Pioneering Preclinical Drug Development with Human Stem Cells

Stephen Minger

Chief Scientist, Cellular Sciences

Life Sciences, GE Healthcare

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

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

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

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Drug Class Withdrawn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

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

Electrophysiology

Cardiomyocytes in Drug Safety

Stem cell

expansion &

differentiation

High Content Analysis

& Image Cytometry

Cardiomyocytes

9 GE Title or job number

7/10/2014

Cytiva™ Plus cardiomyocytes

Troponin I

Hoechst

10 GE Title or job number

7/10/2014

DNA

Morphology

Troponin I

α-actinin

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%

HERG Blockers AP prolongation and torsadogenicity

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

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

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

hESC-Cardiomyocyte Toxicity Assay - Control

DNA Mitochondria Ca2+

DNA α-Actinin

hESC-Cardiomyocyte Toxicity Assay - Amiodarone

DNA Mitochondria Ca2+

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

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

Amiodarone (+ve control) 33.0μM – Mitochondrial count, morphology & viability

Mitochondrial Integrity Calcium Homeostasis Viability

Tyrosine kinase inhibitors Axitinib Tascotinib Vandetanib Sunitinib Imatinib Erlotinib

1.24μM

3.70μM

11.0μM

33.0μM

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

Multi-parameter Profiling HCA Mitochondrial Morphology, Calcium & ATP

100μM 0.05μM

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

Stephen.Minger@ge.com