Measuring ion channel block for CiPA:
Lessons learned and recommendations
Wendy W. Wu, Ph.D.
Division of Applied Regulatory Science
Office of Clinical Pharmacology, Office of Translational Sciences
Center for Drug Evaluation and Research
CSRC CiPA meeting May 21, 2018
This presentation reflects the views of the author and should not be construed to represent FDA’s views or policies
2
1. Multi-ion channel
pharmacology data
2. Computer
modelling of TdP risk
In vitro & in silico components of CiPA
K+
Na+ Ca2+
• Patch clamp data for CiPA reference drugs have been generated for
multiple cardiac ion channels.
• A CiPA myocyte model and a statistical framework for its use
(uncertainty quantification) have been developed and validated.
3
Agenda
1. To show how the two sets of data used to train and
validate the model were generated.
• hERG*
• CaV1.2
• late NaV1.5
2. To show how differences in experimental procedures
for CaV1.2 and late NaV1.5 currents lead to data
variability.
3. To propose how ion channel pharmacology
experiments can be standardized under CiPA.
*Drug binding kinetics
4
Review of ion channel work
Manual patch clampAutomated high throughput
system (HTS)
5
Review of ion channel work
• 12 training drugs1. Manual patch clamp (37°C and RT):
• hERG – drug potency (IC50) and drug block kinetics.
37°C (FDA) and RT (Victor Chang Institute).
– RT data could not resolve drug block kinetics.
• 6 other currents – IC50s taken from a manuscript.
2. HTS at RT (HESI-coordinated 19-site study):• hERG – no useful data generated for block kinetics across all sites for
CiPA protocol; IC50 using conventional protocol varied widely.
• Same for other currents. Only 1 site generated data for all currents.
• 16 validation drugs1. Manual patch clamp studies at 37°C:
• hERG - IC50 and kinetics.
37°C (FDA)
• CaV1.2, late NaV1.5 – IC50 only (Same CRO/publication author).
2. HTS at RT:• Same site generated IC50s for all currents.
RT - room temperature
HTS - high throughput system
37°C left.
1 RT dataset.
6
Review of ion channel work
Used for model training and validation Used for model training and validation
hERG data - 37°C, manual
Other currents - 37°C, manual Other currents - RT, HTS
7
Agenda
1. To show how the two sets of data used to train and
validate the model were generated.
• hERG
• CaV1.2
• late NaV1.5
2. To show how differences in experimental procedures
for CaV1.2 and late NaV1.5 currents lead to data
variability.
3. To propose how ion channel pharmacology
experiments can be standardized under CiPA.
8
Experimental procedure designed to capture drug potency (IC50) and
drug-channel interaction kinetics.
Maintain hERG channel closure
-80 mV
-80 mV
0 mV, 10 s
hERG current
Activate hERG channels
Repeated 10X
Repeated 10X
Assuming drugs do not bind to hERG
channels in the closed state, then one
can infer drug binding kinetics by
analyzing fast hERG current decays in
the presence of drugs.
9
Experimental procedure designed to capture drug potency (IC50) and
drug-channel interaction kinetics.
Maintain hERG channel closure
-80 mV
-80 mV
0 mV, 10 s
hERG current
Activate hERG channels
Repeated 10X
Repeated 10X
Critical factors for these experiments:
1. Background/leak current
2. Baseline stability
3. Cell/recording quality
10
Background/leak current
Baseline stability
Cell/recording quality
Non-hERG current
Background/non-hERG current (biological or artificial) should be
minimized or documented.
• Non-hERG current can affect IC50 and block kinetics assessment.
• Recommendation - a positive control should be performed to
assess the magnitude and profile of the non-hERG current for data
interpretation.
11
Background/leak current
Baseline stability
Cell/recording quality
Baseline stability in control solution
• Both current amplitude and
shape change in control solution
before reaching stability.
• IC50 and block kinetics
affected.
• Recommendation - baseline
stability must be obtained before
drug application.
12
Activate hERG current
Maintain hERG channel closure
-80 mV
-80 mV
0 mV, 10 s
Background/leak current
Baseline stability
Cell/recording quality
Baseline current and input resistance extracted from every current trace
recorded.
• Recommendation - these
parameters should remain stable
throughout the experiments as
they are measurements of cell
health.
Recording quality assessment
13
Agenda
1. To show how the two sets of data used to train and
validate the model were generated.
• hERG
• CaV1.2
• late NaV1.5
2. To show how differences in experimental procedures
for CaV1.2 and late NaV1.5 currents lead to data
variability.
3. To propose how ion channel pharmacology
experiments can be standardized under CiPA.
14
CaV1.2 channel studies
Manual patch clamp data HTS
37°C
Ba2+
RT
Ca2+Quality control steps
Zucotti et al., 2011. Trends in Pharm. Sci.
This state transition does not
occur if using Ba2+
Ca2+-dependent inactivation
15
CaV1.2 channel studies
Manual patch clamp data HTS
37°C
Ba2+
RT
Ca2+
Verapamil 11.2 mM0.2 mM
Quality control steps
10/28 CiPA drugs showed notable IC50 differences
We systematically examined how these procedural differences impact results.
1. Ba2+ vs Ca2+ as the charge carrier
2. Region of analysis
3. Temperature
4. Recording stability
Recommendation
16
Charge carrier on pharmacology
Drug P* 6.9 mM 19.5 mM
*Drugs with QTC signal under multi-channel evaluation at the FDA.
Recommendation - Ca2+ should be used as channel gating is not affected.
Ba2+ vs Ca2+
Region of analysis
Temperature
Recording stability
0.55 mM 2.13 mMVerapamil
IC50
17
Results differ based on analysis region
5.5 mM19.5 mM
Drug M* 36 mM32 mM
Ba2+ vs Ca2+
Region of analysis
Temperature
Recording stability
0.14 mM0.55 mMVerapamil
IC50
0.23 mM2.13 mM
Recommendation - Both regions should be analyzed as they inform
additional mechanism of drug action and can be used for future model
improvement as necessary.
Drug P* 4.4 mM6.9 mM
*Drugs with QTC signal under multi-channel evaluation at the FDA.
18
0.14 mM 0.23 mM0.55 mM 2.13 mMVerapamil
Drug P*
Drug M*
IC50
4.4 mM 5.5 mM6.9 mM 19.5 mM
36 mM32 mM
Ba2+ vs Ca2+
Region of analysis
Temperature
Recording stability Temperature and block potency
19
0.14 mM 0.23 mM0.55 mM 2.13 mMVerapamil
Drug P*
Drug M*
IC50
4.4 mM 5.5 mM6.9 mM 19.5 mM
36 mM32 mM
Temperature may affect % block for some drugs.
Recommendation - 37°C recording preferred; RT recording acceptable.
Temperature and block potency
Ba2+ vs Ca2+
Region of analysis
Temperature
Recording stability
20
• Ca2+ current rundown underestimate IC50.
• Recommendation - wait for baseline stability or capture enough baseline signal for offline rundown correction.
CaV1.2 current rundown
Ba2+ vs Ca2+
Region of Analysis
Temperature
Recording stability
21
Lack of standardized experimental procedures for data acquisition and
analysis.
Adapting a unified experimental approach for ion channel assay
under CiPA is key.
CaV1.2 channel studies
37°C
Ba2+
RT
Ca2+
Verapamil 11.2 mM0.2 mM
Initial current run-down. Correct or not?
Ba2+ vs Ca2+
Region of Analysis
Temperature
Recording stability
10/28 CiPA drugs showed notable IC50 differences
Manual patch clamp data HTS
22
Agenda
1. To show how the two sets of data used to train and
validate the model were generated.
• hERG
• CaV1.2
• late NaV1.5
2. To show how differences in experimental procedures
for CaV1.2 and late NaV1.5 currents lead to data
variability.
3. To propose how ion channel pharmacology
experiments can be standardized under CiPA.
23
Late NaV1.5 current studies
37°C
Veratridine
RT
ATXII
Manual patch clamp data HTS
Agonist to enhance late NaV1.5 current
24
Late NaV1.5 current studies
37°C
Veratridine
RT
ATXII
Manual patch clamp data HTS
ATX-II binding sites
Veratridine binding sites
Choice of agonist may affect subsequent drug-binding to NaV1.5 channels.
Quality control method
Recording solution
25
Late NaV1.5 current studies
37°C
Veratridine
RT
ATXII
Manual patch clamp data HTS
Quality control method
Recording solution
14/28 CiPA drugs showed notable IC50 differences
We systematically examined how these procedural differences impact results.
1. Recording stability, given recording solution used2. Temperature3. Veratridine vs. ATXII4. Region of analysis
Clozapine 2.2 mM73.6 mM
26
“Run-up” of late NaV1.5 current
Recording stability
Temperature
Veratridine vs. ATXII
Region of analysis
Whole cell dialysis period
Start of recording
• Late Na+ current run-up because agonist effect is activity-
dependent
• Recommendations – 1) new solution recipe; 2) baseline
stability needs to be established prior to drug application.
overestimate IC50.
27
Agonist affects IC50 calculation
11 mM65 mMLidocaine
IC50
Ranolazine 121 mM 21 mM
Recording stability
Temperature
Veratridine vs. ATXII
Region of analysis
• ATXII gives lower IC50 values than veratridine. Binding site.
6.1 mMDrug P*
*Drugs with QTC signal
under multi-channel
evaluation at the FDA.24.5 mM
4.7 mMDrug M* 51.1 mM
28
Agonist affects IC50 calculation
65 mM 11 mMLidocaine
Ranolazine
Drug P*
Drug M*
IC50
121 mM 21 mM
Recording stability
Temperature
Veratridine vs. ATXII
Region of analysis
24.5 mM 6.1 mM
51.1 mM 4.7 mM
• ATXII gives lower IC50 values than veratridine. Binding site.
• Recommendation - ATXII should be used as it does not bind to the
channel pore.
*Drugs with QTC signal
under multi-channel
evaluation at the FDA.
29
65 mM 11 mM510 mM 32 mMLidocaine
Ranolazine
Drug P*
Drug M*
IC50
121 mM 21 mM128 mM 31 mM
Recording stability
Temperature
Veratridine vs. ATXII
Region of analysis
24.5 mM 6.1 mM23.3 mM 16.3mM
51.1 mM 4.7 mM24.5 mM 29 mM
*Drugs with QTC signal
under multi-channel
evaluation at the FDA.
Results differ based on analysis region
30
65 mM 11 mM510 mM 32 mMLidocaine
Ranolazine
Drug P*
Drug M*
IC50
121 mM 21 mM128 mM 31 mM
Recording stability
Temperature
Veratridine vs. ATXII
Region of analysis
24.5 mM 6.1 mM23.3 mM 16.3mM
51.1 mM 4.7 mM24.5 mM 29 mM
*Drugs with QTC signal
under multi-channel
evaluation at the FDA.
Results differ based on analysis region
31
65 mM 11 mM510 mM 32 mMLidocaine
Ranolazine
Drug P*
Drug M*
IC50
121 mM 21 mM128 mM 31 mM
Recording stability
Temperature
Veratridine vs. ATXII
Region of analysis
24.5 mM 6.1 mM23.3 mM 16.3mM
51.1 mM 4.7 mM24.5 mM 29 mM
*Drugs with QTC signal
under multi-channel
evaluation at the FDA.
Results differ based on analysis region
Recommendation - Both regions should be analyzed as they inform
additional mechanism of drug action and can be used for future model
improvement as necessary.
32
Late NaV1.5 current studies
37°C
Veratridine
RT
ATXII
Lack of standardized experimental procedures for data acquisition and
analysis.
Adapting a unified experimental approach for ion channel assay
under CiPA is key.
14/28 CiPA drugs showed notable IC50 differences
Clozapine 2.2 mM73.6 mM
Manual patch clamp data HTS
Quality control method
Recording solution
33
Presentation summary
1. Multi-ion channel data for 28 CiPA drugs were generated by the FDA
and others using manual patch clamp method and HTS.
Human-collected; can be high quality robot-generated; what industry uses
34
Presentation summary
1. Multi-ion channel data for 28 CiPA drugs were generated by the FDA
and others using manual patch clamp method and HTS.
Human-collected; can be high quality robot-generated; what industry uses
High
Intermediate
Low
Torsade Metric Score (qNet averaged 1 to 4 Cmax)
35
Presentation summary
1. Multi-ion channel data for 28 CiPA drugs were generated by the FDA
and others using manual patch clamp method and HTS.
2. After seeing the ion channel data variability, FDA launched an internal
investigation and obtained evidence that differences in experimental
procedures (recording, analysis, quality control) associated with these
datasets directly translate into data variability.
3. Our recommendations for ion channel assay standardization will be
discussed with interested parties at the workshop tomorrow.
Recommendations for standardizing ion channel assay for CiPA
Human-collected; can be high quality robot-generated; what industry uses
To avoid the need to retrain the model, patch clamp ion channel studies need
to be standardized for data consistency and replicability.
36
Summary of recommendationsFor all 3 ion channels:
1. Data should be generated with unified voltage/experimental protocols, recording solutions, and analyzed using unified methods.
2. Achieving baseline stability prior to drug application is the key.
3. Background/contaminating current should be addressed with positive controls.
4. Cell health/recording integrity should be documented throughout the experiment.
Additionally,
1. hERG channel studies for drug potency and block kinetics should be performed at 37°C or elevated temperature so that the current activation time is not rate-limiting for measuring drug-mediated block.
2. CaV1.2 channel studies should be performed using Ca2+ as the charge carrier.
3. Late NaV1.5 studies should be performed with ATXII as the agonist.
37
Acknowledgement
FDA Contributors
• Norman Stockbridge
• Christine Garnett
• David Strauss
• Zhihua Li
• Wendy Wu
• Sara Dutta
• Phu Tran
• Jiangsong Sheng
• Kelly Chang
• Kylie Beattie
• Min Wu
• Aaron Randolph
• Richard Gray
• Jose Vicente
• Lars Johannesen
CiPA Steering Committee
Ayako Takei, Bernard Fermini, Colette Strnadova,
David Strauss, Derek Leishman, Gary Gintant, Jean-
Pierre Valentin, Jennifer Pierson, Kaori Shinagawa,
Krishna Prasad, Kyle Kolaja, Natalia Trayanova,
Norman Stockbridge, Philip Sager, Thomas Colatsky,
Yuko Sekino, Zhihua Li, Gary Mirams
CiPA Working groups
• Ion Channel working group
• In silico working group
• Cardiomyocyte working group
• Phase 1 ECG working group
• Public-private partnerships: HESI, SPS, CSRC
• Regulatory Agencies: FDA, EMA, PMDA/NIHS,
Health Canada
• Many pharmaceutical, CRO, and laboratory
device companies
• Academic collaborators
FDA Cellular
Electrophysiology Group
39
Back-up slides
40
Activate hERG current
Maintain hERG channel closure
-80 mV
-80 mV
0 mV, 10 s
Background/leak current
Baseline stability
Cell/recording quality
Baseline current and input resistance extracted from every current trace
recorded.
Baseline current vs RMP
41
Late NaV1.5 current analysis
42
Late NaV1.5 current analysis
43
High
Intermediate
Low
95%CI of each drug’s
2000 scores are shown
as error bars
Torsade Metric Score (qNet averaged 1 to 4 Cmax)
Torsade metric score and threshold
• Flexibility of the model was tested by training and validating with two
patch clamp datasets. Data for two currents were generated using
different experimental procedures and showed considerable differences.
• Nonetheless, model achieved high performance levels across both
datasets.
44
High
Intermediate
Low
95%CI of each drug’s
2000 scores are shown
as error bars
Torsade Metric Score (qNet averaged 1 to 4 Cmax)
Torsade metric score and threshold
To avoid the need to retrain the model, patch clamp ion channel studies need
to be standardized for data consistency and replicability.
hERG, CaV1.2, late NaV1.5 have predominant influence over model output.
Top Related