Manual patch clamp evaluation of herg channel 37 C and next steps

Transcription

1 Manual patch clamp evaluation of herg channel 37 C and next steps Wendy Wu, Ph.D. Division of Applied Regulatory Science Office of Clinical Pharmacology Office of Translational Sciences Center for Drug Evaluation and Research December 6, 2016

2 Patch clamp studies in CiPA initiative 1. In vitro assessment of drug effects on multiple ionic currents 2. In silico reconstruction of human ventricular cardiomyocyte electrophysiology 3. In vitro effects on human stem cell-derived ventricular cardiomyocytes 4. Evaluation of unanticipated effects in clinical phase 1 studies 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. 2

3 Patch clamp studies in CiPA initiative 1. In vitro assessment of drug effects on multiple ionic currents 2. In silico reconstruction of human ventricular cardiomyocyte electrophysiology 3. In vitro effects on human stem cell-derived ventricular cardiomyocytes 4. Evaluation of unanticipated effects in clinical phase 1 studies 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. 3

4 Patch clamp studies in CiPA initiative 1. In vitro assessment of drug effects on multiple ionic currents 2. In silico reconstruction of human ventricular cardiomyocyte electrophysiology 3. In vitro effects on human stem cell-derived ventricular cardiomyocytes 4. Evaluation of unanticipated effects in clinical phase 1 studies Na V 1.5 Ca V 1.2 K V cardiac ion channels were selected for manual patch clamp experiments 1. herg channels (K V 11.1) 2. L-type Ca 2+ channels (Ca V 1.2 subtype) 3. Na + channels (Na V 1.5 subtype; peak and late components) 4

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 drugchannel interaction kinetics. Some details: Milnes et al., 2010 at 37 C; 80% series resistance compensation; one concentration per cell; drugs application started only after baseline stability is reached. 5

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 drugchannel interaction kinetics. Some details: Milnes et al., 2010 at 37 C herg current larger herg current activates faster Sotalol block more potent 6

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 drugchannel 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 7

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 drugchannel 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 Recording temperature affects pharmacology outcomes. 8

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 drugchannel interaction kinetics. Some details: Milnes et al., % series resistance compensation; one concentration per cell; drugs application started only after baseline stability is reached. 9

10 How are the data used? Kinetics of drugchannel interaction Accumulation/relief from block Concentrationresponse curve for potency assessment (IC 50 values) 10

11 herg channel pharmacology IC 50 /C max 11

12 herg channel pharmacology IC 50 /C max A ratio of 30:1 between a drug s IC 50 and C max 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. 12

13 Kinetics of block development Kinetics of drugchannel interaction 13

14 Kinetics of block development 14

15 Kinetics of block development 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. 15

16 Next steps Manual patch clamp experiments on Ca V 1.2 channels at 37 C are ongoing. Na V 1.5 channels next. 5 of 12 CiPA training drugs completed for Ca V 1.2 channels. Ca V 1.2 current exhibits prominent run-down in every cell studied. Baseline stability is crucial prior to drug application. 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. 16

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. 17

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). Ca V 1.2 channel pharmacology underway, using manual patch clamp method and at 37 C. Na V 1.5 channel pharmacology next. All ion channel pharmacology studies are being repeated using an automated patch clamp system. 18

19 Acknowledgement 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 Jiansong Sheng Phu Tran 19

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21 Patch clamp studies in CiPA initiative 1. In vitro assessment of drug effects on multiple ionic currents herg channels Na V 1.5 Ca V 1.2 K V 11.1 Na V 1.5 channels Na V 1.5 channels Ca V 1.2 channels 3 cardiac ion channels were selected for manual patch clamp experiments 1. herg channels (K V 11.1) 2. L-type Ca 2+ channels (Ca V 1.2 subtype) 3. Na + channels (Na V 1.5 subtype; peak and late components) 21

22 Why evaluate multiple cardiac ion channels? 1. In vitro assessment of drug effects on multiple ionic currents Block of herg channels (dofetilide) broadens ventricular action potentials. Na V 1.5 Ca V 1.2 K V 11.1 Martin et al.,

23 Why evaluate multiple cardiac ion channels? 1. In vitro assessment of drug effects on multiple ionic currents Block of herg channels (dofetilide) broadens ventricular action potentials. Na V 1.5 Ca V 1.2 K V 11.1 Martin et al., 2004 Concomitant block of Ca V 1.2 channels (nifedipine) or Na V 1.5 channels (lidocaine) mitigates the effect of herg channel block and normalizes ventricular action potentials. 23

24 Why evaluate multiple cardiac ion channels? 1. In vitro assessment of drug effects on multiple ionic currents Block of herg channels (dofetilide) broadens ventricular action potentials. Na V 1.5 Ca V 1.2 K V 11.1 Martin et al., Ca V 1.2 channel block + Na V 1.5 channel block 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. 24

25 CiPA training drug set 12 drugs with no major metabolites were chosen based on different torsadogenic potential. High Risk Quinidine anti-arrhythmic Bepridil treats angina Dofetilide anti-arrhythmic Sotalol anti-arrhythmic Intermediate Risk Chlorpromazine - antipsychotic Cisapride - gastroprokinetic Terfenadine - antihistamine Ondansetron prevents nausea No/Low Risk Diltiazem treat hypertension and angina Mexiletine anti-arrhythmic Ranolazine treat angina Verapamil treat hypertension and antiarrhythmic 25

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). IC 24C = 13.8 nm; h = IC 37C = 10.1 nm; h =