pioneering preclinical drug development with human stem cells · pioneering preclinical drug...
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Pioneering Preclinical Drug Development with Human Stem Cells
Stephen Minger
Chief Scientist, Cellular Sciences
Life Sciences, GE Healthcare
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GE Healthcare
53,000 employees
$17 billion revenue
$1 billion R&D
Medical diagnostic X-ray, CT, MRI, ultrasound and PET imaging
Imaging contrast agents and molecular diagnostics
Integrated healthcare IT systems
Drug discovery and biopharmaceutical manufacturing technologies • Reduce drug discovery cycles – bring new, more effective products to market
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Life Sciences - Enabling Development & Production of Future Medicines
RESEARCH
INSTRUMENTS
&
CONSUMABLE
S
REGENERATIVE
MEDICINE &
CELL
TECHNOLOGIES
BIOPROCESSING
CAPACITY &
CAPABILITY
MOLECULAR &
IN-VITRO
DIAGNOSTICS
CLINICAL IMAGING
DISCOVERY
PRE-CLINICAL &
CLINICAL
DEVELOPMENT
APPROVAL &
COMPANION
DIAGNOSTICS
THERAPY
SELECTION &
MONITORING
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Hartung 2009 Nature 460 (7252):208-12
Current issues
Using animal models to reflect
human responses
Testing toxicity using maximum
chemical dosage
Testing multiple endpoints leading
to false-positives
Toxicology has evolved through
addition and replacement without
integration
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Drug Cardiotoxicity
Drug Class Withdrawn
Terfenadine Antihistamine 1998
Sertindole Antipsychotic 1998
Astemizole Antihistamine 1999
Grepafloxacin Antibiotic 1999
Cisapride Prokinetic 2000
Droperidol Tranquilizer 2001
Levomethadyl Opiate Dependence 2003
Rofecoxib NSAID 2004
Tegaserod Prokinetic 2007
Sibutramine Appetite Suppressant 2010
Rosiglitazone Antidiabetic 2010
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Cardiomyocyte production
Expansion Differentiation
0 5 10 15 20 25 30
Media 1 Media 2 Growth Factors Feed
H7 hESC Cardiomyocytes
5 x 109 hES cells
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Electrophysiology
Cardiomyocytes in Drug Safety
Stem cell
expansion &
differentiation
High Content Analysis
& Image Cytometry
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Cardiomyocytes
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9 GE Title or job number
7/10/2014
Cytiva™ Plus cardiomyocytes
Troponin I
Hoechst
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10 GE Title or job number
7/10/2014
DNA
Morphology
Troponin I
α-actinin
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11 GE Title or job number
7/10/2014
Cardiomyocyte Electrophysiology Ion channel currents and action potentials
Ca2+
Na+
K+
Planar patch clamping
Voltage clamped whole cell currents
Ventricular
Nodal
Atrial
APD90 102.0±6.9ms
APD90 350.5±16.8ms
Manual perforated patch clamping
Current clamped action potentials
17.6%
81.6%
0.8%
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HERG Blockers AP prolongation and torsadogenicity
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Electrophysiology Comparative Pharmacology of APD90 Prolongation
Compound Rabbit PF Canine PF hESC-VM HEK-hERG
IC50 (µM)
Terfenadine Positive at 1.0µM False Negative Positive at 0.03µM 0.004
Quinidine Positive at 1.0µM Positive at 1.0µM Positive at 0.3µM 0.830
Cisapride Positive at 0.1µM Positive at 0.1µM Positive at 0.01µM 0.026
Sotalol Positive at 10µM Positive at 100µM Positive at 10µM 268
Verapamil Mixed effect at 10µM Mixed effect at 1.0µM Mixed effect at 1.0 µM 0.125
Chromanol 293B False Negative False Negative Positive at 300µM 10.7
E-4031 N/A Positive at 0.1µM Positive at 0.1µM 0.011
Nifedipine N/A Positive at >10µM Positive at 0.03µM N/A
The Action Potential and Comparative Pharmacology of Stem Cell-Derived Human Cardiomyocytes
Peng, S. et.al., Journal of Pharmacological and Toxicological Methods 61 (2010) 277–286
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IN Cell Analyzer GE Healthcare HCA systems
IN Cell Analyzer 2200 Flexible widefield HCA system
with on-board image restoration
(deconvolution)
IN Cell Analyzer 6000 Laser-based Confocal system
with sCMOS detection
and variable aperture technology
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High Content Analysis information Nuclei
Nuc/Cell Intensity
Nuc Area
Nuc cg X
Nuc cg Y
Nuc Elongation
Nuc 1/(Form Factor)
Nuc Displacement
Nuc Intensity
Nuc Intensity CV
Cell Intensity
Compactness
Light Flux (relative)
Chord ratio
Intensity (N+C)
Integrated Intensity (Nuc)
Integrated Intensity (Cell)
Integrated Intensity (Whole Cell)
Nuclei coordination
Spacing (SOI)
Neighbor count (SOI)
Spacing (MIN)
Neighbor count (MIN)
Spacing (Gabriel)
Neighbor count (Gabriel)
Spacing (Lune)
Neighbor count (Lune)
Cells
Nuc/Cell Intensity
Cell/Bckg Intensity
Cell Area
Nuc/Cell Area
Nuc Intensity
Nuc Intensity CV
Cell Gyration Radius
Cell Intensity
Cell Elongation
Cell 1/(Form Factor)
Intensity Spreading
Cell cg X
Cell cg Y
Cell Intensity CV
Background Intensity
Light Flux (relative)
Intensity (N+C)
Integrated Intensity (Nuc)
Integrated Intensity (Cell)
Integrated Intensity (Whole Cell)
Organelles
Count
Spacing
Neighbor Count
Mean Area
1/(Form Factor)
Elongation
Distance to Nuc
Inclusion/Cell Intensity
Intensity
Total Area
Inclusion/Bckg Intensity
Example data from multi-
target analysis module
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hESC-Cardiomyocyte Toxicity Assay - Control
DNA Mitochondria Ca2+
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DNA α-Actinin
hESC-Cardiomyocyte Toxicity Assay - Amiodarone
DNA Mitochondria Ca2+
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Cardiotoxicity & Anticancer Drugs
Data from: Drug pipeline: Q411. Mak H.C. 2012
Nature Biotechnol. 30,15
Toxicity in Drug Development
Hepatotoxicity
Nephrotoxicity
Cardiotoxicity
Rhabdomyolysis
Other
Data from; Wilke RA et al. Nature Reviews Drug
Discovery 2007 6, 904-916
Drug Pipelines
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Cardiotoxicity HCA Workflow
7 doses; n=3 replicate wells; 24h, 48h, 72h pre-incubations
GEHC Cardiomyocytes, live-cell format assay in 384-well plates
4 Fluors: Hoechst, TMRM, TOTO-3, Fluo-4
IN Cell Analyzer 2000, large-chip CCD camera, 40x/0.6NA
objective
IN Cell Investigator – MTA & Developer : 19 Parameters Image Analysis
2o Analysis &
Visualization
Cardiomyocytes
Compounds*
Fluorescent probes
Imaging
Data
Management IN Cell Miner
IN Cell Investigator – Spotfire DecisionSiteTM
*Compounds blinded until data analysis completed
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Amiodarone (+ve control) 33.0μM – Mitochondrial count, morphology & viability
Mitochondrial Integrity Calcium Homeostasis Viability
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Tyrosine kinase inhibitors Axitinib Tascotinib Vandetanib Sunitinib Imatinib Erlotinib
1.24μM
3.70μM
11.0μM
33.0μM
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Compound 33μM Cluster
Compound 33μM Cluster
Nifedipine 1 Entinostat 4
Tasocitinib 1 LY 294002 4
Axinitib 2 NVP-BEZ235 4
Mubritinib 2 PIK 90 4
Pazopanib (Votrient)
2 SB 202190 4
PD 98059 2 Dasatinib (Sprycell) 5
Tyrphostin 2 PD 184352 5
U 0126 2 Amiodarone* 6
VX 702 2 Imatinib (Gleevec)* 6
GECT-Y 3 Lapatinib (Tyverb)* 6
PD 325901 3 Rapamycin 6
Pimecrolimus 3 Sorafenib (Nexavar) 6
Vatalanib 3 Sunitinib (Sutent)* 6
Temsirolimus 6
Vandetanib (Zactima) 6 *Reported Clinical Cardiotoxicity
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Multi-parameter Profiling HCA Mitochondrial Morphology, Calcium & ATP
100μM 0.05μM
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Future toxicity cell models
45% 37%
18%
Cardiotoxic Hepatotoxic Other
Drug Withdrawals 1994-2006
• Development of hepatocytes,
neurons & other key cell types
• Full genomic, epigenomic &
mitochondrial sequences for each
cell line/cell type by BGI
• Expanded genomic diversity by
importation of ~100 hES cell line
from all over the world
• Translation of existing HCA assays
to improved cell models