Cell-Based Ion Channel and Cardiac Safety Assays
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Transcript of 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
3 EVERY STEP OF THE WAY
ION CHANNELS ARE RESPONSIBLE FOR THE ELECTRICAL ACTIVITY OF THE HEART
4 EVERY STEP OF THE WAY
Ca2+
hERG
KvLQT1/minK
Kv4.3Nav1.5
Cav1.2Kir1.2
ION CHANNELS ARE RESPONSIBLE FOR THE ELECTRICAL ACTIVITY OF THE HEART
5 EVERY STEP OF THE WAY
hERG
DRUG-INDUCED TORSADE DE POINTES (TDP)
6 EVERY STEP OF THE WAY
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
7 EVERY STEP OF THE WAY
Normal sinus rhythm
Torsade de Pointes
Ferimini and Fossa, Nature Reviews: Drug Discovery, Volume 2, 2003
8 EVERY STEP OF THE WAY
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)
9 EVERY STEP OF THE WAY
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 .
10 EVERY STEP OF THE WAY
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Date Withdrawn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
THIS NEW STRATEGY HAS PROVEN TO BE SUCCESSFUL
12
Ca2+ Na+
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
• Blocks hERG• Not Very Potent Inhibitor of KvLQT1/mink or Kir2.1• Blocks Cav1.2
LOGISTIC REGRESSION MODELS
13 EVERY STEP OF THE WAY
Kramer et al., Sci Rep. 2013;3:2100.
THIS NEW STRATEGY HAS PROVEN TO BE SUCCESSFUL
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Action Potentials
Field Potentials
Stem Cell Derived Cardiomyocytes
↓ Action Potential Duration
↓ Field Potential Duration
THIS NEW STRATEGY HAS PROVEN TO BE SUCCESSFUL
15 EVERY STEP OF THE WAY
• Package Insert = No TdP Risk• Does Not Prolong The QT Interval
Blocks hERG and Cav1.2
Ca2+
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
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)
16 EVERY STEP OF THE WAY
Confirmation of Electrophysiology Data using Cardiomyocytes
Function Effects on Multiple Cardiac
Currents
In Silico Models
Ca2+
ProarrhythmiaAssessment
MECHANISM OF ACTION
17 EVERY STEP OF THE WAY
Cardiovascular
MECHANISM OF ACTION
18 EVERY STEP OF THE WAY
Cardiovascular
Nav1.5
X
MECHANISM OF ACTION
19 EVERY STEP OF THE WAY
Cardiovascular
Nav1.5
X
MECHANISM OF ACTION
20 EVERY STEP OF THE WAY
Cardiovascular
Nav1.5
X
Kir3.1/3.4
X
MECHANISM OF ACTION
21 EVERY STEP OF THE WAY
Cardiovascular
Nav1.5
X
Kir3.1/3.4
X
MECHANISM OF ACTION
22 EVERY STEP OF THE WAY
Cardiovascular
Nav1.5
X
Kir3.1/3.4
XKir6.2/SUR2AX
CHANNEL PANELS
23 EVERY STEP OF THE WAY
hERG (Kv11.1)
Cav1.2/β2/α2δ1
Cardiovascular
Kir6.2/SUR2ANav1.5
Kir3.1/3.4
CHANNEL PANELS
24 EVERY STEP OF THE WAY
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
25 EVERY STEP OF THE WAY
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
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
Pain/Inflammation
CHANNEL PANELS
26 EVERY STEP OF THE WAY
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
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
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
Seizure/ConvulsionCardiovascular
Pain/Inflammation
CHANNEL PANELS
27 EVERY STEP OF THE WAY
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
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
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
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
28 EVERY STEP OF THE WAY
Jessica Brimecombe, PhDLuke Armstrong, PhDCarlos Obejero-Paz, MD, PhDAndrew Bruening–Wright, PhDYuri-Kuryshev, PhDBob Ostroski, BSKerri Uffman, BS