Cell-Based Assays for Hepatotoxicity:
Cell-Based Assays for Hepatotoxicity:
Cell-Based Assays for Hepatotoxicity:
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<strong>Cell</strong>-<strong>Based</strong> <strong>Assays</strong> <strong>for</strong> <strong>Hepatotoxicity</strong>:<br />
New ideas and lessons learnt<br />
MBC Drug Discovery Breakfast Meeting<br />
December 18 2008<br />
Stella Redpath Ph.D<br />
Global Program Manager – Discovery Toxicology<br />
Millipore Corporation, Bioscience Division
Early microscopy
Now: High Content Analysis<br />
Automated fluorescent<br />
microscope<br />
bioin<strong>for</strong>matics
The need <strong>for</strong> new assays and techniques to<br />
predict drug toxicity<br />
90% of compounds fail<br />
in drug development 1 …<br />
Compounds<br />
Successfully<br />
Marketed<br />
Failed<br />
Compounds<br />
• Factors causing failure of drug<br />
candidates2<br />
– 45% Poor ADME properties<br />
– 11% In vivo toxicity<br />
– 10% Adverse clinical<br />
effects<br />
– 28% Insufficient efficacy<br />
– 6% Commercial reasons<br />
… Resulting in an estimated $70 M spend per drug failure on ADME / Toxicity 3<br />
1 Drug Discovery World (Summer 2005) – Conley, “High content screening: emerging importance of novel reagents/probes and pathway analysis”<br />
2 Business Insights Report, “Predictive ADME and Toxicology Strategies” (2006)<br />
3 D&MD Report (2007)
Inability to accurately screen <strong>for</strong> toxicity effects candidates is<br />
a key issue with current techniques.<br />
Traditional Focus <strong>for</strong> ADME/ Toxicity<br />
1000s<br />
compounds<br />
100s<br />
compounds<br />
Development of a high content analysis<br />
assay <strong>for</strong> hepatotoxicity<br />
‣In vivo testing, assessed by<br />
histopathology<br />
is the traditional toxicology tool<br />
‣HCA can identify histopathological<br />
endpoints.<br />
‣Millipore & <strong>Cell</strong>umen co-developed<br />
the <strong>Cell</strong>Ciphr panel <strong>for</strong> hepatotoxicity<br />
in human HepG2 cells .<br />
‣This kit contains antibodies, dyes &<br />
reagents and data analysis software.
Development of a high content analysis<br />
assay <strong>for</strong> hepatotoxicity<br />
Eleven hepatotoxicity endpoints in human HepG2 cells are measured using the<br />
<strong>Cell</strong> Ciphr Assay<br />
‣ <strong>Cell</strong> Loss<br />
‣ <strong>Cell</strong> Cycle Arrest<br />
‣ DNA Degradation/Apoptosis<br />
‣ Nuclear Size<br />
‣ Oxidative Stress<br />
‣ Stress Kinase Activation<br />
‣ DNA Damage<br />
‣ Mitochondrial Membrane Potential<br />
‣ Mitochondrial Mass<br />
‣ Mitotic Arrest<br />
‣ Cytoskeletal Integrity.
Development of a high content analysis<br />
assay <strong>for</strong> hepatotoxicity<br />
<strong>Cell</strong>Ciphr Cytotoxicity Profile<br />
‣Using cells to rank compounds according to toxicity<br />
‣Using cells to detect subtle changes indicative of compound<br />
toxicity at low concentration doses and earlier time points<br />
‣Identifies potential toxicity be<strong>for</strong>e expensive pre-clinical<br />
animal testing<br />
‣Enables prioritization of lead compounds
Development of a high content analysis<br />
assay <strong>for</strong> hepatotoxicity<br />
Control HepG2 (O)<br />
HepG2 cells treated with Paclitaxel (P)<br />
Blue – Nuclei;<br />
Green – Microtubules;<br />
Red – Mitochondria;<br />
Magenta – Phospho-Histone H3.<br />
‣ The kit includes all reagents required to measure 11 cellular features at 3 time points<br />
<strong>for</strong> 16 compounds in 6 x 384-well microplates.<br />
‣ Each measurement consists of an EC50 value calculated from replicate 10 point dose<br />
response curves
HepG2 cells treated with Campothecin (24h)<br />
<strong>Cell</strong> count Oxidative stress DNA damage
HepG2 cells treated with Campothecin<br />
(30mins – 72h)<br />
<strong>Cell</strong> loss<br />
<strong>Cell</strong> Count<br />
<strong>Cell</strong> Number (n=2, well mean +/- SD)<br />
30min<br />
24hr<br />
72hr<br />
30 mins<br />
24h<br />
72h<br />
1000<br />
0<br />
1.00E-06 1.00E-05 1.00E-04 1.00E-03 1.00E-02 1.00E-01 1.00E+00 1.00E+01<br />
[Camptothecin, + control] (uM on log scale)<br />
Campothecin (uM)
Acute<br />
Early<br />
Chronic<br />
mM μM nM<br />
<strong>Cell</strong>Ciphr Profiles of<br />
Initial Library - Clustered<br />
Staurosporine<br />
Anisom ycin<br />
Cam ptothecin<br />
Vinblastine<br />
P a c lita x e l<br />
Nocodazole<br />
M ethotrexate<br />
Mevastatin<br />
Lovastatin<br />
Terfenadine<br />
Etoposide<br />
CCCP<br />
Furazolindone<br />
Astemizole<br />
Ketoconazole<br />
Am iodarone<br />
C hlorprom azine<br />
Bupivacaine<br />
Buspirone<br />
Im ip ra m in e<br />
Indom ethacin<br />
Tacrine<br />
Paroxetine<br />
M enadione<br />
Q u in id in e<br />
Propranalol<br />
Chloroquine<br />
Diclofenac<br />
V a lp ro ic a c id<br />
Sulindac
Development of high content analysis<br />
assays <strong>for</strong> hepatotoxicity<br />
‣ This multiplex approach monitors multiple functions, time<br />
points and doses<br />
‣ Fully leverages the sensitivity and throughput of HCA<br />
and is relevant to human hepatotoxicity<br />
‣ May deliver insights on mechanism of action<br />
‣ Next generation of assays may include assays <strong>for</strong><br />
primary hepatocytes, stem cell-derived hepatocytes, and<br />
CYP450-competent transfected cell lines
Lessons learnt from designing<br />
hepatotoxicity assays:<br />
Development of cell-based assays<br />
to detect toxicity in neuronal cultures
<strong>Cell</strong>ular Targets <strong>for</strong> Neurotoxicity
Development of HCA assays <strong>for</strong> neurotoxicity<br />
• <strong>Assays</strong> <strong>for</strong> neurite<br />
outgrowth in neurons<br />
• <strong>Assays</strong> <strong>for</strong> detecting<br />
toxicity in astrocytes<br />
• <strong>Assays</strong> <strong>for</strong> detecting<br />
synaptogenesis in<br />
neurons<br />
• <strong>Assays</strong> <strong>for</strong> detecting<br />
toxicity in co-cultures of<br />
astrocytes and neurons
<strong>Assays</strong> <strong>for</strong> detecting toxicity in astrocytes<br />
Primary rat hippocampal astrocytes<br />
<strong>Cell</strong>s treated with two different toxins,<br />
paclitaxel and nocodazole<br />
Both toxins cause increased expression<br />
of GFAP as the levels of toxin rise.<br />
Different toxins cause different effects<br />
on the expression of GFAP<br />
0.4% DMSO CONTROL (4hr)<br />
1µM Paclitaxel TOXIN (4hr)
HCS221 Astrocyte toxicity<br />
Measurement of more than one parameter in a single assay<br />
A<br />
A: <strong>Cell</strong> Area<br />
As toxin levels increase, the size of the cells also<br />
Increases as apoptosis begins<br />
B<br />
B:Levels of GFAP expression<br />
As toxin levels increase, the expression of GFAP<br />
decreases, as apoptosis begins<br />
C<br />
C: <strong>Cell</strong> number<br />
As toxin levels increase, the cell numbers decrease as the cells<br />
die off when toxins are too high<br />
Primary rat hippocampal astrocytes<br />
All cells treated with K252a neurotoxin
Detecting changes in neuronal and astrocyte<br />
biomarkers due to toxins<br />
Neurons & astrocyte co-culture<br />
Untreated cells (Control)<br />
Neurons & astrocyte co-culture<br />
Treated with 10mM Acrylamide<br />
(toxin)
HCS222<br />
Detecting changes in neuron biomarkers due to toxins<br />
Neurons & astrocyte co-culture<br />
untreated (Control)<br />
blue = nuclei<br />
green = III-tubulin (neurons)<br />
red = GFAP (astrocytes)<br />
Neurons & astrocyte co-culture treated<br />
with H 2<br />
O 2<br />
(toxin)<br />
Significant damage to neurons<br />
blue = nuclei<br />
green = III-tubulin (neurons)<br />
red = GFAP (astrocytes)<br />
1mM H 2 O 2
Assessment of toxin effects on synapses (synaptogenesis)<br />
CONTROL<br />
Blue = nuclei<br />
Red = synaptophysin (synapse protein)<br />
Green = bIII tubulin (neurite protein)<br />
<strong>Cell</strong>s treated with acrylamide (toxin)<br />
<strong>Cell</strong>s treated with hydrogen<br />
Peroxide (toxin)
Assessment of acrylamide toxin effects in rat cells<br />
Rat primary<br />
Hippocampal cells<br />
Rat PC12 cell line<br />
Loss of synaptophysin expression<br />
as level of toxin increases<br />
Reduction in cells number<br />
as level of toxin increases
Preliminary data: comparison of HCA methods with biochemical<br />
assays <strong>for</strong> toxicity<br />
MPP+ toxin:<br />
Toxicity detected by HCA assay (neurite outgrowth parameter)<br />
Toxicity effect detected with MTT and LDH assay
Preliminary data: comparison of HCA methods with biochemical<br />
assays <strong>for</strong> toxicity<br />
Kainic acid:<br />
No toxic effect on neurons
Preliminary data: comparison of HCA methods with biochemical<br />
assays <strong>for</strong> toxicity<br />
6 Hydroxydopamine:<br />
Toxicity detected by HCA assay (neurite outgrowth parameter)<br />
Toxicity effect NOT detected with MTT or LDH assay
Preliminary data: comparison of HCA methods with biochemical<br />
assays <strong>for</strong> toxicity<br />
Nocodazole and K-252a:<br />
Toxicity detected by HCA assay (neurite outgrowth parameter)<br />
Toxicity effect NOT detected with MTT or LDH assay
Summary<br />
<strong>Cell</strong>-based assays <strong>for</strong> hepatotoxicity and neurotoxicity<br />
• <strong>Assays</strong> <strong>for</strong> hepatotoxicity using HCA have been adopted <strong>for</strong> HepG2 cells.<br />
Early indications are that the assays can be used as a “filter” to detect toxic<br />
effects earlier in drug discovery process<br />
• <strong>Assays</strong> <strong>for</strong> neurotoxicity using HCA appear to show better sensitivity than<br />
traditional biochemistry assays <strong>for</strong> cytotoxicity. More data required to understand<br />
various mechanisms of action and also develop predictive indices<br />
HCA assays <strong>for</strong> toxicology may identify potential toxicity be<strong>for</strong>e expensive preclinical<br />
animal testing. These assays will also enable prioritization of lead<br />
compounds during screening.
Acknowledgements<br />
Andrew Ball<br />
Janet Anderl<br />
Rocky Mowry<br />
Zaheda Farzin<br />
Matthew Hsu<br />
Anna Waters<br />
Rich Sullivan<br />
Stella Redpath<br />
Michele Hatler<br />
Jeff Till<br />
David Hayes<br />
Rick Ryan<br />
Dennis Harris
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