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 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


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


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


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.


Assay

Labels: Nuclei - green; Phospholipids - red

Hepato-Phospholipid Accumulation assay


Assay


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




� 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


Neutral lipids: Measured by neutral lipid dye

� Detected neutral lipid accumulation (steatosis/cholestasis)

� Robust signal



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


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


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




� 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)


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

K Marcoe In Cell User Ge Meeting 2008

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