Metrion Biosciences: the ion channel specialists
Validation of an impedance-based phenotypic screening assay able
to detect multiple mechanisms of chronic cardiotoxicity in human stem
cell-derived cardiomyocytes
Marc Rogers, CSO
www.metrionbiosciences.com

Drug-induced cardiomyocyte toxicity• CiPA is designed to address drug-induced cardiac arrythmia (TdP)• Acute effects of drug discovery compounds (minutes – hours)• Focus on plasmalemmal ion channels that underlie the cardiac action potential• However, new and existing drugs can also cause structural cardiotoxicity• Produced by a diverse set of chemical compounds and primary target mechanisms• Chronic effects can appear after days, weeks or months; not all are reversible• Classic examples include chemotherapy agents used for clinical oncology••••

Anthracyclines such as Doxorubicin (breast cancer)
Tyrosine kinase inhibitors (TKI) such as the ‘nibs
Proteosome inhibitors such as Bortezomib
HDAC inhibitors such as the ‘stats (some of which may also cause QTc prolongation)• Safety pharmacology testing needs a reliable & predictive assay for chronic cardiotox

Cardiotoxicity:

CiPA:

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Anti-cancer drugs with clinical cardiotoxicity effects
NameTypeIndication

Possible cardiovascular damage

Doxorubicin
Sunitinib

SMSM

Anthracycline - solid tumor (breast cancer)
Multi-targeted TKI (kidney cancer)

Congestive heart failure, Acute myocarditis, left ventricular dysfunction
Arrhythmias

Dasatinib

SM

Multi-targeted TKI

QT prolongation

Ponatinib

SM

TKI inhibitor

Myocardial infarction, congestive heart failure (HF), cardiac arrhythmias

ibrutinib

SM

Bruton’s tyrosine kinase

AF + other arrhythmias

Trametinib

SM

MEK inhibitor (MEK1, MEK2)

Cardiomyopathy

Bortezomib
Trastuzumab
Bevacizumab

SMAb
Ab

Proteasome inhibitor
antineoplastic ErbB2-targeted therapies
VEGFA monoclonal antibody

Left ventricular dysfunction and atrioventricular block
Decline left ventricular ejection fraction
Congestive heart failure

RituximabAb

Non-Hodgkin’s lymphoma

Arrhythmias (link to cytokine release by lymphocyte B?)

Vorinostat

SM

HDAC inhibitor

QT prolongation, thromboembolism

AmiodaroneSM

AF Class III antiarrhythmic

TdP, heart block, sinus bradycardia, CHF, VF

Paclitaxel

SM

Anti-microtubule agent

Arrhythmias (sinus bradycardia, ventricular tachycardia)

CapecitabineSM

Antimetabolite

Myocardial ischemia + arrhythmias• A wide and growing list of chemotherapy agents produce a variety of serious cardiac
side-effects in cancer patients, only some of which reverse after drug washout• Non-traditional, non-CiPA related cardiac liabilities are a challenge for current in vitro assays• As well as safety, iPSC cardiomyocyte assays could enable cardiac disease modelling3

Chemotherapy-induced chronic cardiotoxicity• Various cell-based screening assays and platforms can be used to monitor
cardiomyocyte health and function as part of safety pharmacology testing• High content image analysis (sarcomeres, nucleus, cell morphology)• Functional imaging (Mitochondrial dyes, LDH, ATP, MTT, DNA damage)• Gene expression profiling• Secreted or expressed biomarkers are also gaining traction (translation to clinic)• However, many of these assays require fixation of cells (end-stage assays)• Other assays require labelling of cells, which adds expense and potential artefacts• Impedance is useful as it is cheap, label-free, and physiologic over long time-course
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CiPA screening:• Dual mode platform: impedance &microelectrode array (MEA)

Chronic cardiotox:• Long-term monitoring of beat rate(arrythmia) and base impedance(growth and formation of a stablesyncytium, cell viability)• Beat rate and impedance stabilise after
1-5 days
5• Chronic effects assayed over hours-days

CardioExcyte 96

Nanion CardioExcyte 96: Chronic Impedance assay

Cardiotoxicity assay: Sunitinib
Beat RateBase Impedance• Broad spectrum tyrosine kinase inhibitor (TKI) used to treat kidney cancer• Known cardiac side-effect is mostly ventricular arrhythmia• Other TKIs also produces congestive heart failure, hypertension, and myocardial ischemia

Results:• Complex dose-dependent increase (cell hypertrophy?) followed by profound decreasein base impedance (cardiotoxicity) over 24 hours• Effects on beat rate occurred quickly and at lower concentrations than impedance (QT)
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Cardiotoxicity assay: Trastuzumab (Herceptin)
Beat Rate

Base Impedance• Monoclonal antibody against Her2/ErbB2 receptor, used to treat breast cancer• Known cardiac side-effects are CHF and changes in left ventricular ejection fraction

Results:• Very slow, dose-independent decrease in base impedance (cardiotoxicity) > 72 hours• No effects on beat rate suggest little occurrence of arrythmias or QT changes

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A.

Lidocaine (Nav1.5)• Compound effects on ion channels are usually only
studied over short periods for acute toxicity• We decided to see if CiPA drugs could also
produce long-term effects in a chronic toxicity
assay

B.

C.

Nifedipine (Cav1.2)

Dofetilide (hERG)• Along with beat rate and contraction amplitude,
we could monitor base impedance over several
days on the CE96 platform to assess effects of ionchannel blockers on cell viability and sustained
cardiotoxicity• Lidocaine (Nav1.5) had no effect on cell viability• Nifedipine (Cav1.2) produced time- and dosedependent decreases in iPSC-CM viability• High concentrations of Dofetilide (hERG) also
decreased iPSC-CM base impedance
8Chronic impedance

Cardiotoxicity assay: Ion channel modulators

Cardiotoxicity assay: DoxorubicinA.

1 μM Doxorubicin

B.

C.

10 Ω1s1h24 h• Broad spectrum anti-cancer medication belonging to Anthracycline family• DNA intercalating agent• Known cardiac side effects:
48 h72 h• Acute atrial and ventricular arrhythmia• Chronic cardiomyopathy• Congestive heart failure• Concentration-dependent decrease of base impedance after 24 hours
9Chronic impedancePredrugCardiotoxicity disease modelling: Doxorubicin• Not all breast cancer patients show chronic cardiotoxicity in response to Doxorubicin• Stanford group explored this phenomena using patient-derived iPSC cardiomyocytes• Clear differences in cardiotoxicity measures in sensitive vs insensitive patients• Cell damage markers, functional contractility and arrythmia, gene expression profiles, etc
No toxTox• Impedance-based cardiotoxicity personalised medicine assays could be used to:• Model other types of patient-specific drug cardiotoxicity• Select patients for safest type of chemotherapy and other drug treatments10HL-1 mouse atrial cardiomyocytes: Chronic atrial cardiotox• HL-1 cells are an immortalised mouse ‘atrial’ CM cell line
• Exhibit immature physiology and de-differentiated morphology• A subset of cells demonstrate spontaneous contractility for a few passages• Express atrial-like ion channel expression profile (incl IKACh)• We wanted to test them as a model for chronic atrial cardiotoxicity• Assess acute and long-term changes in cell impedance as a measure of cell toxicity• Assess any changes in spontaneous contractility (beat rate)

Results:• HL-1 cells have similar sensitivity to a wide range of chronic cardiotoxicity agents as humanventricular iPS cardiomyocytes• Ion channel antagonists (esp mixed blockers like Amiodarone)• Doxorubicin• ‘nib kinase inhibitors• HL-1 cells have greater sensitivity to drugs causing atrial-specific damage• Bortezomib

11Human iPSC-CM(ventricular)

HL-1 cells(atrial CM)• Bortezomib is a proteasome inhibitor with known cardiotoxicity (incl AV block)• Large time-dependent decrease in impedance, indicating significant cardiotoxicity• We saw slower effects with lower sensitivity in ventricular human iPSC-CMs,suggesting that HL-1 atrial cells may be a useful model for chronic toxicity studies ofagents such as Bortezomib with atrial-specific side-effects
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Cardiotox studies

Bortezomib: Oncology agent with atrial cardiotoxicity

Acknowledgments• Metrion Biosciences
Said el Haou
Sarah Williams
John Ridley
Louise Webdale
Kathy Sutton
Robert Kirby
Edd Humphries

Thanks•••••••• Nanion Technologies• Sonja Stölzle-Feix• Krisztina Juhasz• Ulrich Thomas• This project received funding from the Eurostars-2 joint program withco-funding from the European Union Horizon 2010 research andinnovation program

13A.

1 μM Amiodarone

B.

C.

10 ΩPredrug

1s1h

24 h

48 h

• Class III antiarrhythmic widely used for treatment of atrial fibrillation• Known cardiac side effects:
72 h• Arrhythmia: Torsade de Pointe, VF and sinus bradycardia• Congestive Heart Failure• Base impedance decreased only after five hours drug exposure
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Chronic impedance

Cardiotoxicity assay: Amiodarone

Molecular mechanism

Base impedance

Impedance amplitude

Beat rate• Tendency of increased basal impedance, suggestive of hypertrophy effect?
• We have seen other compounds also produce chronic increases in impedance• Decreased beat rate as expected with SR Ca2+ store depletion after SERCA block• These data may support the contention that spontaneous beating in iPSC-CMs is due toCa2+ leak from SR, and is a sign of Ca2+ signalling immaturity

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Cardiotox studies

Cardiotoxicity assay: Thapsigargin (Ca2+ pump)Aspirin
Beat Rate

Base Impedance

Amplitude16

Molecular mechanism

Base impedance• Doxorubicin is a DNA alkylating agent used to treat breast cancer• Large time- and dose-dependent decrease in human iPSC cardiomyocyte
impedance, suggesting significant cardiotoxicity• We saw similar effects and dose-dependency in ventricular human iPSC-CMs,suggesting that HL-1 atrial cells may be a useful model for chronic toxicity studies
17Cardiotox studies

Doxorubicin: Breast cancer agent with cardiotoxicity

Molecular mechanism

Base impedance• Sunitinib is a tyrosine kinase inhibitor used to treat kidney cancer• Large time- and dose-dependent decrease in iPSC-CM impedance, indicating
significant toxicity at high doses (steep dose-dependence)• We saw similar time-course and dose-dependency in ventricular human iPSC-CMs,suggesting that HL-1 atrial cells may be a useful model for TKI toxicity studies
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Cardiotox studies

Sunitinib: Kidney cancer agent with cardiotoxicity

Molecular mechanism

Base impedance• Amiodarone is a non-selective ion channel antagonist, class III anti-arrythmic• Small but non-significant reduction in impedance, suggesting some toxicity at higher
concentrations• We saw larger effects in ventricular human iPSC-CMs, suggesting that either theexpression levels or mixture of ion channels in HL-1 atrial cells is different
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Cardiotox studies

Amiodarone: Mixed Nav, Cav and Kv inhibitor