Post on 23-Aug-2020
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
6
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
7
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)
11
hERG channel pharmacology IC50/Cmax
12
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.
13
Kinetics of drug-channel interaction
Kinetics of block development
14
Kinetics of block development
15
• 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
16
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.
17
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.
18
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.
19
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
22
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?
23
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?
24
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
25
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
26
• 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.