Development of an Adverse Outcome Pathway for cardiotoxicity mediated by the blockade of L-type calcium channels

Luigi Margiotta-Casaluci

NC3Rs Strategic Award Holder

NC3Rs/Unilever Workshop London, 10-11 December 2018

Cardiac cycle

NC3Rs Strategic Award Going beyond hERG

O’Hara et al. 2011. PLoS Comput Biol 7(5): e1002061 Image adapted from Gintant et al. 2016 Nature Reviews Drug Discovery 15, 457–471

Cardiotoxicity was identified as an area of potential interest for AOP development at the NC3Rs workshop in 2014 ‘Applying pathways-based approaches across the biosciences’.

Several follow up events Strategic Award 2017

L-type calcium channel blockade as molecular initiating event (MIE)

Calcium ions play a vital role in cellular and organism physiology

• E.g. mediation of muscle contraction, hormone secretion, and neuronal transmission

L-type calcium channels are responsible for the excitation-contraction coupling of skeletal, smooth, and cardiac muscle

Perturbation of calcium dynamics in the heart may impair organ function and health

Image adapted from Gintant et al. 2016 Nature Reviews Drug Discovery 15, 457–471

LTCC structure and tissue distribution

Striessnig et al. 2014; Wiley InterdiscipRev Membr Transp Signal. 3(2): 15–38. Zamponi et al. 2015 Pharmacol Rev 67:821–870

LTCCs and cardiac electromechanical coupling

Stokke et al., 2012

Adverse Outcome in HumansHeart Failure

Adverse Outcome in HumansHeart Failure

Cost to the US health system: $32 billion in 2012

Cardiotox AOP - Project workflow – 6 (+3) months

Literature review and data extraction

Review papersGenetic manipulation studies

CCBs exposure studies------------

In vivo, ex vivo, in vitro

148 experimental papers + reviews

Plausibility, essentiality, & weight of evidence analysis (e.g. dose response concordance, reproducibility, etc)

AOP development

Outputs: • Database of LTCC-mediated effects• #3 AOPs on the AOPWiki• #1 manuscript in preparation

Literature review and data extraction Empirical evidence

CCBs exposure studies

AOP developed using data extracted from exposure studies involving 10 CCBs and healthy biological models (i.e. non-disease models)

Drug Class Total no. of data points Affinity to LTCC (1C) (Lowest Ki, nM)* Species Data

sourceNifedipine Dihydropyridine CCB 345 0.5 Rat ChEMBL

Amlodipine Dihydropyridine CCB 114 20 Rat ChEMBLFelodipine Dihydropyridine CCB 14 0.053 Rat ChEMBLNisoldipine Dihydropyridine CCB 2 0.476 Rat ChEMBLNimodipine Dihydropyridine CCB 2 0.156 Rat ChEMBLNitrendipine Dihydropyridine CCB 1 0.246 Rat ChEMBL

Diltiazem Benzothiazepine CCB 123 16 nM Rat ChEMBLVerapamil Phenylalkylamine CCB 272 12 nM Rat ChEMBL

Fendiline Phenylalkylamine/non-selective CCB 17 17000

(**IC50, Ki n/a) Rat ChEMBL

Mibefradil Non selective CCB 44 156 nM(**IC50, Ki n/a) Human ChEMBL

Most common model species

050

100150200250300

No.

of d

ata

entr

ies

Model species

Target compartments and assay type

050

100150200250300350

No.

of d

ata

entr

ies

Assay type No. of data entries

In vivo 81

Ex vivo 153

In vitro 373

Example of extracted dataReference, Species, Model info, Drug, Details of genetic manipulation, Effect/No Effect concentration, Dose-response concordance, Quantification method, Exposure duration

Effect direction and confidence assessmentof bi-directional KEs

Effect direction and confidence assessment of non bi-directional KEs

AOP261: Disruption of cardiac contractility

AOP262: Disruption of cardiac electrophysiology

0

0.25

0.5

0.75

1ICa,L inhibition (M)

Peak ICa,L density(M)

Pulse-end current(M)

H2O2-inducedcytosolic Ca2+…

ICa,L decaykinetics (L)

Peak ICa,Lamplitude (L)

Rate of Ica,Linactivation (L)

Time-dependentICa,L (L)

ICa,L (H)

Peak ICa,L (H)

ICa,L amplitude (H) Responsiveness

KE: Calcium current, Decrease

Total no. of data points = 91Nifedipine: 37Verapamil: 25Diltiazem: 17Fendiline: 6Felodipine: 3 Amlodipine: 2Semotiadil: 1

0

0.25

0.5

0.75

1

Beat-to-beat APduration variability

(M)Plateau potential

(L)Second plateau

response (L)

AP duration (H)

AP cycle length (L)

Threshold potential(L)

Maximum diastolicpotential (L)AP overshoot (M)

Maximum rate ofdepolarization (L)

AP amplitude (M)

Resting membranepotential (L)

Slope of Phase 4depolarization (L)

AP upstroke (L) Responsiveness

KE: Action potential, Disruption

Total no. of data points = 65Nifedipine: 43Amlodipine: 12 Verapamil: 10

Influence of disease state on the KEs

Healthy model

Disease model

Res

pons

iven

ess Up

Down

Healthy model

Disease model

Res

pons

iven

ess Up

Down

Empirical support+

Weight of Evidence

Identification of modulating factors

+300 data points

Influence of disease state on the KEs

Disease type

• Arrhythmia• Brugada syndrome phenotype• Cerebral ischemia• Coronary artery ligation • Endotoxemia • Long QT syndrome type 4• Myocardial infarction• Multiple organ dysfunction syndrome• Tachycardia• Terminally failing human hearts• Tetralogy of Fallot

Influence of disease state on the KEs

Disease type

• Alcohol dependence• Balloon injury of the carotid

artery• Chronic atrioventricular block• Heart failure• High salt diet• Hypertension• Iron overload• Ischemia• Myocardial infarction• Rapid atrial pacing• Patients undergoing coronary

angiography with or without percutaneous coronary interventions

• SHR hydronephrotic model

Influence of disease state on the KEs

Iron overload (mice)

Systolic blood pressure decrease

Effect rescued by LTCC blockade

Iron metabolism as potential

modulating factor

Genetic manipulation studies and essentiality assessment

Genetic manipulation studies Chemical exposure studies (exposure to CCBs)

AOP

CCB-induced effects + 146 data points describing the effects of genetic manipulations of various component of the pathway:

• Cav1.2, Cav1.3• Calmodulin • Calmodulin kinase II• Cardiac Troponin T

Cav1.2 and disease

Target Relationship type Disease

CACNA1C

contributes to Attention deficit hyperactivity disordercontributes to Autism spectrum disordercontributes to Bipolar disorder

has phenotype Brugada syndromeis marker for Brugada syndrome 3is marker for Hypertensionis marker for Hypoglycaemia

likely_pathogenic_for_condition Idiopathic ventricular fibrillation, non Brugadatype

contributes to Malignant exocrine pancreas neoplasmcontributes to Schizophreniais marker for Timothy syndromecontributes to Unipolar depression

Cav1.2 and Timothy syndrome

Timothy syndrome is a rare disorder that affects many parts of the body including the heart, digits (fingers and toes), and the nervous system.

Characterised by Long QT syndrome, which causes the cardiac muscle to take longer than usual to recharge between beats, and by 2 : 1 functional atrioventricular conduction block.

Napolitano et al., 2011Circulation Research;108:607-618

Consequences of genetic manipulation of Cav1.2Silencing/Deletion/Inactivation

This data confirms the KE identified during the mining of empirical evidence

Consequences of genetic manipulation of Cav1.2Overexpression of beta-subunits

All Beta subunits increased the native cardiac whole-cell L-type Ca2+ channel current density, but produced distinctive effects on channel inactivation kinetics.

Order of potency: β2a ≈ β4 > β1b > β3

AOP1: Disruption of cardiac contractility

KERs Plausibility/WoE Assessment - Summary

KE Upstream KE Downstream Plausibility WoE

LTCC blockade Ca2+ current (↓) Strong Strong

Ca2+ current (↓) Binding to Troponin C (↓) Strong Strong

Binding to Troponin C (↓) Contractile response (↓) Strong Strong

Contractile response (↓) Ejection fraction (↓) Strong Strong

Ejection fraction (↓) Heart failure Strong Strong

Ca2+ current (↓) Intracell. Ca2+ mobiliz.(Disruption)

Strong Strong

Intracell. Ca2+ mobiliz.(Disruption)

Sarcomere assembly (Disruption)

Strong Moderate

Sarcomere assembly (Disruption)

Contractile response (↓) Strong Strong

KEs Essentiality Assessment - Summary

KE Upstream KE Downstream Essentiality

LTCC blockade (MIE) Ca2+ current (↓) Strong

Ca2+ current (↓) Binding to Troponin C (↓) Strong

Binding to Troponin C (↓) Contractile response (↓) Strong

Contractile response (↓) Ejection fraction (↓) Strong

Ejection fraction (↓) Heart failure (AO) Strong?

KE Upstream KE Downstream Essentiality

Ca2+ current (↓) Intracell. Ca2+ mobiliz.(Disruption) Strong

Intracell. Ca2+ mobiliz.(Disruption) Sarcomere assembly (Disruption) Moderate

Sarcomere assembly (Disruption) Contractile response (↓) Moderate

Intracell. Ca2+ mobiliz.(Disruption) Contractile response (↓) Moderate

Cardiac Failure Review 2017;3(1):7–11.

Ejection faction Heart failure

Impact of AOPs on the 3Rs

MIE

KE1

KE2

KE3

AO

Identification of highly predictive safety biomarker suitable for in vitro testing

Tailored in vivo pre-clinical

study design

Empirically supported AOP for healthy modelsNetwork perspective

Future developmentsIn silico modelling of the network

Land et al. (2017) Journal of Molecular & Cellular Cardiology 106: 68–83

Contractility pathwayElectrophysiology pathway

Passini et al. 2016 Journal of Molecular and Cellular Cardiology 96: 72–81

Future developmentsIn vitro/in silico profiling of predictive KEs

and animal test replacement

Intracellular Ca2+ mobilization (Disruption)

Contractile response

(-)

Action potential (Disruption)

In vitro (hiPSC-CM)

+ In silico

Challenge 13: InPulseLed by: Prof Chris Denning

Acknowledgments

Hanna Dusza

Ines Moreira

Philip Marmon

Dr Matthew Winter Professor Chris Denning

Dr Helen Prior