Uses beads loaded with different concentrations of 2 dyes.
 Each bead has it’s own unique spectral signature (100 possible), antibodies are
derivitized to unique bead
 Beads are incubated with test sample
 Sandwich assay performed with a biotinylated second antibody (mouse)
 Streptavidin labeled with phycoerythrin (PE) used for detection
 Beads are run individually (Flow) through a laser which detects the exact bead and
then determines whether PE is associated

20. Multiplexed In vitro Hepato-Cytokine Secretion
Assay (HepG2)
Multiplexed Hepato-Cytokine Secretion Assay
 Biomarker secretion, as markers of inflammation
 Nuclear count, analyte normalization to cell number
 HepG2 cells seeded into 96-well Collagen I coated optical plates incubated
24 hrs
 Cells treated with LPS, TNFα, IL-1β and acetaminophen serially diluted ½
log over 8 concentrations incubated 48 hrs
 Post 48 hrs incubation supernatants collected, cytokine detection was
carried out using Luminex xMAP™ technology
 To quantify cell proliferation the monolayer of HepG2 cells remaining in each
plate was immediately stained with nuclear dye for normalization
 Images were collected using a GE Healthcare INCell Analyzer 1000

21. Multiplexed In vitro Hepato-Cytokine Secretion
Assay
 HepG2 cells treated with LPS, TNFα,
IL-1β and acetaminophen HepG2 cells
 Screened for the secretory presence of
30 human inflammatory markers:
LPS, TNFα,
IL-1β and
acetaminophen
IL-1α, IL-1β, IL-2, IL-4, IL-5,
IL-6, IL-8, IL-10, IL-12p40, IL-
12-70, IL-13, INFγ, INFα2a, Fibrinogen, Apo AI, Apo AII, Apo B,
IP-10, GM-CSF, G-CSF, CRP, Haptoglobin, Apo CII, Apo CIII and
MCP-1, MIP-1α, MIP-1β, SAA Apo E
TNFα, IL-1 receptor
antagonist

22. Multiplexed In vitro Hepato-Cytokine
Secretion Assay (HepG2)

23. Early Safety Screening for Mechanisms
of Hepatotoxicity
Conclusion:
 We have developed a robust and rapid throughput screening system using HepG2
cells that allows early assessment of acute and chronic mechanisms of hepatotoxicity
 Compounds with known hepatotoxicities tested in validating the capabilities of this
multiparametric HCS system in identifying and quantifying toxicities relevant to cell
proliferation, apoptosis, cell cycle, steatosis/cholestasis and phospholipidosis
demonstrated high concordance with reported hepatotoxic profile for each compound
tested
 Evaluation of cytokine secretion in HepG2 cells to identify measurable biomarkers of
inflammation demonstrated significant secretion levels for 6 of the cytokines tested
thus validating this multiplexed approach for quantifying indications of hepatic
inflammation
 These hepatotoxicity screening assays are sensitive and reproducible and provide
results that previously only have been attainable in more complex in vivo models
 Our cost-effective in vitro multiplexed HCS platform offers comprehensive predictive
information allowing pre-selection of drug scaffold designs with long-term
hepatotoxicity considerations and may even have more relevance when performed in
normal primary hepatocytes