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

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)

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

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

Vehicle Vinblastine

Labels: Nuclei - green; Apoptotic cells - blue; Mitotic cells - red

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Cell Proliferation Apoptosis Induction Cell Cycle Block

HepG2, 72 hr assay

Primary human hepatotoxicity assay:

MTS and HCS comparison

Amiodarone Valproic acid Amitriplyline

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MTS

HCS

24 hour compound treatment of human primary hepatocytes; High Content Screening approach (HCS)

Primary human hepatotoxicity assay:

MTS and HCS comparison

Compound MTS Viability IC50

(microM)

HCS % of attached

live cells IC50

(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)

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 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.

Multiplexed In vitro Hepato-Lipid

Accumulation Assay (HepG2)

HepG2, 48 hr assay

Multiplexed In vitro Hepato-Lipid

Accumulation Assay (HepG2)

Labels: Nuclei - green; Neutral lipids - red

Hepato-Neutral Lipid Accumulation Assay

HepG2, 48 hr assay

Multiplexed In vitro Hepato-Lipid

Accumulation Assay (HepG2)

HepG2, 48 hr assay

In vitro Hepato-Lipid Accumulation Assay

using primary human hepatocytes in 384 WP

In vitro Hepato-Lipid Accumulation Assay

using primary human hepatocytes in 384 WP Amiodarone Amitriplyline

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 (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

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:

Multiplexed In vitro Hepato-Cytokine

Secretion Assay (HepG2)

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