The document summarizes an in vitro high content screening platform for assessing drug-induced hepatotoxicity through multiple mechanisms. The platform utilizes HepG2 human hepatocellular carcinoma cells in assays to measure cell proliferation, apoptosis, phospholipidosis, steatosis, cell cycle effects, and inflammatory cytokine secretion. Assays are performed in a multiplexed format using automated fluorescent microscopy and xMAP technology. The platform provides quantitative data on toxicity mechanisms and allows ranking of compounds based on their hepatotoxic potential to help guide drug development.

1. Screening for Mechanisms of
Hepatotoxicity: Phospholipidosis,
Steatosis, Apoptosis and
Inflammatory Markers
IN Cell User Meeting
May 14, 2008
Karen Marcoe
MDS Pharma Services

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 hepatocyte models (human and rat primary cells)
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
Minimal amount of compound for testing (1 - 2 mg)

4. MDS 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
IT Support
XLFit Curve-fitting software/template (embedded macros for handling multiple plates)
AIM automated Data Analysis and Report Generation

5. MDS in vitro 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. Non-contact dispensing automation system for
compound addition, cell fixing and immunostaining
Titertek
Multidro
p
Titertek Multidrop
ultidrop
T itertek M
asher V-Spin Centrifuge
05 w
ELx4 LiCONiC CO2
Biote
k
incubator Labcyte®
Echo™ 550
De-lidder
Carousel w/ 12 hotels, 16
slots each (not shown)
Velocity 11 BioCel® 1200 system

7. MDS AIM automated Data Analysis and
Report Generation
Includes curve fitting, data visualization, quality and statistical assessment
Concentration Relative cell Apoptosis (fold Mitosis (fold
(microM) count (%) induction) induction)
Mean StdDev Mean StdDev Mean StdDev
3.18E-03 96.9 2.6 0.6 0.6 1.1 0.2
1.01E-02 92.9 5.8 1.7 0.6 1.4 0.1
3.18E-02 95.3 6.3 1.6 1.1 0.9 0.1
1.00E-01 93.5 3.6 1.2 0.6 0.9 0.0
3.17E-01 94.2 8.3 0.8 0.2 1.0 0.1
1.00E+00 100.2 12.5 1.0 0.8 0.9 0.0
3.17E+00 89.3 3.6 2.0 0.7 0.8 0.0
1.00E+01 25.2 1.3 4.1 0.8 0.6 0.1
3.16E+01 18.1 2.5 10.4 7.9 0.5 0.2
1.00E+02 7.9 1.5 67.0 8.9 0.6 0.1
Blue indicates that values meet the statically significant
response criteria specified in the data interpretation
chapter.

8. 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

9. 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

10. Multiplexed In vitro Hepatotoxicity Assay
Data Analysis
Total (masked) fluorescent intensities instead of individual cell counts results in
− Higher throughput (reading time savings: 4X, 20 min/plate vs 20X, 2 hrs/plate)
− Lower CVs (more cells analyzed)
For relative cell counts, percent of control (POC), a ratio of the fluorescent intensity of
treated wells to intensity from the control wells, is used for normalization
Nx/Nc = POC
For activated Caspase-3 and phospho-Histone-3, each intensity is first normalized to the
nuclear intensity in their prospective wells. Then this ratio of each well is normalized to the
control wells
(Cx/Nx) / (Cc/Nc) = Fold induction over vehicle
Cx=Caspase intensity for well X Nx= Nuclear intensity for well X
Cc=Caspase intensity for control well Nc=Nuclear intensity for control well

11. Advantages of Multiplex Hepatotoxicity Assay
Data Output
Relative cell number quantified by total nuclear Intensity
Advantageous over cell count due to cells that were hard to mask (over lapping cells)
Allowed use of 4X objective to capture more cells (less imaging time with better statistics)
Used less image storage space
Output: EC50 / IC50
− Relative cell count IC50 = test compound concentration that produces 50% of the cell
proliferation inhibitory response or 50% cytotoxicity level
− Relative cell count EC50 = test compound concentration that produces 50% of the
maximum effective response, accounts for cells not killed (curve inflection point)
Apoptosis: Measured by activated Caspase-3 antibody
Detected a wide range of dying and dead cells (from early to late apoptosis)
Robust signal
Output: [ ] at 5-fold over background
Cell Cycle: Measured by phospho-Histone-3 antibody
Measure of cells in mitotic phase
Could also detect buildup in G1/S by decrease in mitotic signal from that of control
Output: [ ] at 2-fold over background or 2-fold below background

12. 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 0
-13 -12 -11 -10 -9 -8 -7 -6 -13 -12 -11 -10 -9 -8 -7 -6
[Vinblastine], M [Vinblastine], M
[Vinblastine], M

13. Multiplexed In Vitro Hepatotoxicity Assay
Hepatotoxicity assay parameters for compounds tested, (n = 3)
Inhibition of
Apoptosis
Mitosis mitosis (G1/S
Relative cell Relative cell induction
cell cycle block cell cycle
count count [ ] at 5-fold
Compound [ ] at 2-fold over block) [ ] at 2-
IC50 EC50 over
background fold below
(microM) (microM) background
(microM) background
(microM)
(microM)
Propranolol 62.81 ± 5.23 61.38 ± 5.38 55.28 ± 5.43 − −
Staurosporine 0.036 ± 0.005 0.027 ± 0.003 0.036 ± 0.005 − 0.144 ± 0.018
Cyclosporin A 7.71 ± 0.76 5.43 ± 0.30 9.99 ± 0.29 − −
Vinblastine 0.002 ± 0.000 0.002 ± 0.000 0.003 ± 0.001 0.002 ± 0.000 −
Erythromycin > 100 > 100 − − −
All values are given as the mean ± s.e.m.

14. 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

15. 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.

16. Multiplexed In vitro Hepato-Lipid Accumulation
Assay
Hepato-Phospholipid Accumulation assay
Labels: Nuclei - green; Phospholipids - red

17. Multiplexed In vitro Hepato-Lipid Accumulation
Assay

18. Multiplexed In vitro Hepato-Lipid Accumulation
Assay
Hepato-Neutral Lipid Accumulation Assay
Labels: Nuclei - green; Neutral lipids - red

19. Advantages of Multiplexed In vitro Hepato-
Lipid Accumulation Assay
Data Output
Relative cell number quantified by total nuclear Intensity
Advantageous over cell count due to cells that were hard to mask (over lapping cells)
Allowed use of 4X objective to capture more cells (less imaging time with better statistics)
Used less image storage space
Output: EC50 / IC50
− Relative cell count IC50 = test compound concentration that produces 50% of the cell
proliferation inhibitory response or 50% cytotoxicity level
− Relative cell count EC50 = test compound concentration that produces 50% of the
maximum effective response, accounts for cells not killed (curve inflection point)
Phospholipidosis: Measured by fluorescently labeled phospholipid
Detected phospholipid accumulation
Robust signal
Output: [ ] at 5-fold over background
Neutral lipids: Measured by neutral lipid dye
Detected neutral lipid accumulation (steatosis/cholestasis)
Robust signal
Output: [ ] at 5-fold over background

20. Multiplexed In vitro Hepato-Lipid Accumulation
Assay
Hepato-lipid accumulation assay parameters for
each compound tested, (n = 3)

21. 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

22. 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

23. Multiplexed In vitro Hepato-Cytokine Secretion
Assay
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

24. 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

25. Advantages of Multiplexed In vitro Hepato-
Cytokine Secretion Assay
Data Output
Relative cell number quantified by total nuclear Intensity
Advantageous over cell count due to cells that were hard to mask (over lapping cells)
Allowed use of 4X objective to capture more cells (less imaging time with better
statistics)
Used less image storage space
Output: percent of control (POC)
− Relative cell count POC, ratio of the fluorescent intensity in treated wells to
intensity from control wells, used for normalization
POC = Nx/Nc
Nx= Nuclear intensity for well X Nc=Nuclear intensity for control well
Inflammatory marker secretion: multiplexed Luminex xMAP™ technology
Detected biomarker secretion
Normalized by nuclear intensity POC as a measure of relative cell count
Robust signal
Output:
− [ ] of biomarker secretion at 3-fold over background
− Emax (maximum [ ] of secreted biomarker)

26. Multiplexed In vitro Hepato-Cytokine Secretion
Assay

27. 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

28. Acknowledgements
Cell Biology Team Automation
Christine O’Day Robert Keyser
Yulia Ovechkina
Phuong TB Nguyen IT
Rod Shively Mike Harges
Jenny Mulligan Mark Taylor
Cheryl Bogucki Ed Gonterman

29. Automated compound addition using non-contact
acoustic based system, Labcyte® Echo™ 550
Inverted receiving
plate with cells
Source plate with
compound in
DMSO solution
Piezoelectric
transducer
(a). Piezoelectric (b). Compound in (c). When the cell
transducer produces DMSO sitting at assay plate is inverted
focused acoustic the meniscus of back to its up-right
waves to transfer cell media after position, compound in
compound. transfer. DMSO diffuses
toward the cell
monolayer. One well
of 384 well plate is
shown.

30. Customized cell image quantitation protocols are
constructed with IN Cell Developer software
Wide range of quantitative measures
Flexible, multifunctional and user- friendly protocol editor
Fast analysis
Batch capabilities