Comprehensive in vitro Proarrhythmia Assay (CiPA) Using Cor.4U - - PowerPoint PPT Presentation

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Comprehensive in vitro Proarrhythmia Assay (CiPA) Using Cor.4U - - PowerPoint PPT Presentation

Mar 06, 2024 191 likes •567 views

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

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  • Presented for:

Financials Cooperation Products &Services Marketing Company

Comprehensive in vitro Proarrhythmia Assay (CiPA) Using Cor.4U Cardiomyocytes with the FDSS in a Calcium Transient Assay

09.06.2016 Hamamatsu User Meeting Barcelona, Spain

  • Dr. Ralf Kettenhofen
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  • Presented for:

Characterization Product/Format Service Company

Content

The CiPA Initiative - Short Introduction Factors Influencing the Calcium Transient Assay Customer Report - Drug Development Support

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  • Presented for:

Comprehensive in vitro Proarrhythmia Assay CiPA - Initiative

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SLIDE 4
  • Presented for:

CiPA Members

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SLIDE 5
  • Presented for:

CiPA - Overview of Working Groups

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SLIDE 6
  • Presented for:

CiPA Phase 1 - Pilot Study

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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
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SLIDE 7
  • Presented for:

CiPA Phase II - Validation Study

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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

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SLIDE 8
  • Presented for:

CiPA Phase II - Validation Study

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SLIDE 9
  • Presented for:

Characterization Product/Format Service Company

Excitation-Contraction Coupling

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Time Amplitude

Currents Action Potential

Na+ Ca2+ L-type Ca2+ T-type Na+/Ca2+-exchanger K+ Ito K+ IKs K+ IKr K+ IKur

A) B)

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SLIDE 10
  • Presented for:

Financials Cooperation Products &Services Marketing Company

High Throughput Kinetic Plate Reader Assays

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SLIDE 11
  • Presented for:

Characterization Product/Format Service Company

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Hamamatsu FDSS µCell

Setup A Pipettor Head

96 well EFS head

Setup B EFS Head

Hamamatsu FDSS 7000EX

Both systems can be equipped with a temperature control

Plate Reader System - Hamamatsu

Data generated in collaboration with Hamamatsu

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SLIDE 12
  • Presented for:

Characterization Product/Format Service Company

Plating Efficiency of Cor.4U Cardiomyocytes on a 384 Well Plate

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Data generated in collaboration with Hamamatsu

Recording of Cor.4U cardiomyocytes with the FDSS 7000EX using Cal520 dye (AAT Bioquest).

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  • Presented for:

Assay Optimisation

Important Factors Influencing the Calcium Transient Assay with hiPSC- derived Cardiomyocytes

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SLIDE 14
  • Presented for:

Characterization Product/Format Service Company

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)

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SLIDE 15
  • Presented for:

Characterization Product/Format Service Company

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Dye-induced Morphological Differencesand Changes of Cor.4U Cardiomyocytes’ Calcium Transients

Cal520 (AAT Bioquest) Calcium 5 Assay Kit (Molecular Devices) ACTOne (Codex)

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  • Presented for:

Characterization Product/Format Service Company

Results - Cal-520

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30 min 10 µM 5 µM 2.5 µM 1.0 µM 0.5 µM

  • 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).

ACT One FLIPR Calcium 5

time windows: 4s Bent fast rise of the calcium transient and prolonged peak width Physiological fast rise of the calcium transient and short peak width

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SLIDE 17
  • Presented for:

Characterization Product/Format Service Company

Results - Cal-520

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90 min 10 µM 5 µM 2.5 µM 1.0 µM 0.5 µM

At higher concentrations of Cal-520 the slope at 80% to 100% starts to slow as well.

ACT One FLIPR Ca 5

time windows: 4s Bent fast rise of the calcium transient

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SLIDE 18
  • Presented for:

Characterization Product/Format Service Company

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Quantitative Analysis of Non-Wash 
 Cal-520 Calcium Transients Recorded from Cor.4U Cardiomyocytes

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SLIDE 19
  • Presented for:

Characterization Product/Format Service Company

Results - Cal-520

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10 20 30 40 50 60

10000 5000 2500 1000 100 1:6 FLIPR Calcium 5 0.3x ACTOne bea$ng frequency [bpm] concentra$ons [nM]

30 min 45 min 60 min 90 min 120 min 180 min 240 min

Beat Rate

  • 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.
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SLIDE 20
  • Presented for:

Characterization Product/Format Service Company

Results - Cal-520

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Peak Width (PW) 80%

  • 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?).

100 200 300 400 500 600 700 800 900 1000 10000 5000 2500 1000 100 1:6 FLIPR Calcium 5 0.3x ACTOne

average peak width 80% [ms]

concentr8ons [nM]

30 min 45 min 60 min 90 min 120 min 180 min 240 min

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SLIDE 21
  • Presented for:

Characterization Product/Format Service Company

Results - Cal-520

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2000 4000 6000 8000 10000 12000 14000 16000 18000

10000 5000 2500 1000 100 1:6 FLIPR Calcium 5 0.3x ACTOne

average AMP [RFU]

concentra3ons [nM]

30 min 45 min 60 min 90 min 120 min 180 min 240 min

Calcium Transient Amplitude

  • 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.
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SLIDE 22
  • Presented for:

Characterization Product/Format Service Company

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Wash Assay Using
 Cal-520TM , AM (AAT Bioquest) and ACTOne (Codex)

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SLIDE 23
  • Presented for:

Characterization Product/Format Service Company

Results

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Calcium Transient Amplitude

  • Amplitude of Cal-520 calcium transients is absolutely stable during after 4 hours.
  • ACTOne amplitudes are decreased after 3 hours.

0.00 500.00 1000.00 1500.00 2000.00 2500.00 3000.00 3500.00 4000.00

2.5 µM Cal-520 1.0 µM Cal-520 0.5 µM Cal-520 0.5x ACTOne 0.3x ACTOne 0.25x ACTOne average AMP [RFU]

Calcium Dyes concentra;ons [nM]

60 min 90 min 120 min 150 min 180 min 210 min 240 min

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SLIDE 24
  • Presented for:

Characterization Product/Format Service Company

Results

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Peak Width (PW) 80%

  • 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.

0.00 200.00 400.00 600.00 800.00 1000.00 1200.00 1400.00 2.5 µM Cal-520 1.0 µM Cal-520 0.5 µM Cal-520 0.5x ACTOne 0.3x ACTOne 0.25x ACTOne

average peak width 80% [ms]

Calcium Dyes concentra=ons [nM]

60 min 90 min 120 min 150 min 180 min 210 min 240 min

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SLIDE 25
  • Presented for:

Characterization Product/Format Service Company

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Dye Effect on GPCR Agonist Pharmacology with Cor.4U Cardiomyocytes

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  • Presented for:

Characterization Product/Format Service Company

Results

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  • Right shift of isoproterenol increased beat rate with the Calcium 5 Assay Kit dye
  • Cal520: More physiological isoproterenol effect

0.00 20.00 40.00 60.00 80.00 100.00 120.00 1000 333 111 37 12.3 4.12 1.37 0.46 0.15 0.05 Control bea$ng frequency [bpm] concentra$on [nM] baseline 5 min 10 min 15 min 20 min 25 min 30 min

Calcium 5 Assay Kit Dye

10 20 30 40 50 60 70 80 90 vehicle control 30 100 300 1000 beat rate [bpm] concentra.on [nM]

Beat Rate

Baseline 5 min 15 min 30 min

Cal520

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  • Presented for:

Characterization Product/Format Service Company

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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

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SLIDE 28
  • Presented for:

Support of Pharma Drug Development

  • Dr. Thomas Licher, Sanofi Frankfurt, Germany

http://axiogenesis.com/resources/presentations/webinar.html

10.02.2016 Axiogenesis webinar

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  • Presented for:

Characterization Product/Format Service Company

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10.02.2016 Axiogenesis webinar, Dr. Thomas Licher,

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  • Presented for:

Characterization Product/Format Service Company

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10.02.2016 Axiogenesis webinar, Dr. Thomas Licher,

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SLIDE 31
  • Presented for:

Characterization Product/Format Service Company

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10.02.2016 Axiogenesis webinar, Dr. Thomas Licher,

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  • Presented for:

Characterization Product/Format Service Company

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10.02.2016 Axiogenesis webinar, Dr. Thomas Licher,

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  • Presented for:

Characterization Product/Format Service Company

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10.02.2016 Axiogenesis webinar, Dr. Thomas Licher,

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  • Presented for:

Characterization Product/Format Service Company

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10.02.2016 Axiogenesis webinar, Dr. Thomas Licher,

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Thank you!

www.axiogenesis.com info@axiogenesis.com

  • rder@axiogenesis.com

Axiogenesis AG

Nattermannallee 1 50829 Cologne Germany tel: +49 221 99 88 18 - 0 fax: +49 221 9988 18 -10