This document describes multiplexed in vitro assays that can screen for mechanisms of hepatotoxicity, including phospholipidosis, steatosis, apoptosis, and inflammatory markers. The assays utilize human hepatocyte cells (HepG2) in high content screening to measure multiple parameters related to cell proliferation, apoptosis, cell cycle, lipid accumulation, and cytokine secretion. This allows for a more robust and rapid screening platform to detect various drug-induced hepatotoxicity mechanisms earlier in the drug development process. Primary human hepatocytes may also be used in these assays in the future.

1. SCREENING FOR MECHANISMS OF HEPATOTOXICITY: PHOSPHOLIPIDOSIS, STEATOSIS,
APOPTOSIS AND INFLAMMATORY MARKERS
K.F. Marcoe, R. Keyser, P. TB. Nguyen, Y. Ovechkina, and C. O’Day
MDS Pharma Services – Bothell, WA, USA
INTRODUCTION MULTIPLEXED HEPATOTOXICITY ASSAY MULTIPLEXED HEPATO-LIPID ACCUMULATION ASSAY
Drug-induced hepatotoxicity is difficult to predict and remains a major cause for failures during drug development. Predictive toxicology Hepato-Phospholipid Accumulation Assay A summary of Hepato-Lipid Accumulation Assay parameters for each compound tested, (n = 3).
screening assays for identifying biomakers and providing mechanistic assessments allow earlier identification of potential liabilities and
can result in recommendations to minimize these risks during lead optimization as well as understand their relevance in provoking clinical Relative cell count Relative cell count Phospholipid induction Neutral lipid induction
hepatotoxicity. Development of an effective in vitro cell-based screening model to assess human hepatotoxicity potential of drugs requires Compound IC50, (microM) EC50, (microM) (microM) (microM)
the use of multiplexed technologies that utilize human hepatocytes and measure parameters at the single cell level, morphological and
biochemical, investigative of pre-lethal cytotoxic effects, representative of different mechanisms of toxicity and suitable for rapid throughput. Propranolol > 100 > 100 11.42 ± 1.13 95.43 ± 5.79
In this study we used multiplexed high content screening (HCS) with automated fluorescence microscopy and image analysis based technology
(GE Healthcare INCell Analyzer 1000) to develop mechanistic cellular assays for assessment of hepatotoxicity in HepG2 cells and multiplexed
Cyclosporin A 19.70 ± 5.66 8.73 ± 0.52 15.19 ± 1.10 9.40 ± 0.86
sandwich immunoassays based on flowmetric Luminex xMap™ technology to measure inflammatory markers in these same cells. These
in vitro cell-based assays provide a robust and rapid screening system for testing compound effects on cell proliferation, apoptosis, cell Vehicle Vinblastine
cycle, steatosis, phospholipidosis and cytokine secretion and other inflammatory markers. This cost-effective extensive multiplexed platform Vehicle Propranolol Erythromycin > 100 > 100 35.10 ± 9.98 —
for predictive assessments delivers a more sensitive approach to detection of end-point-specific drug hepatotoxicities. Use of primary Hepatotoxicity Assay, representative images are shown. Labels: Nuclei - green; Apoptotic cells - blue;
hepatocytes as the cell source for these assays is in development. Mitotic cells - red The relative cell count IC50 (half maximal inhibitory constant) and EC50 (half maximal effective constant) values measure cell proliferation. Compound concentrations are
indicative of 5-fold phospholipid and neutral lipid induction over vehicle background. All values are given as the mean ± s.e.m.
Ce l l Pr ol i f e r at i on Apopt osi s I n d u c t i o n Ce l l C y c l e B l o c k
METHODS
MULTIPLEXED HEPATO-CYTOKINE SECRETION ASSAY
160 100 6
Cell Culture Human hepatocellular carcinoma cell line (HepG2) was grown in MEM, 10% FBS, 1% NEAA, 1% Alanyl-L-Glutamine and 1%
Percent of Control
over Background
over Background
140
Fold Induction
Fold Induction
80
120
sodium pyruvate in tissue culture flasks, 75 mm2 and 225 mm2, in a humidified atmosphere of 5% CO2 at 37oC. Working stocks of log-phase 100
80
60
4
HepG2 cells were cryo-preserved using a standard slow cooling in 10% DMSO protocol. Cells were thawed from working stocks and either 60 40
2 A summary of detected cytokine secretion measured in HepG2 cells, (n =3). HepG2 cells were treated with LPS, TNFα, IL-1β and acetaminophen
40
passage once prior to seeding into plates or directly seeded into plates from cryo-preserved stocks. 20
20
and screened for the secretory presence of 30 human inflammatory markers including IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p40, IL-
0
-10 -9 -8 -7 -6 -5 -4 -3
0
-10 -9 -8 -7 -6 -5 -4 -3
0
-10 -9 -8 -7 -6 -5 -4 -3 Vehicle Erythromycin 12p70, 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,
Multiplexed Hepatotoxicity Assay Caspase-3 activation, a marker of apoptosis, phospho-histone-3, a marker of mitosis, and [Cyclosporin A], M [Cyclosporin A], M [Cyclosporin A], M
Apo AI, Apo AII, Apo B, Apo CII, Apo CIII and Apo E.
nuclear count, an index of cell proliferation, were measured. HepG2 cells were seeded at 2x103 cells per well with complete 160 100 6 Hepato-Phospholipid Accumulation Assay, representative images are shown. Labels: Nuclei - green;
growth media into 384-well Collagen I coated optical plates (BD Sciences) and incubated in a humidified atmosphere of 5%
Percent of Control
over Background
over Background
Phospholipids - red
140
Fold Induction
Fold Induction
5
80
120
CO2 at 37oC . Test compounds were serially diluted 3-fold over 10 concentrations and added 24 hours post cell seeding with 100 60
4
α α
a final assay concentration of 0.5% DMSO. Following an additional 72 hour incubation in the humidified atmosphere of 5%
80 3
60 40
2 Cell Proliferation Phospholipid Induction Neutral Lipid Induction [LPS] µg/ml: where
CO2 at 37oC, cells were fixed and immunolabeled with anti-active caspase-3 for detection of apoptosis and anti-phospho- 40
20
20
1 Cell Proliferation Phospholipid Induction Neutral Lipid Induction cytokine secretion _ _ _ _ _ _
histone-3 for detection of cell cycle and stained with a nuclei dye for cell proliferation quantification. Automated fluorescence 3-fold over background
120 260 60
0 0 0 240
(30 - 0.01 µg/ml)
Percent of Control
over Background
over Background
-10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 120
100 260
220 60
50
Maximum [secreted cytokine],
Fold Induction
Fold Induction
240
microscopy was carried out using a GE Healthcare INCell Analyzer 1000, and images were collected with a 4X objective.
200
_ _ _ _ _ _
Percent of Control
[Propranolol], M [Propranolol], M [Propranolol], M
over Background
over Background
180
00 220
180 50
Fold Induction
Fold Induction
40
80
60
200
160
1 80
140 40
30
pg/ml
[TNFα] ng/ml: where
160
120
60 140
100
40 30
20
Multiplexed Hepato-Lipid Accumulation Assay Intracellular phospholipids, a marker of phospholipidosis, intracellular neutral
180
20
cytokine secretion 0.45 ± 0.08 0.29 ± 0.04 0.40 ± 0.05 0.110 ± 0.01 _ _
α
160 10 0 6 160
00
40
20 20
10
80
40
Percent of Control
over Background
over Background
140
lipid, a marker of steatosis, and nuclear count, an index of cell proliferation, were measured. HepG2 cells were seeded at
60
5 20
3-fold over background
Fold Induction
Fold Induction
80 20
0 10
(90 - 0.04 ng/ml)
120 40
0 0
4 -8 -7 -6 -5 -4 -3 20 -8 -7 -6 -5 -4 -3 -8 -7 -6 -5 -4 -3
100 0 0 0
Maximum [secreted cytokine],
4x103 cells per well with complete growth media into 384-well Collagen I coated optical plates (BD Sciences) and incubated in
60
80 3 -8 -7 [Pro6 ranolol], M
-p -5 -4 -3 -8 -7 [Pro6 ranolol], M
-p -5 -4 -3 -8 -7 [Pro6 ranolol], M
-p -5 -4 -3
10.64 ± 1.75 6454 ± 458 26.19 ± 2.53 87.83 ± 8.31 _ 5955 ± 29
60 40 [Propranolol], M [Propranolol], M [Propranolol], M pg/ml
a humidified atmosphere of 5% CO2 at 37oC for 24 hours. For detection of phospholipid accumulation a fluorescently-labeled
2
[IL-1β] ng/ml: where
40
20
1
20
phospholipid (Invitrogen, H34350) was added to the cells with test compounds serially diluted 3-fold over 10 concentrations cytokine secretion 0.016 ± 0.003 < 0.04 _ 0.002 ± 0.000 0.81 ± 0.45 0.15 ± 0.05
β
0 0 0
-12 -11 -10 -9 -8 -7 -6 -5 -12 -11 -10 -9 -8 -7 -6 -5 -12 -11 -10 -9 -8 -7 -6 -5
120 260 60
240
3-fold over background
Percent of Control
in a final assay concentration of 0.5% DMSO. Following an additional 48 hour incubation in a humidified atmosphere of 5%
over Background
over Background
[Staurosporine], M [Staurosporine], M [Staurosporine], M 120
100 260
220 60
50
Fold Induction
Fold Induction
240
200
(90 - 0.04 ng/ml)
Percent of Control
over Background
over Background
00
180 220
180 50
Maximum [secreted cytokine],
Fold Induction
Fold Induction
40
CO2 at 37oC, cells were fixed and stained with a neutral lipid dye (Invitrogen, H34476) for neutral lipid detection and a nuclei 16.78 ± 0.78 5039 ± 39 _ 259 ± 12 6.64 ± 1.11 8.85 ± 1.29
200
160
80 180
140
pg/ml
60 40
30
160
120
dye for cell proliferation quantification. Automated fluorescence microscopy was carried out using a GE Healthcare INCell 60 140
100
40 30
160 100 6
40
20
120
80
100
60
20
20
[Acetam.] mM: where
Analyzer 1000, and images were collected with a 4X objective.
80
40 10
cytokine secretion _ _ _ _ _ _
Percent of Control
over Background
over Background
140 60
Fold Induction
Fold Induction
80 20
120 0
20 40
0 10
0
100 60
4
0
-8 -7 -6 -5 -4 -3 20 -8
0
-7 -6 -5 -4 -3
0
-8 -7 -6 -5 -4 -3
(1 mM - 450 nM) 3-fold over background
[Eryt6 romyc5n], M -4
-h -i [Eryt6 romyc5n], M -4
-h -i [Eryt6 romyc5n], M -4
-h -i
80 -8 -7 -3 -8 -7 -3 -8 -7 -3
Maximum [secreted cytokine],
Multiplexed Hepato-Cytokine Secretion Assay Cytokine secretion, as markers of inflammation, and nuclear count, as an index of cell 60
40
40
2
[Erythromycin], M [Erythromycin], M [Erythromycin], M
pg/ml
_ _ _ _ _ _
proliferation, were measured in HepG2 cells. HepG2 cells were seeded at 8x103 cells per well with complete growth media into 96-well
20
20
0 0 0
Collagen I coated optical plates (BD Sciences) and incubated in a humidified atmosphere of 5% CO2 at 37oC for 24 hours. Cells were treated -13 -12 -11 -10
[Vinblastine], M
-9 -8 -7 -6 -13 -12 -11 -10
[Vinblastine], M
-9 -8 -7 -6 -13 -12 -11 -10
[Vinblastine], M
-9 -8 -7 -6
Hepato-Phospholipid Accumulation Assay, representative curves for cell proliferation and phospholipid and Concentrations for cytokine induction and secretion are given as the mean ± s.e.m. Lipopolysaccharide (LPS), Tumor necrosis factor (TNF), Interleukin (IL), Interferon
with LPS (30 µg/ml), TNFα (90 ng/ml), IL-1β (90 ng/ml) and acetaminophen (1 mM) serially diluted 3-fold over 8 concentrations. Final assay neutral lipid accumulation are shown. (INF), Interferon-gamma-inducible protein (IP), Granulocyte-macrophage-colony-stimulating factor (GM-CSF), Granulocyte colony-stimulating factor (G-CSF), Monocyte
chemoattractant protein (MCP ), Macrophage inflammatory protein (MIP), C-Reactive Protein (CRP), Serum Amyloid A (SAA), Apolipoprotein (Apo)
concentration of DMSO for acetaminophen was 0.05%. Following an additional 48 hour incubation in a humidified atmosphere of 5% CO2
at 37oC, supernatants were collected and cytokine detection was carried out using multiplexed sandwich immunoassays based on Luminex 160 100 6
Percent of Control
140
Hepato-Neutral Lipid Accumulation Assay
over Background
over Background
xMAP™ technology. To quantify cell proliferation, the monolayer of HepG2 cells remaining in each plate was immediately stained with
Fold Induction
Fold Induction
80
120
4
CONCLUSION
100
Hoechst nuclear dye for normalization, and incubated 15 min at 37°C. Images were collected using a GE Healthcare INCell Analyzer 1000
60
80
60 40
with a 10X objective. 40
20
2
20
0 0 0
Identification of drug-induced hepatotoxic potential early in the drug development cascade can create opportunities for ranking and prioritizing,
Data Analysis For HCS 12 bit tiff images were acquired using the INCell Analyzer 1000 3.2 and analyzed with Developer Toolbox 1.6 software.
-10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3
[Erythromycin], M [Erythromycin], M [Erythromycin], M
or developing alternatives with lower toxicity. We have developed a robust and rapid throughput screening system using HepG2 cells that
EC50 and IC50 values were calculated using nonlinear regression to fit data to a sigmoidal 4 point, 4 parameter One-Site dose response allows early assessment of acute and chronic mechanisms of hepatotoxicity. Compounds with known hepatotoxicities were tested to
model, where: y (fit) = A + [(B – A)/(1 + (C/x) ^ D))]. Cytokine and inducer concentrations were interpolated from standard curves obtained validate the capabilities of this multiparametric HCS system in identifying and quantifying toxicities relevant to cell proliferation, apoptosis,
by 5 point parameter sigmoidal nonlinear regression analysis. Curve-fitting and EC50 and IC50 calculations were performed using XLFit™ Hepatotoxicity Assay, representative curves for cell proliferation, apoptosis induction, and cell cycle block are
shown. cell cycle, steatosis, phospholipidosis. High concordance was found with the reported hepatotoxic profile for each compound tested.
software (IDBS). Curves were generated with GraphPad Prism™ 3 software using a sigmoidal dose-response, variable slope model. Further, we evaluated cytokine secretion in HepG2 cells to identify measurable biomarkers of inflammation. Significant secretion levels
for 6 of the cytokines tested were confirmed thus validating this multiplexed approach for quantifying indications of hepatic inflammation.
Measured Parameters Cell proliferation was measured by the signal intensity of the incorporated nuclear dye. The cell proliferation output A summary of Hepatotoxicity Assay parameters for each compound tested, (n = 3).
Vehicle Cyclosporin A These hepatotoxicity screening assays are sensitive and reproducible and provide results that previously only have been attainable in
was referred to as the relative cell count. To determine the cell proliferation end point, the cell proliferation data output was transformed
more complex in vivo models. Our cost-effective in vitro multiplexed HCS platform offers comprehensive predictive information allowing
to percent of control (POC) using the following formula: Inhibition of
Relative cell count Relative cell count Apoptosis Mitosis pre-selection of drug scaffold designs with long-term hepatotoxicity considerations and may even have more relevance when performed
POC = relative cell count (compound wells) x100 mitosis (G1/S cell Hepato-Neutral Lipid Accumulation Assay, representative images are shown. Labels: Nuclei - green; in normal primary hepatocytes.
Compound IC50 EC50 induction cell cycle block
relative cell count (vehicle wells) cycle block) Neutral lipids - red.
(microM) (microM) (microM) (microM)
(microM)
Relative cell count IC50 is the test compound concentration that produces 50% of the cell proliferation inhibitory response or 50% cytotoxicity
Propranolol 62.81 ± 5.23 61.38 ± 5.38 55.28 ± 5.43 — — Cell Proliferation Phospholipid Induction Neutral Lipid Induction
level. A relative cell count EC50 is the test compound concentration that produces 50% of the maximum effective response that occurs at the
curve inflection point. The output of each biomarker is fold increase over vehicle background normalized to the relative cell count in each Staurosporine 0.036 ± 0.005 0.027 ± 0.003 0.214 ± 0.019 — 0.144 ± 0.018
Cell Proliferation Phospholipid Induction Neutral Lipid Induction REFERENCES
well. Concentrations of test compound that cause a 5 or more fold induction in the caspase-3 signal were determined to induce significant Cyclosporin A 7.71 ± 0.76 5.43 ± 0.30 9.99 ± 0.29 — — 120 260 60 1. Dambach DM, Andrews BA, Moulin F. New technologies and screening srategies for hepatotoxicity: use on in vitro models. Toxicologic
apoptotic induction. When the fold induction of the phospho-histone-3 signal over background is ~1, there was “no effect” on the cell 240
Percent of Control
over Background
over Background
Vinblastine 0.002 ± 0.000 0.002 ± 0.000 0.003 ± 0.001 0.002 ± 0.000 —
120 260
220 60
Pathology, 2005;33:17-26.
Fold Induction
Fold Induction
100
240
Percent of Control
200
over Background
over Background
cycle. Two or more fold increase in phospho-histone-3 signal over vehicle background indicated significant test compound induction of 220
Fold Induction
Fold Induction
180
00 180 40
2. Anderson N, Borlak J. Drug-induced phospholipidosis. FEBS Lett. 2006;580(23):5533-40.
260
1 00
mitotic block. Two or more fold decrease in the phospho-histone-3 signal may indicate G1/S block only when cytotoxicity levels are below Erythromycin > 100 > 100 — — — 80
60
180
140
160
120
140
100
40
3. Nioi P, Perry BK, Wang E, et al. In vitro detection of drug-induced phospholipidosis using gene expression and fluorescent phospholipid-
60
40 180
20 20
the measured relative cell count IC80. When 2 or more fold decrease in the phospho-histone-3 signal are observed at concentrations higher 40
20
160
00
80
40
20
than the relative cell count nuclear IC80, the decrease in mitotic cell counts are most likely due to a more general cytotoxicity effect rather 0
20
60
20
40
0 0 based methodologies. Toxicology Sciences, 2007;99(1):162-173.
The relative cell count IC50 (half maximal inhibitory constant) and EC50 (half maximal effective constant) values measure cell -8 -7 -6 -5 -4 -3 20 -8 -7 -6 -5 -4 -3 -8 -7 -6 -5 -4 -3
4. Stonans I, Stonane E, RuBwurm S, et al. HepG2 human hepatoma cells express multiple cytokine genes. Cytokines, 1999;11(2):151-6.
0 0 0
than a true G1/S phase block. Wells with concentrations higher than the relative cell count nuclear IC80 are eliminated from the phospho- proliferation. Compound concentrations are indicative of a 5-fold apoptosis induction in activated caspase-3 signal and a
-8 -7 [Cyc-l6 spori-5 A], M -4
o n
[Cyclosporin A], M
-3 -8 -7 [Cyc-l6 spori-5 A], M -4
o n
[Cyclosporin A], M
-3 -8 -7 [Cyc-l6 spori-5 A], M -4
o n
[Cyclosporin A], M
-3
histone-3 analysis. Concentrations of test compound that cause a 5 or more fold induction of the labeled-phospholipid signal represent 2-fold change in phospho-histone 3 signal (mitosis marker) over vehicle background. All values are given as the mean ± s.e.m.
significant phospholipidosis induction. Concentrations of test compound which cause a 5 or more fold induction of the neutral lipid signal Hepato-Neutral Lipid Accumulation Assay, representative curves for cell proliferation, phospholipid and
represent significant steatosis induction. Three or more fold increase in cytokine detection represents significant cytokine secretion. neutral lipid accumulation are shown.