Download - K Marcoe In Cell User Ge Meeting 2008
Screening for Mechanisms of
Hepatotoxicity: Phospholipidosis,
Steatosis, Apoptosis and
Inflammatory Markers
Karen Marcoe
MDS Pharma Services
IN Cell User Meeting
May 14, 2008
� 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 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)
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
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)
Non-contact dispensing automation system for
compound addition, cell fixing and immunostaining
Titertek Mult
idropTitertek Multidrop
Titertek Multidrop
Biotek E
Lx405 wa
sher V-Spin CentrifugeLiCONiC CO2 incubator
De-lidder
Carousel w/ 12 hotels, 16
slots each (not shown)
Labcyte®
Echo™ 550
Velocity 11 BioCel® 1200 system
0.10.68.967.01.57.91.00E+02
0.20.57.910.42.518.13.16E+01
0.10.60.84.11.325.21.00E+01
0.00.80.72.03.689.33.17E+00
0.00.90.81.012.5100.21.00E+00
0.11.00.20.88.394.23.17E-01
0.00.90.61.23.693.51.00E-01
0.10.91.11.66.395.33.18E-02
0.11.40.61.75.892.91.01E-02
0.21.10.60.62.696.93.18E-03
StdDevMeanStdDevMeanStdDevMean
Mitosis (fold
induction)
Apoptosis (fold
induction)
Relative cell
count (%)
Concentration
(microM)
Blue indicates that values meet the statically significant
response criteria specified in the data interpretation
chapter.
MDS AIM automated Data Analysis and
Report Generation
� Includes curve fitting, data visualization, quality and statistical assessment
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 hepatotoxicitypotential 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
IN Cell Analyzer 1000
� Images collected with a 4X objective
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
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
Multiplexed In vitro Hepatotoxicity Assay
Vehicle Vinblastine
Labels: Nuclei - green; Apoptotic cells - blue; Mitotic cells - red
-13 -12 -11 -10 -9 -8 -7 -60
2
4
6
[Vinblastine], M
Fold Induction
over Background
-13 -12 -11 -10 -9 -8 -7 -60
20
40
60
80
100
[Vinblastine], M
Fold Induction
over Background
-13 -12 -11 -10 -9 -8 -7 -60
20
40
60
80
100
120
140
160
[Vinblastine], M
Percent of Control
Cell Proliferation Apoptosis Induction Cell Cycle Block
Multiplexed In Vitro Hepatotoxicity Assay
Hepatotoxicity assay parameters for compounds tested, (n = 3)
−−−> 100> 100Erythromycin
−0.002 ± 0.0000.003 ± 0.0010.002 ± 0.0000.002 ± 0.000Vinblastine
−−9.99 ± 0.295.43 ± 0.307.71 ± 0.76Cyclosporin A
0.144 ± 0.018−0.036 ± 0.0050.027 ± 0.0030.036 ± 0.005Staurosporine
−−55.28 ± 5.4361.38 ± 5.3862.81 ± 5.23Propranolol
Inhibition of
mitosis (G1/S
cell cycle
block) [ ] at 2-
fold below
background
(microM)
Mitosis
cell cycle block
[ ] at 2-fold over
background
(microM)
Apoptosis
induction
[ ] at 5-fold
over
background
(microM)
Relative cell
count
EC50(microM)
Relative cell
count
IC50 (microM)
Compound
All values are given as the mean ± s.e.m.
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
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 phospholipidaccumulation 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.
Multiplexed In vitro Hepato-Lipid Accumulation
Assay
Labels: Nuclei - green; Phospholipids - red
Hepato-Phospholipid Accumulation assay
Multiplexed In vitro Hepato-Lipid Accumulation
Assay
Multiplexed In vitro Hepato-Lipid Accumulation
Assay
Labels: Nuclei - green; Neutral lipids - red
Hepato-Neutral Lipid Accumulation Assay
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
Multiplexed In vitro Hepato-Lipid Accumulation
Assay
Hepato-lipid accumulation assay parameters for
each compound tested, (n = 3)
Multiplexed In vitro Hepato-Cytokine Secretion
Assay
Multiplexed Hepato-Cytokine Secretion Assay
IN Cell
Automated
fluorescent
microscopy
imaging ����
cell count
normalization
xMAP™
technology
using
Luminex����
Markers of
inflammation
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
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 ½log 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
HepG2 cells
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, IP-10, GM-CSF, G-CSF, MCP-1, MIP-1α, MIP-1β, TNFα, IL-1 receptor
antagonist
Fibrinogen,
CRP, Haptoglobin,
SAA
Apo AI, Apo AII, Apo B,
Apo CII, Apo CIII and
Apo E
LPS, TNFα, IL-1β and
acetaminophen
Multiplexed In vitro Hepato-Cytokine Secretion
Assay
� HepG2 cells treated with LPS, TNFα, IL-1β and acetaminophen
� Screened for the secretory presence of
30 human inflammatory markers:
Advantages of Multiplexed In vitro Hepato-
Cytokine Secretion Assay
Data OutputRelative 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)
Multiplexed In vitro Hepato-Cytokine Secretion
Assay
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
Acknowledgements
Cell Biology Team
� Christine O’Day
� Yulia Ovechkina
� Phuong TB Nguyen
� Rod Shively
� Jenny Mulligan
� Cheryl Bogucki
Automation
� Robert Keyser
IT
� Mike Harges
� Mark Taylor
� Ed Gonterman
Automated compound addition using non-contact
acoustic based system, Labcyte® Echo™ 550
(c). When the cell
assay plate is inverted
back to its up-right
position, compound in
DMSO diffuses
toward the cell
monolayer. One well
of 384 well plate is
shown.
(a). Piezoelectric
transducer produces
focused acoustic
waves to transfer
compound.
(b). Compound in
DMSO sitting at
the meniscus of
cell media after
transfer.
Inverted receiving
plate with cells
Source plate with
compound in
DMSO solution
Piezoelectric
transducer
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