Nanion Technologies
Complementary HTS Technologies towards a more Rigorous Safety Screening Paradigm
George Okeyo, Ph.D. Application Scientist Nanion Technologies Inc. Agenda
• Company Background Brief history Technologies
• Comprehensive In Vitro Pro-arrhythmia assay Goals Relevant ion channels
• Recording platforms Patchliner CardioExcyte Syncropatch 384/768 Company History & Development ContinuousIt’s a people’s organic business & dynamic growth since 2002 90 Final scale-up 80 to HTS
70 Dedicated staff
60 Entering HTS
50
40 Versatile automation
30
20 First APC instrument 10
0 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015
Management Seed invest Company profitable since 2003 Long experienceGlobal for best presenceBuyout customer service 13 years of patch clamping - Nanion: The Patchliner. Because quality does matter. and more to come
• Unlimited experimental freedom • Best of all worlds: throughput,
The SURFE2R. performance and versatility The SyncroPatch 96. Catch the wave for transporters. • Press one button and walk away: Get more throughput.
- 48 cells in one run • In-depth analysis of transporter • Cost-efficient ion channel protein activity and function screening • Compatible with diverse • Ligand- and voltage-gated membrane sources channels • Multiple targets investigated with The Port-a-Patch. • High throughput and high data The world’s smallest patch clamp rig. one sensor quality • Fast access to high quality patch clamp data The CardioExcyte 96. • Quick evaluation of cells and Bump up your safety screening. The Orbit 16. compounds Instant bilayer – just add protein.
• Ultra-precise impedance • Novel experimental possibilities • 16 parallel bilayers measurements • Low noise, high bandwidth • Real time access to beating recordings parameters • Compatible with your existing • Quick experiments and long term The Vesicle Prep Pro. amplifier observations Liposomes made easy. Slide # 4 • Quick and easy formation of GUVs Measure More • Temperature control • Stable bilayers for ephys Membrane recordings
Smart tools for ion channel and transporter research and screening Agenda
• Company Background Brief history Technologies
• Comprehensive In Vitro Pro-arrhythmia assay Goals Relevant ion channels
• Recording platforms Patchliner CardioExcyte Syncropatch 384/768
Ongoing HESI/FDA initiative: CiPA (Comprehensive In Vitro Pro-arrhythmia assay)
CiPA • A proposal to evaluate the pro-arrhythmic risk of compounds based on mechanistic electrophysiological understanding of this risk
iPS CM play a major role; they express all 5: ILSI Health and Environmental Sciences Institute hERG, Nav1.5, Cav1.2 and KvLQT1, Kir2.1
Ongoing HESI/FDA initiative: CiPA (Comprehensive In Vitro Pro-arrhythmia assay)
CiPA • A proposal to evaluate the pro-arrhythmic risk of compounds based on mechanistic electrophysiological understanding of this risk
Goal • Move the analysis and evaluation of pro-arrhythmia risk earlier in the discovery/development process
• Increase efficiency of development pathway
• Enhance accuracy of current or future drugs labeling
• Increase the number of compounds in development
• Revise ICH S7B guideline, and remove TQT study described in ICH E14 guideline – Proposed timelines: abandon E14 by July 2015, and revise S7B by July 2016
iPS CM play a major role; they express all 5: ILSI Health and Environmental Sciences Institute hERG, Nav1.5, Cav1.2 and KvLQT1, Kir2.1 HTS on the CIPA stipulated ion channels (hERG, Nav1.5, Cav1.2 and KvLQT1, Kir2.1)
Voltage gated channels: Cell lines: BK, Cav1.2,Cav1.2, Cav2.2, Cav3.1, Cav3.2, Cav3.3, hERGhERG,,, 1321 N1, BHK, HEK293, CHO, COS, HeLa, IMR-32, hEAG, KCa1.1, Kv1.3, Kv1.5, KCNQ1,KCNQ1, Nav1.1, Nav1.2, Jurkat, L-tk, ND7-23, NG108-15, PC-12, RBL, S2, S9, hNav1.5,hNav1.5, Nav1.7, hNav1.8, Shaker I, Shaker II SHS5Y5
Ligand gated: Primary cells:
5-HT3, ASIC ,CNG, GABAA, hGlyR α1, HCN, hNAChR BY2 Protoplasts, DRG neurons, erythrocytes, α7, NAChR a3β4, NMDA, P2X2/3, P2X7, TRPA1, hippocampal granule cells, human corneal TRPC1, TRPC3, TRPC5, TRPM2, TRPM3, TRPM7, endothelial cells, human sanoviocytes, human T- TRPM8, TRPV1, TRPV3, TRPV4 lymphocytes, human neutrophils, human vasacular smooth muscle cells, lysosomes, Others: lymphoblasts, mesophyll protoplasts, Kir1.1, Kir7.1, Kir2.1Kir2.1,, rGIRK, kNBCs-1 (NBCe1-A), mitochondria, mitoplasts, rat astrocytes ROMK, TPCN2 Stem cells: Bilayers: hES (undifferntiated), hESC- derived Alamethicin, Bacterial Cytolysin, Connexins, cardiomyocytes (Axiogenesis, Cellectis, CDI,
Gramicidin, IP3, KcsA, Kv1.2, McsL, NaChBac, Geron/GE Health Care), mESC-derived OmpC, OmpF cardiomycytes (Axiogenesis), Primary neuronal stem cells
Measured on Nanion APC Agenda
• Company Background Brief history Technologies
• Comprehensive In Vitro Pro-arrhythmia assay Goals Relevant ion channels
• Recording platforms Patchliner CardioExcyte Syncropatch 384/768 Is your safety program up-to-date? Nanion’s approach to CiPA….
CardioExcyte 96 Non-invasive impedance system for beating cardiac networks
Patchliner SyncroPatch 384/768 High quality whole cell patch clamp High quality patch clamp & 100% HTS recordings
Patchliner
Automated patch clamp for safety screening Patchliner®. Unlimited experimental freedom.
Rapid solution exchange Heatable pipette Physiological temperature
nAChR a7 TRPV3
Efficient CRC generation Internal exchange
Primary rat astrocyte
Current clamp recordings
+ accurate pharmacology!
Stem cell derived cardiomyocytes Pharmacology of hERG at physiological temperature
Pharmacology can be altered at physiological temperature
Erythromycin is 10X more potent at physiological temperature vs RT
Polunchuk, L. Frontiers in pharmacology, 2012,Vol 3, 1-7 Patchliner offers all required features for screening of stem cell-derived cells
• Current clamp – automated current clamp recordings • GigaOhm seals even with primary /stem cells • Temperature control – stable physiological temperatures or temperature jumps (<70°C) • Minimized cell usage – higher cost efficiency • Internal solution exchange – allows modulation of the ion channels on the cytosolic membrane side Action potential are shortened by the • Fast solution exchange - >20 ms solution switch presence of nifedepine, an L-type calcium channel blocker. time • Brief compound exposure – allows short compound exposure times down to 500 ms
CardioExcyte 96
label-free cardiac safety screening CardioExcyte96 CardioExcyte96
• Combined Impedance and MEA-like extracellular field potential (EFP) measurements non-invasive
• 96-well format, fully parallel readout from stem cell derived cardiomyocytes
• enables cost efficient safety pharmacology and contractility assays
• Efficient measurements inside the incubator or with climate control chamber
Replacing the Incubator – CardioExcyte Incubation chamber
• Temperature control up to 40°C
• 5 % CO2 • 95 % humidity
→ Minimal space requirements
→ Easy experimental setup for assessment of temperature- dependent effects Impedance and Extracellular Field Potential (EFP) measurements recorded in a combined mode
A
Impedance CardioExcyte96 Extracellular Impedance Ion-channel field FPD modulation effects: B potential Na+,Ca2+, K+, hERG Cell Electric Field Potential Electrical monolayer conduction contractility Comprehensive safety (i.e. Long QT EFP syndrome Amplitude assays
I EFPI
CaL Ks
INa IKr
C Impedance and EFP recordings 1 can be recorded successively. A. Human ventricular action potential
B Extracellular electric field potential (EFP) and cardiac ion channel currents that contribute to iPSC-CM potentials.
C Surface electrocardiogram (ECG).1
1. Front. Pharmacol., 2011 doi: 10.3389/fphar.2011.00078 Extended Applications: cell adhesion and initiation of rhythmic activity
cell attachment cells start beating Synchronized beating network of cells, monolayer
Base Impedance, Amplitude and Beat Rate during the attachment/growth of cardiac cells (iCell® Cardiomyocytes) (T = 0: cell seeding, dotted lines: Medium exchange). Combined impedance and EFP recordings Cardiac Pharmacology: Dofetilide – single point addition
Impedance EFP
Baseline
Dofetilide
nM 100 100 Detection and quantification of secondary beats
Impedance recordings
~35 min pre-addition
Compound addition
Impedance Impedance (Ω) Automatic detection of secondary beats
~1h 45 min post-addition
Impedance Impedance (Ω)
3nM Dofetilide Impedance (Ω)
~9h 45 min post-addition
Impedance Impedance (Ω) Cardiac Pharmacology: Nifedipine
Impedance EFP
data
Raw
response
- Dose Compound profiles
Impedance EFP
Amp Rate PW50 BRRI Amp Rate FPD BRRI
Dofetilide 100 nM hERG blocker 1 1 1 1 1 0 2 1
Cisapride 1 uM hERG blocker 1 1 1 1 1 1 3 1
E-4031 1 nM hERG blocker 1 1 1 1 1 1 1 1
Sotalol 1 uM hERG blocker 1 1 1 1 1 1 1 1
Astemizole 1 nM hERG blocker 0 0 1 1 1 0 1 1
Quinidine 30 uM hERG,Ca,Na blocker 1 1 1 1 0 0 2 1
Terfenadine 1 uM hERG,Na blocker 0 1 1 1 1 1 1 1
Isoproterenol 30 uM β-adrenergic agonist 1 1 1 1 1 1 1 1
Nifedipine 300 nM Ca blocker 1 1 1 1 1 1 1 1
BAYK8644 1 nM Ca activator 1 1 1 1 1 1 1 1
increase decrease Nanion Technologies
SyncroPatch 384 & 768 PE The PatchEngine – 100 % integration in HTS environment:
Fits into commercially available liquid handler Up to two PE’s per robot Open design allows integrations into fully robotic environments Used successfully with Beckman Coulter’s Biomek Cybio’s Felix
The PatchEngine – the core of the SyncroPatch 384PE Current-Voltage (I/V) Relationship of hERG (CHO)
Wellplate overview Close up selectable traces
One well highlighted
Experiment statistics
84 % > 500 Mohm Success rates of hERG expressed in CHO cells
250
200
150 100 counts 100 Start of experiment 80 50 End of experiment
0 60 <5 5-10 10-15 15-20 20-25 >25
C slow [pF] cells[%] 40
300 20 250
0
200 disabled (no <100 100 - 500 >500 150 catch) R seal [MΩ] counts 100
50
0 <5 5-10 10-15 15-20 20-25 R series [MΩ]
Ongoing project: HTS screening of CDI/Cor.4U cells!
Cor.4U iPSC Derived Human Cardiomyocytes Low Cell consumption: ~300 cells per well One T-75 Flask with 1.5 Mio cells is sufficient for 10 x 384-patch clamp plates Summary: HTS on the CIPA stipulated ion channels
KvLTQ
Cav1.2
Kir2.1 hERG Nav1.5
Cor.4U iPSC Derived Human Cardiomyocytes All 6 targets recorded with high success rate CaV1.2 In stem cells on Pimozide 5.9nMWe have MexiletineAstemizol cooperations 81µM 7.3nM AmitriptylinewithDofetilide Millipore 5µM 70nM , ChanTestLidocaineQuinidine 295µM 658nM CisaprideTetracaine 50nM 4.5µM and Anaxon – choose your cell Patchlinerline! IV on SyncroPatch 96hERG NaV1.5 pharmacology pharmacology onCurrent SyncroPatch on clamp SyncroPatch 384PE 384PE Thank you!
• Company intro • Overall platforms • CIPA intro • PL • CE • Syncro • Relate to CIPA Compound list slide