Measuring ion channel block for CiPA:

Transcription

1 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 In vitro & in silico components of CiPA 1. Multi-ion channel pharmacology data 2. Computer modelling of TdP risk K + Na + Ca 2+ 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. 2

3 Agenda 1. To show how the two sets of data used to train and validate the model were generated. herg* Ca V 1.2 late Na V 1.5 *Drug binding kinetics 2. To show how differences in experimental procedures for Ca V 1.2 and late Na V 1.5 currents lead to data variability. 3. To propose how ion channel pharmacology experiments can be standardized under CiPA. 3

4 Review of ion channel work Manual patch clamp Automated high throughput system (HTS) 4

5 Review of ion channel work 12 training drugs 1. Manual patch clamp (37 C and RT): herg drug potency (IC 50 ) and drug block kinetics. 37 C (FDA) and RT (Victor Chang Institute). RT data could not resolve drug block kinetics. 6 other currents IC 50 s taken from a manuscript. 2. HTS at RT (HESI-coordinated 19-site study): RT - room temperature HTS - high throughput system 37 C left. herg no useful data generated for block kinetics across all sites for CiPA protocol; IC 50 using conventional protocol varied widely. Same for other currents. Only 1 site generated data for all currents. 1 RT dataset. 16 validation drugs 1. Manual patch clamp studies at 37 C: herg - IC 50 and kinetics. 37 C (FDA) Ca V 1.2, late Na V 1.5 IC 50 only (Same CRO/publication author). 2. HTS at RT: Same site generated IC 50 s for all currents. 5

6 Review of ion channel work herg data - 37 C, manual Other currents - 37 C, manual Other currents - RT, HTS Used for model training and validation Used for model training and validation 6

7 Agenda 1. To show how the two sets of data used to train and validate the model were generated. herg Ca V 1.2 late Na V To show how differences in experimental procedures for Ca V 1.2 and late Na V 1.5 currents lead to data variability. 3. To propose how ion channel pharmacology experiments can be standardized under CiPA. 7

8 herg current Experimental procedure designed to capture drug potency (IC 50 ) and drug-channel interaction kinetics. 0 mv, 10 s Repeated 10X -80 mv Activate herg channels -80 mv Maintain herg channel closure 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. 8

9 herg current Experimental procedure designed to capture drug potency (IC 50 ) and drug-channel interaction kinetics. 0 mv, 10 s Repeated 10X -80 mv Activate herg channels -80 mv Maintain herg channel closure Repeated 10X Critical factors for these experiments: 1. Background/leak current 2. Baseline stability 3. Cell/recording quality 9

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 IC 50 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. 10

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. IC 50 and block kinetics affected. Recommendation - baseline stability must be obtained before drug application. 11

12 Background/leak current Baseline stability Cell/recording quality Recording quality assessment Baseline current and input resistance extracted from every current trace recorded. 0 mv, 10 s -80 mv Activate herg current -80 mv Maintain herg channel closure Recommendation - these parameters should remain stable throughout the experiments as they are measurements of cell health. 12

13 Agenda 1. To show how the two sets of data used to train and validate the model were generated. herg Ca V 1.2 late Na V To show how differences in experimental procedures for Ca V 1.2 and late Na V 1.5 currents lead to data variability. 3. To propose how ion channel pharmacology experiments can be standardized under CiPA. 13

14 Ca V 1.2 channel studies Manual patch clamp data HTS 37 C Ba 2+ Quality control steps RT Ca 2+ Ca 2+ -dependent inactivation This state transition does not occur if using Ba 2+ Zucotti et al., Trends in Pharm. Sci. 14

15 Ca V 1.2 channel studies Manual patch clamp data HTS 37 C Ba mm Quality control steps Verapamil RT Ca mm 10/28 CiPA drugs showed notable IC 50 differences We systematically examined how these procedural differences impact results. 1. Ba 2+ vs Ca 2+ as the charge carrier 2. Region of analysis 3. Temperature 4. Recording stability Recommendation 15

16 Ba 2+ vs Ca 2+ Region of analysis Temperature Recording stability Charge carrier on pharmacology IC 50 Verapamil 0.55 mm 2.13 mm Drug P* 6.9 mm 19.5 mm *Drugs with QT C signal under multi-channel evaluation at the FDA. Recommendation - Ca 2+ should be used as channel gating is not affected. 16

17 Ba 2+ vs Ca 2+ Region of analysis Temperature Recording stability Results differ based on analysis region IC 50 Verapamil Drug P* 0.55 mm 6.9 mm 0.14 mm 4.4 mm 2.13 mm 19.5 mm 0.23 mm 5.5 mm *Drugs with QT C signal under multi-channel evaluation at the FDA. Drug M* 32 mm 36 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. 17

18 Ba 2+ vs Ca 2+ Region of analysis Temperature Recording stability Temperature and block potency IC 50 Verapamil 0.55 mm 0.14 mm 2.13 mm 0.23 mm Drug P* 6.9 mm 4.4 mm 19.5 mm 5.5 mm Drug M* 32 mm 36 mm 18

19 Ba 2+ vs Ca 2+ Region of analysis Temperature Recording stability Temperature and block potency IC 50 Verapamil 0.55 mm 0.14 mm 2.13 mm 0.23 mm Drug P* 6.9 mm 4.4 mm 19.5 mm 5.5 mm Drug M* 32 mm 36 mm Temperature may affect % block for some drugs. Recommendation - 37 C recording preferred; RT recording acceptable. 19

20 Ba 2+ vs Ca 2+ Region of Analysis Temperature Recording stability Ca V 1.2 current rundown Ca 2+ current rundown underestimate IC 50. Recommendation - wait for baseline stability or capture enough baseline signal for offline rundown correction. 20

21 Ba 2+ vs Ca 2+ Region of Analysis Temperature Recording stability Ca V 1.2 channel studies Manual patch clamp data HTS 37 C Ba mm Initial current rundown. Correct or not? Verapamil RT Ca mm 10/28 CiPA drugs showed notable IC 50 differences Lack of standardized experimental procedures for data acquisition and analysis. Adapting a unified experimental approach for ion channel assay under CiPA is key. 21

22 Agenda 1. To show how the two sets of data used to train and validate the model were generated. herg Ca V 1.2 late Na V To show how differences in experimental procedures for Ca V 1.2 and late Na V 1.5 currents lead to data variability. 3. To propose how ion channel pharmacology experiments can be standardized under CiPA. 22

23 Late Na V 1.5 current studies Manual patch clamp data HTS 37 C Veratridine RT ATXII Agonist to enhance late Na V 1.5 current 23

24 Late Na V 1.5 current studies Manual patch clamp data HTS 37 C Veratridine Quality control method Recording solution RT ATXII ATX-II binding sites Veratridine binding sites Choice of agonist may affect subsequent drug-binding to Na V 1.5 channels. 24

25 Late Na V 1.5 current studies Manual patch clamp data HTS 37 C Veratridine 73.6 mm Quality control method Recording solution Clozapine RT ATXII 2.2 mm 14/28 CiPA drugs showed notable IC 50 differences We systematically examined how these procedural differences impact results. 1. Recording stability, given recording solution used 2. Temperature 3. Veratridine vs. ATXII 4. Region of analysis 25

26 Recording stability Temperature Veratridine vs. ATXII Region of analysis Run-up of late Na V 1.5 current Whole cell dialysis period Start of recording Late Na + current run-up because agonist effect is activitydependent overestimate IC 50. Recommendations 1) new solution recipe; 2) baseline stability needs to be established prior to drug application. 26

27 Recording stability Temperature Veratridine vs. ATXII Region of analysis Agonist affects IC 50 calculation IC 50 Lidocaine 65 mm 11 mm Ranolazine 121 mm 21 mm Drug P* Drug M* 24.5 mm 51.1 mm 6.1 mm 4.7 mm *Drugs with QT C signal under multi-channel evaluation at the FDA. ATXII gives lower IC 50 values than veratridine. Binding site. 27

28 Recording stability Temperature Veratridine vs. ATXII Region of analysis Agonist affects IC 50 calculation IC 50 Lidocaine Ranolazine Drug P* Drug M* 65 mm 11 mm 121 mm 21 mm 24.5 mm 6.1 mm 51.1 mm 4.7 mm *Drugs with QT C signal under multi-channel evaluation at the FDA. ATXII gives lower IC 50 values than veratridine. Binding site. Recommendation - ATXII should be used as it does not bind to the channel pore. 28

29 Recording stability Temperature Veratridine vs. ATXII Region of analysis Results differ based on analysis region IC 50 Lidocaine Ranolazine Drug P* Drug M* 510 mm 65 mm 32 mm 11 mm 128 mm 121 mm 31 mm 21 mm 23.3 mm 24.5 mm 16.3mM 6.1 mm 24.5 mm 51.1 mm 29 mm 4.7 mm *Drugs with QT C signal under multi-channel evaluation at the FDA. 29

30 Recording stability Temperature Veratridine vs. ATXII Region of analysis Results differ based on analysis region IC 50 Lidocaine Ranolazine Drug P* Drug M* 510 mm 65 mm 32 mm 11 mm 128 mm 121 mm 31 mm 21 mm 23.3 mm 24.5 mm 16.3mM 6.1 mm 24.5 mm 51.1 mm 29 mm 4.7 mm *Drugs with QT C signal under multi-channel evaluation at the FDA. 30

31 Recording stability Temperature Veratridine vs. ATXII Region of analysis Results differ based on analysis region IC 50 Lidocaine Ranolazine Drug P* Drug M* 510 mm 65 mm 32 mm 11 mm 128 mm 121 mm 31 mm 21 mm 23.3 mm 24.5 mm 16.3mM 6.1 mm 24.5 mm 51.1 mm 29 mm 4.7 mm *Drugs with QT C signal under multi-channel evaluation at the FDA. Recommendation - Both regions should be analyzed as they inform additional mechanism of drug action and can be used for future model improvement as necessary. 31

32 Late Na V 1.5 current studies Manual patch clamp data HTS 37 C Veratridine 73.6 mm Quality control method Recording solution Clozapine RT ATXII 2.2 mm 14/28 CiPA drugs showed notable IC 50 differences Lack of standardized experimental procedures for data acquisition and analysis. Adapting a unified experimental approach for ion channel assay under CiPA is key. 32

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 33

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 C max ) 34

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. 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. 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. Recommendations for standardizing ion channel assay for CiPA 3. Our recommendations for ion channel assay standardization will be discussed with interested parties at the workshop tomorrow. 35

36 Summary of recommendations For 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. Ca V 1.2 channel studies should be performed using Ca 2+ as the charge carrier. 3. Late Na V 1.5 studies should be performed with ATXII as the agonist. 36

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 FDA Cellular Electrophysiology Group 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 37

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39 Back-up slides 39

40 Background/leak current Baseline stability Cell/recording quality Baseline current vs RMP Baseline current and input resistance extracted from every current trace recorded. 0 mv, 10 s -80 mv Activate herg current -80 mv Maintain herg channel closure 40

41 Late Na V 1.5 current analysis 41

42 Late Na V 1.5 current analysis 42

43 Torsade metric score and threshold 95%CI of each drug s 2000 scores are shown as error bars High Intermediate Low Torsade Metric Score (qnet averaged 1 to 4 C max ) 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. 43

44 Torsade metric score and threshold 95%CI of each drug s 2000 scores are shown as error bars High Intermediate Low Torsade Metric Score (qnet averaged 1 to 4 C max ) To avoid the need to retrain the model, patch clamp ion channel studies need to be standardized for data consistency and replicability. herg, Ca V 1.2, late Na V 1.5 have predominant influence over model output. 44