Introduction to CiPA

The International Council on Harmonization (ICH) S7B and E14 regulatory guidelines were introduced in 2005 to evaluate the proarrhythmic liability of new drugs. They were implemented in response to the discovery that inhibition of the rapid delayed rectifier potassium current (I_Kr), which is encoded by the human ether-à-go-go related gene (K_V11.1), is associated with prolongation of the QT interval and the potentially deadly arrhythmia, Torsades de Pointes.

The guidelines utilise hERG inhibition and QT interval prolongation as surrogate markers of proarrhythmic liability, which are highly sensitive and have proven effective at preventing proarrhythmic drugs from reaching the market. However, these markers have low specificity, with only a modest correlation between hERG inhibition, QT prolongation and proarrhythmic liability. Therefore, to address these limitations, the Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative was launched by the Food and Drug Administration (FDA) in July 2013. The CiPA initiative aims to improve the accuracy and reduce the cost of predicting cardiac liability using three ‘pillars’:

We have developed a comprehensive panel of CiPA compliant assays and additional assays that provide an excellent in vitro evaluation of cardiac risk.

Comprehensive ion channel panel

An active participant in the CiPA ion channel HTS sub-team, we are also a member of the Health and Environmental Sciences Institute (HESI) cardiac committee. We worked closely with HESI to help reduce data variability between screening sites and to provide validation data to support efforts to generate predictive in silico models. Through these activities, Metrion has validated assays for a wide range of different human ventricular ion channels, which includes the full CiPA panel: hERG, peak and late Na_v1.5, Ca_v1.2, KCNQ1/KCNE1, K_ir2.1 and K_v4.3 (Table 1). We have also developed a dynamic hERG assay, the data from which can be used to improve the accuracy of the FDA model.

Screening services against HCN4 and K_v1.5, which play important roles in controlling human heart rate and atrial repolarisation, respectively, are also provided.

Table 1. Assays for a wide range of different human ventricular ion channels.

Potency assessments against each cardiac ion channel

We provide potency assessments against each cardiac ion channel using single-point or four-point concentration-response assays using:

1. The QPatch48 automated patch-clamp platform.
2. Gold standard manual patch-clamp methodology.

The potency data derived from these high-fidelity platforms are suitable for use in in silico action potential models, which is the second ‘pillar’ of the CiPA initiative.

Assays for hERG, Na_V1.5 and Ca_V1.2

Cardiac screening assays have been developed against hERG, Na_V1.5 and Ca_V1.2 on the Qube, a 384 well automated patch-clamp platform. A number of different assay configurations are available. 

Dynamic hERG assay

New screening assays can be created from existing cell lines. As part of our efforts to create an expanded panel of CiPA-compliant cardiac safety screening assays, we have developed and validated an optimised version of the very challenging ‘Milnes’ dynamic hERG voltage protocol suitable for automated patch clamp.

Translational cardiac safety assays

Clinical QTc/QRS prediction using hiPSC derived cardiomyocytes

QTc and QRS liabilities are a serious concern when developing novel clinical compounds. Assessment of ion channel activity provides a robust method to highlight potential risks, however it may not be sufficient to capture all potential mechanisms that could induce QTc, QRS or arrhythmia issues. In such cases an integrated system such as the hiPSC cardiomyocyte (hiPSC-CM) model can be a valuable model.

We can assess compounds for such liabilities in hiPSC-CMs in a higher throughput 96-well plate-based format. Using a voltage sensitive fluorescent dye, we can simultaneously measure action potential waveforms with high fidelity across all wells using the Lumencor Volta high frequency (10kHz) plate reader. This allows us to accurately capture endpoints such as action potential duration (e.g. APD₉₀), rise time and beat rate. Moreover, this system allows for the assessment of compounds over extended time periods (up to 72h) in serum free conditions.

A key aspect of this model is its ability predict a compounds propensity to generate a prolongation in the clinical QTc interval. Moreover, that assay can help predict the free clinical exposure of a novel compound that would be associated with a 10 ms change in clinical QTc. Similarly, this model can also define the probability of a QRS clinical liability.

Further reading: Characterization of a high throughput human stem cell cardiomyocyte assay to predict drug-induced changes in clinical electrocardiogram parameters, European Journal of Pharmacology, Volume 912, 2021.

iPSC-derived cardiomyocyte screening using conventional manual patch-clamp

Services to evaluate the effect of compounds on action potentials recorded from iPSC-derived cardiomyocytes using conventional manual patch clamp methodology are also provided. Spontaneous or evoked action potentials can be recorded and used to determine the effect of compounds on a range of action potential parameters. The recordings are stable for >30 minutes, which allows the cumulative application of multiple concentrations of each compound.

The manual patch clamp assay generates high fidelity recordings that allow the detection of even subtle changes to the action potential waveform. This helps to successfully discern between compounds with low, medium and high proarrhythmic risk profiles. For example, the figures below show mean data generated using 50 nM dofetilide, which reveals a significant prolongation of all measured APD values.

Chronic cardiotoxicity assay – hiPSC derived cardiomyocytes

Base impedance, an indicator of cell viability, can be used to non-invasively identify structural and functional cardiotoxicity over a chronic time course. We have developed a chronic cardiotoxicity assay using human iPSC-derived cardiomyocytes, which has been validated with a number of cardiotoxicants. For example, doxorubicin, a member of the anthracycline family that is used to treat breast cancer, is associated with a number of cardiac side effects, which includes acute atrial and ventricular arrhythmias, chronic cardiomyopathy and congestive heart failure. Our chronic cardiotoxicity assay recapitulates doxorubicin’s cardiotoxic effect by producing a concentration-dependent decrease of base impedance that develops following a 24 hour exposure period.

This protocol is designed to measure the potential for test compounds to become trapped inside the hERG channel pore, and it is the first to be validated on an automated patch clamp platform.

Technologies used for CiPA screening

• Qube
• QPatch 48
• Gold standard manual patch-clamp

Cardiac Safety Screening Resource Library