cell-based ion channel and cardiac safety assays

CELL-BASED ION CHANNEL AND CARDIAC SAFETY ASSAYSDrug Development “Boot Camp”: Practical Aspects of Positioning Your Research

EVERY STEP OF THE WAY

EVERY STEP OF THE WAY

THE ECG IS THE ELECTRICAL ACTIVITY MEASURED FROM AN ENTIRE HEART

2 EVERY STEP OF THE WAY

THE ACTION POTENTIAL IS THE ELECTRICAL ACTIVITY MEASURED FROM AN INDIVIDUAL CARDIOMYOCYTE


ION CHANNELS ARE RESPONSIBLE FOR THE ELECTRICAL ACTIVITY OF THE HEART


Ca2+

hERG

KvLQT1/minK

Kv4.3Nav1.5

Cav1.2Kir1.2

ION CHANNELS ARE RESPONSIBLE FOR THE ELECTRICAL ACTIVITY OF THE HEART


hERG

DRUG-INDUCED TORSADE DE POINTES (TDP)


Normal sinus rhythm

Torsade de Pointes

Ferimini and Fossa, Nature Reviews: Drug Discovery, Volume 2, 2003

TDP INCIDENCE TOO LOW TO DETECT IN CLINICAL TRIALSTERFENADINE: TDP IN 1/25,000 PRESCRIPTIONS


Normal sinus rhythm

Torsade de Pointes

Ferimini and Fossa, Nature Reviews: Drug Discovery, Volume 2, 2003


hERG

Prolonged QT Interval

DRUGS THAT BLOCK HERG AND PROLONGS THE QT INTERVAL MAY CAUSE TDP

GUIDANCE FOR INDUSTRY ICHS7B NONCLINICAL EVALUATION OF THE POTENTIAL FOR DELAYED VENTRICULAR REPOLARIZATION (QT INTERVAL

PROLONGATION)


Evaluation of torsadogenic risk determined by effects on only 2 surrogates of TdP: 1. In Vivo QT Interval Prolongation2. hERG Inhibition

THIS STRATEGY HAS PROVEN TO BE SUCCESSFUL .


Drug Class hERG Blocker, QT Prolonger

Date Withdrawn

Terfenadine Antihistamine √ 1998Sertindole Antipsychotic √ 1998Astemizole Antihistamine √ 1999

Grepafloxacin Antibiotic √ 1999Cisapride Prokinetic √ 2000Droperidol Antipsychotic √ 2001

Levomethadyl Opiate Dependence √ 2003

THIS NEW STRATEGY HAS PROVEN TO BE SUCCESSFUL

11

0.01 0.1 1 10 100 1000 10000Concentration (µM)

Verapamil

hERG IC50

Cav1.2 IC50

Nav1.5 IC50

KvLQT1/minK IC50

Kir2.1 Est. IC50

Plasma Level

Ca2+

• Blocks hERG• Not Very Potent Inhibitor of KvLQT1/mink or Kir2.1


12

Ca2+ Na+


Verapamil

hERG IC50

Cav1.2 IC50

Nav1.5 IC50

KvLQT1/minK IC50

Kir2.1 Est. IC50

Plasma Level

• Blocks hERG• Not Very Potent Inhibitor of KvLQT1/mink or Kir2.1• Blocks Cav1.2

LOGISTIC REGRESSION MODELS


Kramer et al., Sci Rep. 2013;3:2100.


14

Action Potentials

Field Potentials

Stem Cell Derived Cardiomyocytes

↓ Action Potential Duration

↓ Field Potential Duration



• Package Insert = No TdP Risk• Does Not Prolong The QT Interval

Blocks hERG and Cav1.2

Ca2+


Verapamil

hERG IC50

Cav1.2 IC50

Nav1.5 IC50

KvLQT1/minK IC50

Kir2.1 Est. IC50

Plasma Level

Vicente J, Johannesen L et al. Comprehensive T wave morphology assessment in a randomized clinical study of dofetilide, quinidine, ranolazine, and verapamil. J Am Heart Assoc. 2015 Apr 13;4(4).

Blocks hERG and Cav1.2

COMPREHENSIVE IN VITRO PROARRHYTHMIA ASSAY (CIPA)


Confirmation of Electrophysiology Data using Cardiomyocytes

Function Effects on Multiple Cardiac

Currents

In Silico Models

Ca2+

ProarrhythmiaAssessment

MECHANISM OF ACTION


Cardiovascular

MECHANISM OF ACTION


Cardiovascular

Nav1.5

X

MECHANISM OF ACTION


Cardiovascular

Nav1.5

X

Kir3.1/3.4

X

MECHANISM OF ACTION


Cardiovascular

Nav1.5

X

Kir3.1/3.4

XKir6.2/SUR2AX

CHANNEL PANELS


hERG (Kv11.1)

Cav1.2/β2/α2δ1

Cardiovascular

Kir6.2/SUR2ANav1.5

Kir3.1/3.4

CHANNEL PANELS


hERG (Kv11.1)Kv4.3KvLQT1/mink (Kv7.1)Kv1.5

Cav3.2HCN2 and HCN4Kir2.1, Kir3.1/3.4Nav1.5Kir6.2/SUR2A

Cav1.2/β2/α2δ1 BK, IK and SK3, ENaCTRPC1, TRPC4, TRPC6,TRPM4, TRPV1 and TRPV4NCX1, nAChRα7 and P2X4

Cardiovascular

CHANNEL PANELS


hERG (Kv11.1)

Kv1.3, Kv1.4,Kv4.2/KChiP2.2, KCNQ2/3, KCNQ2/4Cav2.1/β4/α2δ1,Cav2.2/β3/α2δ1 and Cav3.2NR1/NR2B, HCN1nAChRα7, GABA (α3β3γ2)P2X1, P2X2, P2X3, P2X4 and P2X7Nav1.1, Nav1.2, Nav1.3, Nav1.7 and Nav1.8/β3TRPA1, TRPC4, TRPM4, TRPM8, TRPV1 and TRPV4NMDA (NR1/NR2ABK and IK, ASIC1a, ASIC2a and ASIC3

Kv4.3KvLQT1/mink (Kv7.1)Kv1.5



Cardiovascular

Pain/Inflammation

CHANNEL PANELS


hERG (Kv11.1)


CLC-2, Cav2.1/β4/α2δ1 and Cav3.2HCN1 and HCN2GABAA (α1β2γ2, α2β2γ2, α5β2γ2NR1/NR2A, NR1/NR2B)BK, IK, SK2 and SK3Kv1.1, Kv4.2/KChiP2.2KCNQ2/3, KCNQ2/4, KCNQ3/5Nav1.1, Nav1.2, Nav1.3 and Nav1.6




Seizure/ConvulsionCardiovascular

Pain/Inflammation

CHANNEL PANELS


hERG (Kv11.1)


CLC-2, Cav2.1/β4/α2δ1 and Cav3.2HCN1 and HCN2GABAA (α1β2γ2, α2β2γ2, α5β2γ2NR1/NR2A, NR1/NR2B)BK, IK, SK2 and SK3Kv1.1, Kv4.2/KChiP2.2KCNQ2/3, KCNQ2/4, KCNQ3/5Nav1.1, Nav1.2, Nav1.3 and Nav1.6




Seizure/ConvulsionCardiovascular

Pain/InflammationPsychiatric Disorder Cav1.3/β3/α2δ

(GABA (α1β3γ2, α2β3γ2, α3β3γ2,α4β3γ2 and α5β3γ2), nACHR (α4/β2, α7, α3β4α5, α6/3β2β3)NMDA (NR1/NR2A,NR1/NR2B, NR1/NR2Cand NR1/NR2D))KCNQ2/3 and KCNQ3/5SK1, SK2 and SK3

ACKNOWLEDGEMENTS


Jessica Brimecombe, PhDLuke Armstrong, PhDCarlos Obejero-Paz, MD, PhDAndrew Bruening–Wright, PhDYuri-Kuryshev, PhDBob Ostroski, BSKerri Uffman, BS

cell-based ion channel and cardiac safety assays

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