Development of an Adverse Outcome Pathway for ......Development of an Adverse Outcome Pathway for...
Transcript of Development of an Adverse Outcome Pathway for ......Development of an Adverse Outcome Pathway for...
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