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

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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.

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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

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Review of ion channel work

Manual patch clampAutomated high throughput

system (HTS)

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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.

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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

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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.

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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.

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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

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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.

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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.

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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

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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.

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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

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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

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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

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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.

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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

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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

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• 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

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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.

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Late NaV1.5 current studies

37°C

Veratridine

RT

ATXII

Manual patch clamp data HTS

Agonist to enhance late NaV1.5 current

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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

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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

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“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.

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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

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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.

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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

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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

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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

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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

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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)

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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.

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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.

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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

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Back-up slides

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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.

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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.