Manual patch clamp evaluation of hERG channel pharmacology @ 37°C

and next steps

Wendy Wu, Ph.D.Division of Applied Regulatory Science

Office of Clinical PharmacologyOffice of Translational Sciences

Center for Drug Evaluation and Research

December 6, 2016

2

4. Evaluation of unanticipated

effects in clinical phase 1 studies

1. In vitroassessment of drug effects on multiple ionic

currents

2. In silicoreconstruction of human ventricular

cardiomyocyteelectrophysiology

3. In vitro effects on human stem

cell-derived ventricular

cardiomyocytes

Patch clamp studies in CiPA initiative

www.fda.gov

Goals are: 1. to generate reliable and reproducible patch clamp data on select human cardiac ion

channels using reference drugs that have different levels of torsadogenic potential.2. to identify simple voltage protocols and practical data quality standards that

minimize lab-to-lab variability.

3

4. Evaluation of unanticipated

effects in clinical phase 1 studies

1. In vitroassessment of drug effects on multiple ionic

currents

2. In silicoreconstruction of human ventricular

cardiomyocyteelectrophysiology

3. In vitro effects on human stem

cell-derived ventricular

cardiomyocytes

Patch clamp studies in CiPA initiative

www.fda.gov

Manual patch clamp recordings performed at 37C:◦ to benchmark data quality;◦ to determine how to best translate from the industry’s room temperature and

automated patch clamp machine ‘best practice” to a physiological in silico model.

4

4. Evaluation of unanticipated

effects in clinical phase 1 studies

2. In silicoreconstruction of human ventricular

cardiomyocyteelectrophysiology

3. In vitro effects on human stem

cell-derived ventricular

cardiomyocytes

www.fda.gov

3 cardiac ion channels were selected for manual patch clamp experiments1. hERG channels (KV11.1)2. L-type Ca2+ channels (CaV1.2 subtype)3. Na+ channels (NaV1.5 subtype; peak and late components)

1. In vitroassessment of drug effects on multiple ionic

currents

NaV1.5

CaV1.2

KV11.1

Patch clamp studies in CiPA initiative

5

Overview: hERG channel experiments• Recordings were performed:

– on a HEK293 cell line that overexpresses the hERG1a proteins;– used a simple protocol to generate information regarding drug potency and drug-

channel interaction kinetics.

• Some details: – at 37°C;– 80% series resistance compensation;– one concentration per cell;– drugs application started only after baseline stability is reached.

Milnes et al., 2010

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Overview: hERG channel experiments• Recordings were performed:

– on a HEK293 cell line that overexpresses the hERG1a proteins;– used a simple protocol to generate information regarding drug potency and drug-

channel interaction kinetics.

• Some details: Milnes et al., 2010

at 37°C • hERG current larger• hERG current activates faster• Sotalol block more potent

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Overview: hERG channel experiments• Recordings were performed:

– on a HEK293 cell line that overexpresses the hERG1a proteins;– used a simple protocol to generate information regarding drug potency and drug-

channel interaction kinetics.

• Some details: Milnes et al., 2010

at 37°C • hERG current larger• hERG current activates faster• Sotalol block more potent• Faster development of block

8

Overview: hERG channel experiments• Recordings were performed:

– on a HEK293 cell line that overexpresses the hERG1a proteins;– used a simple protocol to generate information regarding drug potency and drug-

channel interaction kinetics.

• Some details: Milnes et al., 2010

Recording temperature affects pharmacology outcomes.

at 37°C • hERG current larger• hERG current activates faster• Sotalol block more potent• Faster development of block

9

Overview: hERG channel experiments• Recordings were performed:

– on a HEK293 cell line that overexpresses the hERG1a proteins;– used a simple protocol to generate information regarding drug potency and drug-

channel interaction kinetics.

• Some details: – 80% series resistance compensation;– one concentration per cell;– drugs application started only after baseline stability is reached.

Milnes et al., 2010

10

Kinetics of drug-channel interaction

How are the data used?

Accumulation/relief from block Concentration-

response curve for potency assessment(IC50 values)

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hERG channel pharmacology IC50/Cmax

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hERG channel pharmacology IC50/Cmax

• A ratio of 30:1 between a drug’s IC50 and Cmax at therapeutic doses is taken as a lack of torsadogenic potential (Redfern et al., 2003)

• This ratio does not separate the 12 CiPA training drugs into accurate risk categories.

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Kinetics of drug-channel interaction

Kinetics of block development

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Kinetics of block development

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• Kinetics of hERG channel block also does not separate CiPA training drugs into accurate torsadogenic risk categories.

• Drug effects on multiple cardiac ion channels need to be considered.

Kinetics of block development

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Next steps• Manual patch clamp experiments on CaV1.2 channels at 37°C are

ongoing. NaV1.5 channels next.

• hERG channel experiments are being repeated using an automated patch clamp system (a part of the HESI-coordinated effort)

– To understand advantages, drawbacks, and limitations;– To develop separate recording criteria and data quality standards for this

platform.

• 5 of 12 CiPA training drugs completed for CaV1.2 channels.

• CaV1.2 current exhibits prominent run-down in every cell studied. Baseline stability is crucial prior to drug application.

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Take home messages• Completed hERG channel pharmacology using 12 CiPA training drugs

and manual patch clamp rigs. • Applied strict data quality criteria and compared results generated by 3

electrophysiologists using different styles of manual patch clamp rigs.– Many factors can impact data quality hence reproducibility.

• Cell health-related (resting membrane potential, holding current, input resistance);

• Recording quality-related (seal resistance, magnitude and stability of series resistance, stability of baseline recording).

– Lessons learned: • With the simple protocol used and cell lines, the most important factor

that minimizes variability amongst experimenters and systems is baseline stability of ion channel activity prior to drug application(we obtained time course plots illustrating that run-up or run-down process has stabilized for every cell).

• CiPA initiative is global and collaborative. Cardiac ion channel pharmacology data generated by different laboratories for model calibration purpose should be comparable.

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Take home messages• Completed hERG channel pharmacology using 12 CiPA training drugs

and manual patch clamp rigs. • Applied strict data quality criteria and compared results generated by 3

electrophysiologists using different styles of manual patch clamp rigs.– Many factors can impact data quality hence reproducibility.

• Cell health-related (resting membrane potential, holding current, input resistance);

• Recording quality-related (seal resistance, magnitude and stability of series resistance, stability of baseline recording).

– Lessons learned: • With the simple protocol used and cell lines, the most important factor

that minimizes variability amongst experimenters and systems is baseline stability of ion channel activity prior to drug application(we obtained time course plots illustrating that run-up or run-down process has stabilized for every cell).

• CaV1.2 channel pharmacology underway, using manual patch clamp method and at 37°C. NaV1.5 channel pharmacology next.

• All ion channel pharmacology studies are being repeated using an automated patch clamp system.

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Acknowledgement

Jiansong Sheng

Phu Tran

Members of the Ion Channel Working Group, In Silico Working Group, and FDA colleagues involved in the CiPA initiative.

ORISE scholars involved in the patch clamp ion channel pharmacology effort: Jiansong Sheng, Min Wu, and Phu Tran.

Min Wu

21www.fda.gov

3 cardiac ion channels were selected for manual patch clamp experiments1. hERG channels (KV11.1)2. L-type Ca2+ channels (CaV1.2 subtype)3. Na+ channels (NaV1.5 subtype; peak and late components)

1. In vitroassessment of drug effects on multiple ionic

currents

NaV1.5

CaV1.2

KV11.1

Patch clamp studies in CiPA initiative

NaV1.5 channels

hERG channels

NaV1.5 channels

CaV1.2 channels

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1. In vitroassessment of drug effects on multiple ionic

currents

www.fda.gov

NaV1.5

CaV1.2

KV11.1

Martin et al., 2004

Block of hERG channels (dofetilide) broadens ventricular action potentials.

Why evaluate multiple cardiac ion channels?

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1. In vitroassessment of drug effects on multiple ionic

currents

www.fda.gov

NaV1.5

CaV1.2

KV11.1

Martin et al., 2004

Concomitant block of CaV1.2 channels (nifedipine) or NaV1.5 channels (lidocaine) mitigates the effect of hERG channel block and normalizes ventricular action potentials.

Block of hERG channels (dofetilide) broadens ventricular action potentials.

Why evaluate multiple cardiac ion channels?

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1. In vitroassessment of drug effects on multiple ionic

currents

www.fda.gov

NaV1.5

CaV1.2

KV11.1

Martin et al., 2004

Block of hERG channels (dofetilide) broadens ventricular action potentials.

Why evaluate multiple cardiac ion channels?

To make accurate predictions regarding the torsadogenic potential of drugs, one must know drug effects on multiple key cardiac ion channels that shape the ventricular action potentials.

+ CaV1.2 channel block + NaV1.5 channel block

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12 drugs with no major metabolites were chosen based on different torsadogenic potential.

CiPA training drug set

High RiskQuinidine – anti-arrhythmic

Bepridil – treats anginaDofetilide – anti-arrhythmic

Sotalol – anti-arrhythmic

Intermediate RiskChlorpromazine - antipsychotic

Cisapride - gastroprokineticTerfenadine - antihistamine

Ondansetron – prevents nausea

No/Low RiskDiltiazem – treat hypertension and angina

Mexiletine – anti-arrhythmicRanolazine – treat angina

Verapamil – treat hypertension and antiarrhythmic

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• Note the long baseline recording for this cell (~40 min).

• Illustrated traces are the averages of the last 5 consecutively recorded traces (in the boxed region).

• IC50 @ 24C = 13.8 nM; h = 0.83. IC50@ 37C = 10.1 nM; h = 0.73.