CiPA - Comprehensive in vitro Proarrhythmia Assay

The Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative aims to replace the preclinical hERG current assay required under the ICH S7B safety pharmacology guidelines and clinical TQT study, which provides a surrogate marker of Proarrhythmia, with more translationally relevant assessments of proarrhythmic risk (Sager et al., 2014). CiPA intend to achieve this by evaluating proarrhythmic risk of evolving drug candidates based on an understanding of the electrophysiologic mechanisms responsible for proarrhythmia linked to Torsades de pointes (TdP) and QT prolongation.

The CiPA workstreams

The Comprehensive in Vitro Proarrhythmia Assay (CiPA) paradigm was initiated in 2013. The Steering Team is comprised of partners from the US FDA, HESI, CSRC, SPS, Japan NIHS, PMDA, EMA and Health Canada. A number of participating organizations, amongst them Nanion Technologies, build the backbone of the initiative. The CiPA work streams include: In Silico, Myocyte, Ion Channel, and Clinical Translation working groups.

Nanion is a committee member of the Ion Channel HTS Team and a participant in the Myocyte Working Group. Nanion has a long-standing interest and extensive experience in automated patch clamp screening of cardiac ion channels. Label-free contractility and extracellular field potential recordings of stem cell-derived cardiomyocytes (iPSC-CMs) is also available in our portfolio. Our instruments are used for safety screening by major pharmaceutical companies and CROs worldwide and we are happy to assist you in setting up your CiPA assays.

Results of Phase I Studies

The Ion Channel Work Group finalized its phase I study in 2017. Besides further external sites, Nanion Technologies in Germany, USA and Japan participated with the Patchliner and the SyncroPatch 384PE in this study.

The image on the left hand side displays the results of the blocking effect of 12 compounds on hERG.
The results are in good agreement with manual patch clamp data (Crumb et al., 2016).

Nanion's products in CiPA workstreams

SyncroPatch 384/768PE - Patch clamp meets HTS

The SyncroPatch 384PE (upgradeable to 768PE) is a high throughput patch clamp instrument recording from up to 384 (or 768) cells simultaneously. The SyncroPatch 384/768PE is the highest throughput patch clamp instrument on the market with giga-seal data quality.
The SyncroPatch 384PE is used for CiPA ion channel working group studies.
Request the CiPA assay protocol for the SyncroPatch 384PE here.

Patchliner - Versatile and sophisticated

The Patchliner is a fully automated planar patch clamp instrument recording from up to 8 cells simultaneously. With its vast experimental freedom and gigaseal data quality, the Patchliner is one of the most versatile patch clamp instruments on the market.
The Patchliner is used for CiPA ion channel working group studies.
Request the CiPA assay protocol for the Patchliner here.

Icon CE CardioExcyte 96 - Combined contractility, electrophysiology and cell viability

The CardioExcyte 96 is a hybrid system recording both contractility and electrophysiology of intact cardiomyocyte networks. In addition, the base impedance is continuously and automatically monitored as a measure of acute and chronic cell activity. No subtleties of cytotoxic responses are missed, this includes non-contractile cell types such as hepatocyte-like cells or cancer cells as well as contractile cardiac cells. Impedance and extracellular field potential measurements are performed at high resolution, are non-invasive and label-free. The CardioExcyte 96 is a fully automated device, recording from 96 wells at a time.
The CardioExcyte 96 is used for CiPA myocyte working group studies.
Request the CiPA assay protocol for the CardioExcyte 96 here.

Ion Channel Work Group (ICWG)

ICWG: Description and History

"The Ion Channel Working Group (ICWG), sponsored by the Safety Pharmacology Society (SPS), was established in December 2013. Its primary role is to work in close collaboration with the In Silico Working Group (ISWG) in providing ion channel support for the development of a computer model of the adult human ventricular myocyte, to be used as part of the CiPA initiative in predicting the clinical risk of drug-induced TdP."

"The original remit of the ICWG was: 1) to select key cardiac ion channels to include in the CiPA evaluation; 2) to develop robust, reliable and reproducible voltage clamp protocols required to generate data allowing the training and validation of the in silico model; 3) to define which biophysical and/or pharmacological properties of the channels to study for drug effects in order to optimize the predictivity of the model (e.g., potency (IC₅₀), kinetic of block, rate/use/voltage dependence) and; 4) to define the requirements needed to transition the various ion channel protocols from manual to automated high throughput (HT) patch clamp platforms, in order to adapt to the screening environment present in most pharmaceutical companies."

Selected recombinant human channels: "I_Kr (hERG), I_Ca (L-type; Cav1.2), I_Na (Nav1.5 peak and late current); I_TO (Kv4.3); I_Ks (KCNQ1 + KCNE1), and I_K1 (Kir2.1). Following the selection, the ICWG set out to design standardized voltage clamp protocols for each of these channels."

(Source: Journal of Pharmacological and Toxicological Methods "The Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative — Update on progress. Colatzky et al. (2016) ")

Progress of the Ion Channel Work Group

"Protocols were developed and manual experimental work was completed. The High-Throughput Systems (HTS) studies to evaluate and characterize the automated patch clamp systems is underway. The training compounds have been completed for both manual and automated systems and data analysis initiated. The second phase of the HTS study with the validation compounds is underway. Additional ion channel studies will likely be planned beyond 2017 to further validate the model."

(Source: CiPAproject webpage April 2018 "CiPA Workstream Timelines")

Data, Applications and Publications on the six Cardiac Ion Channels investigated by the "CiPA Ion Channel Work Group"

Myocyte Work Group (MWG)

MWG: Description and History

The role of human cardiac stem cell derived cardiomyocytes (hSC-CMs) within the CiPA paradigm is to confirm in silico reconstructions of the electrophysiologic effects of drugs. This will be accomplished by evaluating the acute effects of drugs on the electrical activity of hSC-CM's using higher throughput approaches that enable more comprehensive and robust assessments earlier in the drug discovery process. Typical techniques involved include assessing changes in the extracellular field potentials of spontaneously active hSC-CMs using multielectrode array (MEA) platforms, or changes in the action potential configuration recorded optically using voltage-sensing dyes (VSD)."

"A pilot study was conducted by the Health and Environmental Sciences Institute (HESI) Cardiac Safety Committee Myocyte Subteam to evaluate reproducibility and variability of electrophysiologic responses across cells, platforms and volunteer study sites for 8 compounds, Mexiletine, Nifedipine, E-4031, JNJ-303, Flecanide, Moxifloxacin, Quinidine and Ranolazine. Overall, most sites detected changes in repolarization consistent with clinical findings, with the greatest source of variability attributed to study site."

"More recently, the HESI Myocyte Subteam was awarded a Broad Area Announcement Grant from the FDA to extend the pilot study to a larger set of 28 compounds categorized according to high, intermediate, and low/no risk of proarrhythmia based on clinical findings. The effects of these CiPA reference standards will be evaluated by a core group in this Phase II Validation study across four MEA and two VSO technology platforms using two commercially viable hSC-CM preparations." Results will be compared with prospective in silico reconstructions (based on voltage clamp studies) and published studies employing hSC-CMs from non-core laboratories and consortia for the same 28 compounds."

(Source: Journal of Pharmacological and Toxicological Methods "The Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative — Update on progress. Colatzky et al. (2016) ")

Progress of the Myocyte Work Group

"The publication detailing the results of the Pilot Study is now available online. The Phase II Validation Study has been completed and the publication detailing the results has been submitted."

(Source: CiPAproject webpage May 2018 "CiPA Workstream Timelines")

Nanion for CiPA:

Data and Applications

NaV1.5 - Late Current Analysis using the CiPA Protocol

SyncroPatch 384/768 PE data and applications:
Cells were kindly provided by Charles River.

Screenshots of the PatchControl 384 software showing Na_V1.5 current traces in response to the CiPA voltage step protocol, measured on the SyncroPatch 384PE using whole cell patch clamp methodology and single-hole chips. The Na_V1.5 late current was activated by the application of 60 nM ATX-II. The IC₅₀ value of Ranolazine of the late Sodium current current was determined as 40.4 µM.

hERG - Pharmacology at Physiological Temperature using the CiPA Protocol

SyncroPatch 384/768 PE data and applications:
Cells were kindly provided by Charles River.

Screenshots of the PatchControl 384 software showing hERG current traces in response to the CiPA voltage step protocol at 35 degree Celsius. Measured on the SyncroPatch 384PE using perforated patch clamp methodology (Escin) and multi-hole chips (4 holes per well). The IC₅₀ value of Erythromycin of the peak current was determined as 60.5 µM.

KV4.3 - Pharmacology of Metropolol Tartrate, using the CiPA Protocol

SyncroPatch 384/768 PE data and applications:
Cells were kindly provided by Charles River.

Screenshots of the PatchControl 384 software showing K_V4.3 current traces in response to the CiPA voltage step protocol, measured on the SyncroPatch 384PE using the whole cell patch methodology and single-hole chips. The IC₅₀ value of Metropolol Tartrate was determined as 128 µM.

CaV1.2 - Pharmacology of Nifedipine, using the CiPA protocol

SyncroPatch 384PE data and applications:
Cells were kindly provided by Charles River.

Screenshots of the PatchControl 384 software showing hCa_V1.2/β2/α2δ1 current traces in response to the CiPA voltage step protocol and the corresponing current-voltage relationship plot. Measured on the SyncroPatch 384PE using perforated patch methodology (Escin) and multi-hole chips (4 holes per well), the success rate of valuable data for the analysis was 94%. The IC₅₀ value of Nifedipine was determined as 106 nM.

hERG - Pharmacology of Cisapride, using the CiPA protocol

Patchliner data and applications:
Cells were kindly provided by Charles River.

The effect of cisapride on hERG currents was investigated, using the CiPA voltage step protocol. Measured on the Patchliner the perforated patch methodology (Escin) and multi-hole chips (4 holes per well) were used. The IC₅₀ value of Cisapride was determined as 112 nM.

hERG - Pharmacology using the CiPA Protocol

SyncroPatch 384/768 PE data and applications:
Cells were kindly provided by Charles River.

Screenshots of the PatchControl 384 software showing hERG current traces in response to the CiPA voltage step protocol. Measured on the SyncroPatch 384PE using whole cell patch clamp methodology and multi-hole chips (4 holes per well). The IC₅₀ value of the following compounds of the peak current was determined as 4.18 µM for Diltiazem, 37.4 nM for Terfenadine, 971 nM for Quinidine, 63 µM for Mexiletine, 431 nM for Verapamil and 4.54 µM for Ranolazine.

hERG - recordings with great stability using the CiPA step ramp protocol

SyncroPatch 384PE data and applications:
Cells were kindly provided by Charles River.

Screenshots of the PatchControl 384 software showing hERG current traces in response to the CiPA voltage step protocol. Measured on the SyncroPatch 384PE using perforated patch clamp methodology (Escin) and multi-hole chips (4 holes per well).

Cardiac Ion Channels - Pharmacology of Vandetanib

Icon CE CardioExcyte 96 and Patchliner data and applications:
Cells were kindly provided by Charles River and Cellular Dynamics.

The image on the left hand side displays the results of the blocking effect of Vandetanib on hERG, Na_V1.5, Ca_V1.2 and K_V4.3. The compound induced arrhythmia when iPSC-CM were exposed to a minimum concentration of 1 µM. Arrhytmic events were both detected in field potential recordings as well as in the impedance based contractility.

Cardiac Ion Channels - Pharmacology of Sotalol

Icon CE CardioExcyte 96 and SyncroPatch 384PE data and applications:
Cells were kindly provided by Charles River and Cellular Dynamics.

The image on the left hand side displays the results of the blocking effect of Sotalol on hERG. The result is in good agreement with manual patch clamp data (Crumb et al., 2016). The compound induced arrhythmia when iPSC-CM were exposed to a minimum concentration of 10 µM. Arrhytmic events were both detected in field potential recordings as well as in the impedance based contractility measurements.

Kir2.1 - Pharmacology of Barium

SyncroPatch 384/768 PE data and applications:
Cells were kindly provided by Charles River.

Screenshots of the PatchControl 384 software showing Kir2.1 current traces in response to a voltage step protocol. Measured on the SyncroPatch 384PE using the whole cell patch methodology and multi-hole chips (4 holes per well), the success rate of valuable data for the analysis was 93%. The IC₅₀ value of Barium was determined as 6.38 µM (Literature: 16.2 µM, Schram et al. Cardiovasc Res. 2003).

KV4.3 - Pharmacology of Quinidine

SyncroPatch 384/768 PE data and applications:
Cells were kindly provided by Charles River.

Screenshots of the PatchControl 384 software showing K_V4.3 current traces in response to the CiPA voltage step protocol. Measured on the SyncroPatch 384PE using the whole cell patch methodology and multi-hole chips (4 holes per well), the success rate of valuable data for the analysis was 95.3%. The IC₅₀ value of Quinidine was determined as 21.2 µM (Literature: 79.3 µM, Crumb et al., J Pharmacol Toxicol Methods. 2016).

hERG - Application of "Sticky Compounds"

Patchliner data and applications:
hERG expressing HEK293 cells were kindly provided by Cytomyx/Millipore.

Even sticky compounds pose no problem for the Patchliner. IC₅₀ measurements of well known sticky substances were determined on the Patchliner: Terfenadine IC₅₀ = 11.0 ± 3 nM, Flunarizine IC₅₀ 163.7 ± 19 nM and Cisapride IC₅₀8.9 ± 3 nM.

NaV1.5 - Stable Access Resistance

Patchliner data and applications:
Cells were kindly provided by Millipore.

The I/V-characteristics of Na_V1.5 currents (HEK293) are shown together with the repeated dose dependent block by TTX (lower panel). Five concentrations of TTX (0.3, 1, 3, 10, 30 μM) were applied, followed by washout with antagonist-free buffer and re-application of the same TTX concentrations.

hERG - Efficient Screening

Patchliner data and applications:
Cells were kindly provided by Cytomyx/Millipore.

The effects of six different blockers (terfenadine, cisapride, E4031, astemizole, propafenone, quinidine) on hERG currents (HEK293 cells) were investigated. Expected IC₅₀ values for the different compounds were obtained. In two days, 119 full dose response curves were collected by a single person. Data was analyzed using Nanion’s Data Analysis Package, a very efficient and convenient data analysis tool!

hERG - Simple Data Analysis

Patchliner data and applications:

With our analysis tools, especially programmed routines in Igor make dose response curves, raw data and current time courses easily accessible. Also, creating average dose response curves over multiple experiments - even conducted on different days - remains easy.

hERG - Block at Physiological Temperature

Patchliner data and applications:
Cells were kindly provided by Cytomyx/Millipore, UK.

The effects of erythromycin on hERG currents were tested at different temperatures. Erythromycin has been shown to block hERG channels at physiological temperature with an IC₅₀ of approx. 40 µM. However, at RT erythromycin is much less potent. For more details on these experiments please refer to the Application Note.

hERG - Stable Recordings with Accurate Pharmacology

SyncroPatch 384PE data and applications:
Cells were kindly provided by Charles River Laboratories.

Current-voltage relationship of hERG (Kv11.1) expressed in HEK293 is shown along with pharmacology of 4 hERG-active compounds. The current-voltage relationships for all 384 wells (top) using perforated patch (Escin) and multi-hole chips (4 holes per well) are shown. In all 384 wells, a hERG-mediated current was observed with peak amplitude >700 pA at -20 mV. Using a pharmacology voltage protocol, experiments were stable lasting over 20 minutes. Concentration response curves for astemizole, pimozide, cisapride and terfenadine revealed IC₅₀ values consistent with those found in the literature.

hERG - Stable Recordings

Patchliner data and applications:
Cells were kindly provided by Cytomyx/Millipore, UK.

A series of drug concentrations can be applied to each cell. The top figures show the original traces and the corresponding average dose-response curve. Five concentrations of Quinidine (0.1, 0.3, 1, 3 and 10 μM) have been applied.

The lower figure shows the corresponding Imax (-40 mV) including a wash out step and an additional application of the blocker to demonstrate the stability of whole cell recordings.

NaV1.5 - Lidocaine Block

Patchliner data and applications:
Cells were kindly provided by Cytomyx/Millipore.

Full dose response curves at different holding potentials were recorded for each cell (hNav1.5 in HEK293). Currents were elicited by a 10 ms voltage step to 0 mV. Plotted are average peak currents as a function of holding potential and lidocaine concentration.

Cardiomyocytes - Data from different cell providers

Icon CE CardioExcyte data and applications:
Cells were kindly provided by Axiogenesis, Cellular Dynamics, GE Healthcare, Pluriomics, ReproCell, Takara Bio Cellartis Clonetech.

The CardioExcyte 96 allows for
• Non-invasive, label-free measurements of beating cardiomyocyte networks
• 96 recording wells in parallel with 1 ms time resolution
• Quick experiments or long-term compound effects on cardiotoxicity
• Real-time access to beating parameters
• Outstanding software for data analysis and export
• Cost effcient consumables - 96-well format

Cardiomyocytes - Tetracaine dose response curves as recorded with Cor.4U cells

Icon CE CardioExcyte data and applications:
Cells were kindly provided by Axiogenesis.

Impedance amplitude is not changed by addition of increasing concentrations of Tetracaine (left panel), while beat rate of Cor.4U^® cells is decreasing. For example, 29.6µM of Tetracaine decreased the beat rate by ~60% when compared to pre-addition values. Cumulative dose-response relationships indicate Tetracaine potency for same-well additions. Representative raw traces for impedance signals (middle panel) clearly indicate a decrease in cell monolayer beat rate with increasing concentrations of Tetracaine.
Extracellular Field Potential (EFP) spike amplitude is decreased by cumulative Tetracaine dose applications to the same monolayer of Cor.4U^® hIPSC-CMs (top right), in agreement with compound mechanism of action. Representative raw traces for EFP signals (bottom graph) clearly indicate a decrease in spike amplitude.

Cardiomyocytes - Nifedipine and its concentration dependent effect on Cor.4U cells

Icon CE CardioExcyte data and applications:
Cells were kindly provided by Axiogenesis.

Nifedpinie is a dihydropyridine calcium channel blocker that primarily blocks L-type calcium channels. Impedance and EFP recordings on Cor.4U cells reveal a concentration dependent effect on impedance amplitude, beat rate and also a shortening of the FPD as expected.

Cardiomyocytes - Effect of E4031 on the impedance and EFP signals on iCell cardiomyocytes

Icon CE CardioExcyte data and applications:
Cells were kindly provided by Cellular Dynamics.

Effect of the specific hERG blocker, E4031, on the impedance and EFP signals. A Impedance signal of 12 wells in control conditions (left) and the same 12 wells after 13 mins incubation in E4031 at the concentrations indicated (right). E4031 (300 nM, 3 mins incubation) induces EAD, shown in the inset, which can lead to potentially fatal ventricular arrhythmias. B EFP signal of 12 wells in control conditions (left) and the same 12 wells after 13 mins incubation in E4031 at the concentrations indicated (right). E4031(300 nM, 30 mins incubation) also causes arrhythmic effects in the EFP mode shown in the inset.

Cardiomyocytes - Effects of sotalol on impedance (left) and EFP (right) signals of Cellartis Cardiomyocytes

Icon CE CardioExcyte data and applications:
Cells were kindly provided by Takara Bio Cellartis Clontech.

Impedance (left) and EFP signals (right) in control conditions (A) and 1 μM sotalol (B) are shown.
Irregular beating can be observed in the presence of sotalol.

Cardiomyocytes - Effects of verapamil on impedance (left) and EFP (right) signals of Cellartis Cardiomyocytes

Icon CE CardioExcyte data and applications:
Cells were kindly provided by Takara Bio Cellartis Clontech.

(A) Impedance (left) and EFP signals (right) in control conditions
(B) Upon application of 10 nM verapamil
(C) Upon application of 100 nM verapamil.
(D) The mean beats for impedance (left) and EFP (right) are shown in D

Cardiomyocytes - Myocyte phase II study: CiPA conform analysis and arrhythmia detection

Icon CE CardioExcyte data and applications:
Cells were kindly provided by Axiogenesis.

Nanion developed a CiPA conform analysis for the Myocyte phase II study. The feature comes along is included in our CiPA analysis routine. Automated arrhythmia detection is just one highlight out of many when it comes to the CardioExcyte 96 software.

CaV1.2 - Stable recording from frozen stock cells

SyncroPatch 384PE data and applications:
Cells were kindly provided by Charles River.

Screenshots of the PatchControl 384 software showing hCa_V1.2β2/α2δ1 current traces in response to a voltage step protocol and the corresponing current-voltage relationship plot. Measured on the SyncroPatch 384PE using perforated patch methodology (Escin) and multi-hole chips (4 holes per well), the success rate of valuable data for the analysis was 100 %. The cells were used from a frozen cell stock (after induction) and recorded stably for more than 20 minutes. The IC₅₀ value of Nifedipine was determined as 21 nM.

KV4.3/KChIP2 - Dose-response curve of Flecanaide

SyncroPatch 384PE data and applications:
Cells were kindly provided by Charles River.

Screenshots of the PatchControl 384 software showing K_V4.3/KChIP2 current traces in response to a voltage step protocol and the corresponing current-voltage relationship plot. Using whole cell mode in combination with multi-hole chips (4 holes per well), stably transfected cells were measured on the SyncroPatch 384PE. The IC₅₀ value of flecanaide was determined as 28.3 µM which is in accordance to literature. The success rate of valuable data for the analysis was 100%.

KV7.1 (KVLQT) - Dose-response curve

SyncroPatch 384PE data and applications:
Cells were kindly provided by Charles River.

Screenshots of the PatchControl 384 software showing K_V7.1/KCNE (KVLQT/minK) current traces in response to a voltage step protocol and the corresponing current-voltage relationship plot. Using the perforated patch methodology (Escin) in combination with multi-hole chips (4 holes per well), stably transfected cells were measured on the SyncroPatch 384PE. The IC₅₀ value of Chromanol 293B was determined as 3.82 µM. The success rate of valuable data for the analysis was 100%.

hERG - Pharmacology of Bepridil, Dofetilide, Cisapride, Diltiazem (Results CiPA Phase I Study)

Patchliner data and applications:
Cells were kindly provided by Charles River.

The effect of four compounds on hERG currents were investigated, using the CiPA voltage step protocol. Measured on the Patchliner the perforated patch methodology (Escin) and multi-hole chips (4 holes per well) were used. The IC₅₀ value of Cisapride was determined as 112 nM, Bepridil as 178 nM, Dofetilide as 33.9 nM and Diltiazem as 14.5 µM.

hERG - Pharmacology of Mexiletine, Quinidine, Ondansetron, Ranolazine (Results CiPA Phase I Study)

Patchliner data and applications:
Cells were kindly provided by Charles River.

The effect of four compounds on hERG currents were investigated, using the CiPA voltage step protocol. Measured on the Patchliner the perforated patch methodology (Escin) and multi-hole chips (4 holes per well) were used. The IC₅₀ value of Mexiletine was determined as 77.3 µM, Quinidine as 1.04 µM, Ondansetron as 1.13 µM and Ranolazine as 11.9 µM.

hERG - Pharmacology of Sotalol, Terfenadine, Verapamil (Results CiPA Phase I Study)

Patchliner data and applications:
Cells were kindly provided by Charles River.

The effect of four compounds on hERG currents were investigated, using the CiPA voltage step protocol. Measured on the Patchliner the perforated patch methodology (Escin) and multi-hole chips (4 holes per well) were used. The IC₅₀ value of Sotalol was determined as 157 µM, Terfenadine as 82.8 nM and Verapamil as 485 nM.

Application Notes

Cardiac Ion Channels - "High Throughput Screening of Cardiac Ion Channels"

SyncroPatch 384PE Patchliner Icon CE CardioExcyte 96 application note (2.3 MB)

Our CiPA Instruments

CiPA hERG Protocol

HTS CiPA hERG Assay

Myocyte & Ion Channel Effects

Gigaseal HTS patch clamp

Gigaseal HTS patch clamp

CardioExcyte 96 screening tool

CiPA - Comprehensive in vitro Proarrhythmia Assay

The CiPA workstreams

Results of Phase I Studies

Nanion's products in CiPA workstreams

Ion Channel Work Group (ICWG)

ICWG: Description and History

Progress of the Ion Channel Work Group

Data, Applications and Publications on the six Cardiac Ion Channels investigated by the "CiPA Ion Channel Work Group"

Myocyte Work Group (MWG)

MWG: Description and History

Progress of the Myocyte Work Group

Nanion for CiPA:

Data and Applications

Application Notes

Publications

Posters

Oral Presentations

Instrument Product Sheets