Cardiac Ion Channel Safety Profiling:
hERG and beyond
G Erdemli
Novartis Institutes for Biomedical Research
Summary
 Introduction to preclinical cardiac ion channel safety profiling
 Overview of automated electrophysiology technologies used at Novartis
• QPatch 16, HT & HTX
• IonWorks Quattro
• IonFlux 16 microfluidics system
 Cardiac ion channel in vitro assays and case studies
• hERG, Nav1.5, Cav1.2 and KCNQ1/minK
 Implementation of preclinical in vitro ion channel safety profiling in the
integrated risk assessment for cardiac safety
 Indirect ion channel modulation, potential mechanisms and implications
in preclinical safety assessment
G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010
ECG and action potential repolarization
Cardiac ion channel profiling cardiac risk assessment
R
T
P
Q
Repolarization
reserve
S QT interval
LQT
+25 mV
Ito (hKv 4.2
1
2
hKv 4.3)
IKs
-0 mV
(hminK + hKvLQT1)
IKr
INa
0
ICa
(KvHERG+hMiRP)
3
IK1 (hK
IR
RMP -80/90 mV
G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010
)
Genetic basis for LQTS
(hERG)
Kaufman. Heart rhythm 2009 vol. 6 (8 Suppl) pp. S51-5
Schwartz et al Circulation 2001
G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010
Cardiac ion channel safety profiling
hERG on Qpatch-HT
 Routine hERG screening
on QPatch-HT
 6 pnt CRC, n=3
 70% success rate
(completed experiments)
G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010
High quality and reproducible results on QPatch-HT
30
28
26
24
22
20
18
16
14
12
10
8
6
4
2
0
Amitriptyline
Aspirin
Astemizole
Bepridil
Cisapride
Diphenhydramine
Droperidol
Erythromycin
Haloperidol
Pimozide
Propranolol
Quinidine
Thioridazine
Verapamil
E-4031
DMSO
QPatch HT IC50 (uM)
QPatch HT hERG assay
reproducibility
Axis Title
G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010
Compound
Amitriptyline
Aspirin
Astemizole
Bepridil
Cisapride
Diphenhydramine
Disopyramide
Droperidol
Erythromycin
Haloperidol
Pimozide
Primaquine
Propranolol
Quinidine
Thioridazine
Verapamil
E-4031
QPatch_HT
IC50 (µM)
4.80
>30
0.05
1.41
0.05
Conventional
EP IC50 (µM)
3.00
14.90
0.33
30.00
0.20
0.05
>30.00
13.20
1.70
1.58
0.82
3.8
7.2
0.1
39
0.18
0.018
>30
7
1
1.2
0.83
0.1
0.06
5
>30
0.021
0.5
0.045
QPatch data analysis: Reaching equilibrium
G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010
hERG Openers
During routine screening hERG channel enhancers from different chemical
series are identified
Incorporated into automated data analysis for alerts
G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010
Effects of temperature in drug-induced hERG inhibition
22°C
G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010
35°C
hERG current on IonFlux-16
Automated patch clamp at physiological temperatures
A.
C
D
G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010
Cardiac ion channel safety profiling
Nav1.5 assay on IonWorks Quattro
Raw traces pre-cpd
Normalized current post-cpd
QC Monitor for seals
 8 pnt CRC, n=4
 ~100% success rate
 Use-dependency
Multi-state IC50 determination
Use-dependence Characterization
G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010
hNav1.5 Pharmacology on Quattro
Manual patch clamp
from literature
G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010
Compound 1
An example of translational value of hNav1.5 assay
 Preclinical cardiosafety data
•
in vitro Nav1.5 IC50 = 15.4 mM
•
Dose dependent prolongation of P, PQ and QRS in dog telemetry, no/minimal QT
interval prolongation
•
Sudden deaths in 13 week dog toxicity study & polymorphic ventricular tachycardia consistent with an extreme sodium channel inhibition, with PR prolongation as the first
sign
 Clinical cardiosafety data
•
Compound 1 at 640 mg (single dose) caused PR interval prolongation that coincided
with the Tmax of the drug’s plasma concentration. NOEL = 1.72 mM (320 mg/kg)
 Clinical studiesstudies terminated due to risk of cardiac conduction
abnormalities
•
Therapeutic margin < 0.8 for PR prolongation
G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010
Compound 1 Preclinical Cardiac Safety Data (Dog)
Dose
(mg/kg)
5@
Cmax
(µM)
2.6
Cmax free F%
(µM)
0.6
23
Nav1.5 IC50
(Nav1.5IC20)
15.8 µM
(3.9µM)
10@
7.9
1.8
IV/IV
QRS
P duration
Index50 duration
(ms)
(IV/IV
(ms)
Index20)
>26
51
40
(>6,5)
9
52
2
25@
12.8
3
5
1
30#
14.7
3.4
4.5
(1)
60#
27
6.2
2.4
(0.6)
 IV/IV Index50: In vitro/In vivo Index: Nav1.5 IC50/Free CMax
 IV/IV Index20: In vitro/In vivo Index: Nav1.5 IC20/Free Cmax
G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010
64
+11
+23%
48
+6
+13%
80
+38
+90%
43
+4
+10%
56
+16
+40%
49
+8
+18%
75
+34
+81%
PQ (PR)
interval
Heart
Rate
QTc
(ms)
(bpm)
78
110
239
83
+5
+6%
108
+30
+38%
101
+18
+22%
142
+59
+71%
130
+25
+24%
150
+45
+43%
135
+39
+41%
150
+55
+57%
239
(ms)
244
233
262
+28
+12%
Compound 1 Clinical Cardiac Safety Data
Dose
Cmax
(mg/kg)
(µM)
Cmax
free
Nav1.5
IC50
IV/Human
QRS
P
Index50 duration duration
(µM)
80mg
0.36
Nav1.5 (IV/Human
IC20
Index20)
0.14 15.8^µM
>110
(3.9µM)
160mg
320mg
640mg
0.95
1.72
3.1
PQ (PR)
interval
Heart
Rate
QTc
(ms)
(ms)
(ms)
(ms)
(bpm)
NC
NC
NC
NC
NC
(>28)
0.37
>43
NC
NC
NC
NC
NC
0.67
(>11)
>24
NC
NC
NC
NC
NC
1.2
(>5.8)
13
NC
NC
~16 msec
NC
NC
(3.3)
NC: No change
G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010
mean
prolongation
Compound 1 – SAD 320mg, 640mg
Maximum PR Interval Increase vs Maximum Plasma Concentration
Maximum PR Increase from Baseline (msec)
40
320 mg
35
640 mg
Baseline
30
Pre-dose
Baseline variability
3 measurements/patient
25
20
15
10
5
0
-5
-10
0
500
(0.93 µM)
1000
(1.9 µM)
G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010
1500
Cmax (ng/mL)
2000
(3.7 µM)
2500
3000
(4.7 µM)
(5.6 µM)
Cardiac Ion Channel Profiling on Automated Systems
A part of preclinical integrated risk assessment
Structure
G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010
Indirect modulation of hERG channels
 An indirect QTc mechanism is invoked if a drug produces:
• An in vivo QTc prolongation despite no in vitro signal indicating direct effect on cardiac ion
channels.
• A clinical QTc prolongation despite no preclinical signal indicating direct effect on cardiac
ion channels
 Potential mechanisms
• Channel trafficking, maturation and degradation
• Hypokalemia - regulation of ventricular repolarization by plasma potassium levels
- Furosemide has no effect on hERG current and APD in rabbit purkinje fiber but causes QT interval prolongation
- Inverse relationship between the plasma potassium levels and QT and QTc interval durations
• Changes in plasma glucose levels
- Hypoglycemia-induced hERG channel inhibition - due to decrease in intracellular ATP
- Hyperglycemia-induced hERG channel inhibition - due to production of reactive oxygen species
• Changes in autonomic tonus
- Adrenergic modulation of hERG channel - Functional coupling of α and β adrenoceptors to hERG channel
- Adrenergic regulation of IKs channel - functional coupling of β adrenoceptors to IKs
• Macromolecular Complexes can produce ↑QTc – Ankyrins, Caveolin-3, Syntrophin - No
direct drug-induced examples, but candidates for off-target interactions
G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010
Indirect modulation of cardiac channels
Definition & decision tree
Negative in Nav1.5, Cav1.2, hERG,
KCNQ1 but QTc prolongation in vivo
PK/PD relationship
Test metabolites
Cmax/AUC driven
PK/PD disconnect
Test on other cardiac ion channels
No effect
Delayed disposition (parent/metabolite) to heart tissue
Accumulation (parent/metabolite) in heart tissue
Modulation of ion channel trafficking/maturation/degradation
Modulation of ion channel gene expression
Hypokalemia
Autonomic nervous system
Glucose homeostasis
Structural macromolecular complexes
G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010
Summary
 Automated electrophysiology has been implemented in routine cardiac ion channel safety
screen in all stages of drug discovery
 Allow thousands of compounds profiling with IC50 values and quick turnaround time
 Assay validation, optimization and setting QC parameters for automated data analysis are key
for successful implementation
 Results show very good correlation with conventional electrophysiology in most cases
 Common reasons for discrepancies between conventional and automated electrophysiology
are differences in
• compound application duration
• recording temperature
 Physicochemical properties of compounds should be taken into consideration for data
analysis – solubility, permeability etc
Overall in vitro preclinical cardiac ion channel profiling data provide high
quality translational information for integrated risk assessment
G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010
Acknowledgments
Novartis
Fluxion
 Xueying Cao
 Chris Penland
 Tony Lee
 Dan Meyers
 Mats Holmqvist
 Tycho Heimbach
 A Golden
 Albert Kim
 Steven Whitebread
 Dmitri Mikhailov
 Karl Chin
 Clayton Springer
 Mark Deurinck
 Bob Pearlstein
 Qin Chen
 Nianzhen Li
 Tanner Nevill
 Juliette Johnson
 Cristian Ionescu-Zanetti
 Jeff Jensen
Sophion
 Berengere Dumotier
 Ali Yehia
 M Traebert
Millipore
 Laszlo Urban
 Duncan Jarman
G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010