Company Cooperation Products &Services Marketing Financials Comprehensive in vitro Proarrhythmia Assay (CiPA) Using Cor.4U Cardiomyocytes with the FDSS in a Calcium Transient Assay 09.06.2016 Hamamatsu User Meeting Dr. Ralf Kettenhofen Barcelona, Spain •Presented for:
Company Characterization Product/Format Service Content The CiPA Initiative - Short Introduction Factors Influencing the Calcium Transient Assay Customer Report - Drug Development Support •Presented for: 2
Comprehensive in vitro Proarrhythmia Assay CiPA - Initiative •Presented for:
CiPA Members •Presented for: 4
CiPA - Overview of Working Groups •Presented for: 5
CiPA Phase 1 - Pilot Study 3 Providers of pluripotent stem cell-derived cardiomyocytes 16 Volunteer sites 12 sites; 3 microelectrode array platforms 4 sites; 4 Voltage-sensing-optical (VSO) platforms 8 blinded test compounds; 4 concentrations, 3 triplicates - Study was accomplished End 2014 - Manuscript for publication is under discussion •Presented for: 6
CiPA Phase II - Validation Study 2 Providers of pluripotent stem cell-derived cardiomyocytes 5 core sites (funded by FDA grant) 2 sites; 4 microelectrode array platforms 3 sites; 3 Voltage-sensing-optical (VSO) platforms Calcium Transient Assay (potential backup assay) 3 sites: Janssen, Axiogenesis, Merck (USA) Compounds: 28 blinded test compounds; 4 concentrations, 6 replicates 4 calibration compounds Volunteer non-core test sites: 12 blinded test compounds + 4 calibration compounds •Presented for: 7
CiPA Phase II - Validation Study •Presented for: 8
Company Characterization Product/Format Service Excitation-Contraction Coupling A) B) Action Potential Currents Amplitude Na + Ca 2+ L-type Ca 2+ T-type Na + /Ca 2+ -exchanger K + I to K + IK s K + IK r K + IK ur Time •Presented for: 9
Company Cooperation Products &Services Marketing Financials High Throughput Kinetic Plate Reader Assays •Presented for:
Company Characterization Product/Format Service Plate Reader System - Hamamatsu Hamamatsu Hamamatsu FDSS µCell FDSS 7000EX Setup A Setup B Pipettor Head EFS Head 96 well EFS head Data generated in collaboration with Hamamatsu Both systems can be equipped with a temperature control •Presented for: 11
Company Characterization Product/Format Service Plating Efficiency of Cor.4U Cardiomyocytes on a 384 Well Plate Data generated in collaboration with Recording of Cor.4U cardiomyocytes with the FDSS 7000EX using Cal520 dye (AAT Bioquest). Hamamatsu •Presented for: 12
Assay Optimisation Important Factors Influencing the Calcium Transient Assay with hiPSC- derived Cardiomyocytes •Presented for:
Company Characterization Product/Format Service Calcium Transient Assay - Important factors The calcium dyes Dye loading time Assay stability over time (assay window) Wash vs. non-wash Signal to noise ratio Medium / buffer Quencher Addition of organic anion transporter (e.g. probenecid) •Presented for: 14
Company Characterization Product/Format Service Dye-induced Morphological Differencesand Changes of Cor.4U Cardiomyocytes’ Calcium Transients Cal520 (AAT Bioquest) Calcium 5 Assay Kit (Molecular Devices) ACTOne (Codex) •Presented for: 15
Company Characterization Product/Format Service Results - Cal-520 30 min 10 µM 5 µM 2.5 µM 1.0 µM 0.5 µM time windows: 4s Bent fast rise of the calcium transient Physiological fast and prolonged FLIPR ACT rise of the calcium peak width transient and short Calcium 5 One peak width - FLIPR Calcium 5 and Codex ACTOne reveal an slowed rise of the calcium transients from 80% to 100%. - There is obviously a changes in calcium transients which potentially indicates the start of toxic events at an early time point. - Calcium transient durations are increased with the FLIPR Calcium 5 dye and the ACTOne dye at concentrations tested compared to Cal-520 dye (see also quantitative analysis). •Presented for: 16
Company Characterization Product/Format Service Results - Cal-520 90 min 10 µM 5 µM 2.5 µM 1.0 µM 0.5 µM time windows: 4s Bent fast rise of the calcium transient ACT FLIPR One Ca 5 At higher concentrations of Cal-520 the slope at 80% to 100% starts to slow as well. •Presented for: 17
Company Characterization Product/Format Service Quantitative Analysis of Non-Wash Cal-520 Calcium Transients Recorded from Cor.4U Cardiomyocytes •Presented for: 18
Company Characterization Product/Format Service Results - Cal-520 Beat Rate 60 50 bea$ng frequency [bpm] 40 30 min 45 min 30 60 min 90 min 20 120 min 180 min 10 240 min 0 10000 5000 2500 1000 100 1:6 FLIPR 0.3x Calcium 5 ACTOne concentra$ons [nM] - Beat rate is higher in Cal-520 Assay compared to the both other dyes, especially at the lowest dye concentration. - Beat rate decreases with increasing dye concentrations. •Presented for: 19
Company Characterization Product/Format Service Results - Cal-520 Peak Width (PW) 80% 1000 average peak width 80% [ms] 900 800 700 30 min 600 45 min 500 60 min 400 90 min 300 120 min 200 180 min 100 240 min 0 10000 5000 2500 1000 100 1:6 FLIPR 0.3x Calcium 5 ACTOne concentr8ons [nM] - Cal-520 calcium transient PW30% and 80% increase over time in the highest concentrations (toxic effect?). - FLIPR Calcium 5 and ACTOne dye PW80% values are almost twice as high compared to the lowest Cal-520 concentration (=> toxic or unphysiological?). •Presented for: 20
Company Characterization Product/Format Service Results - Cal-520 Calcium Transient Amplitude 18000 16000 14000 12000 average AMP [RFU] 30 min 45 min 10000 60 min 8000 90 min 6000 120 min 4000 180 min 2000 240 min 0 10000 5000 2500 1000 100 1:6 FLIPR 0.3x Calcium 5 ACTOne concentra3ons [nM] - Calcium Transient amplitudes from Cal-520 increase over time (max after 3 hours) although no probenecid was added. - FLIPR Calcium 5 and ACTOne dye amplitudes reach a maximum after 60 min. •Presented for: 21
Company Characterization Product/Format Service Wash Assay Using Cal-520 TM , AM (AAT Bioquest) and ACTOne (Codex) •Presented for: 22
Company Characterization Product/Format Service Results Calcium Transient Amplitude 4000.00 3500.00 average AMP [RFU] 3000.00 60 min 90 min 2500.00 120 min 2000.00 150 min 1500.00 180 min 210 min 1000.00 240 min 500.00 0.00 2.5 µM 1.0 µM 0.5 µM 0.5x 0.3x 0.25x Cal-520 Cal-520 Cal-520 ACTOne ACTOne ACTOne Calcium Dyes concentra;ons [nM] - Amplitude of Cal-520 calcium transients is absolutely stable during after 4 hours. - ACTOne amplitudes are decreased after 3 hours. •Presented for: 23
Company Characterization Product/Format Service Results Peak Width (PW) 80% average peak width 80% [ms] 1400.00 1200.00 60 min 1000.00 90 min 800.00 120 min 600.00 150 min 400.00 180 min 200.00 210 min 0.00 2.5 µM 1.0 µM 0.5 µM 0.5x 0.3x 0.25x 240 min Cal-520 Cal-520 Cal-520 ACTOne ACTOne ACTOne Calcium Dyes concentra=ons [nM] - 0.5x ACTOne peak width at 80% are doubled compared to 2.5 µM Cal-520 (and also 0.25x ACTOne) and almost 3x the values of 0.5 µM Cal-520. •Presented for: 24
Company Characterization Product/Format Service Dye Effect on GPCR Agonist Pharmacology with Cor.4U Cardiomyocytes •Presented for: 25
Company Characterization Product/Format Service Results Cal520 Calcium 5 Assay Kit Dye Beat Rate 120.00 90 100.00 80 bea$ng frequency [bpm] beat rate [bpm] 70 80.00 baseline 60 50 5 min 60.00 Baseline 40 10 min 30 5 min 15 min 40.00 20 20 min 15 min 10 20.00 25 min 0 30 min 30 min vehicle control 30 100 300 1000 0.00 Control 0.05 0.15 0.46 1.37 4.12 12.3 37 111 333 1000 concentra.on [nM] concentra$on [nM] - Right shift of isoproterenol increased beat rate with the Calcium 5 Assay Kit dye - Cal520: More physiological isoproterenol effect •Presented for: 26
Company Characterization Product/Format Service Conclusion Choice of the right calcium dye is important Cal520 at low concentrations revealed to be the most physiologic dye Long-term stability (assay window) calcium transient and beating parameters No quencher is required for Cal520 when the right assay medium/ buffer is chosen Washout is required for Cal520 •Presented for: 27
Support of Pharma Drug Development Dr. Thomas Licher, Sanofi Frankfurt, Germany http://axiogenesis.com/resources/presentations/webinar.html 10.02.2016 Axiogenesis webinar •Presented for:
Company Characterization Product/Format Service 10.02.2016 Axiogenesis webinar, Dr. Thomas Licher, •Presented for: 29
Company Characterization Product/Format Service 10.02.2016 Axiogenesis webinar, Dr. Thomas Licher, •Presented for: 30
Company Characterization Product/Format Service 10.02.2016 Axiogenesis webinar, Dr. Thomas Licher, •Presented for: 31
Company Characterization Product/Format Service 10.02.2016 Axiogenesis webinar, Dr. Thomas Licher, •Presented for: 32
Company Characterization Product/Format Service 10.02.2016 Axiogenesis webinar, Dr. Thomas Licher, •Presented for: 33
Company Characterization Product/Format Service 10.02.2016 Axiogenesis webinar, Dr. Thomas Licher, •Presented for: 34
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