InPulse

Background

Cardiovascular (CV) safety liabilities are one of the most common causes of drug attrition in both the nonclinical and clinical setting. These liabilities manifest in a variety of ways that include structural injuries and derangements in CV function. Changes in contractility may lead to clinical heart failure and even death. Important classes of drugs, such as anti-neoplastic tyrosine kinase inhibitors, are currently facing particular safety challenges with this effect.

iPS cell-derived cardiomyocytes (iPSC-CMs) have shown early promise for replacing animal use in cardiovascular research and many pharmaceutical companies and academic researchers are developing assay platforms to identify potential electrophysiological and structural liabilities earlier in development (i.e. ‘pre-animal’). A similar capability for contractility assessments would be very useful from a scientific and 3Rs perspective.

Historically, there has been a heavy reliance on highly invasive, technically demanding, surgically instrumented telemetered animal models in preclinical studies that provide indirect measures of contractility in vivo. More recently, imaging (e.g. echocardiography) has been used as an investigative and mechanistic tool both preclinically and clinically but it is low throughput and labour intensive. The only in vitro/ex vivo models that are available are perfused whole heart models from animals and isolated cardiac cells from animals and humans. There is no currently validated/qualified human-derived in vitro system for assessing drug-induced changes in contractility under varying levels of physiologic load.

The ability to study changes in cardiac contractility in vitro would allow the investigation of potential causes of long QT syndrome that are both hERG and non-hERG dependent. This would allow a more accurate prediction of potential drug-induced cardiac toxicity in preclinical and clinical settings; thus help to reduce attrition. Mechanistic understanding of changes in cardiac contractility from in vitro models would also be relevant to efficacy studies earlier in the drug discovery process.

The rapid development of human iPS cell technologies and materials sciences provides an opportunity to develop a dynamic, human-relevant assay system to screen and characterize novel drug candidates for cardiac contractility liabilities. Recent publications have demonstrated that the extracellular matrix, morphology and orientation of cardiac cells are important for cardiac contractility and calcium signalling in cardiac myocytes and there have been significant advances in substrates for monitoring contractile tension.However, there is the need for a robust in vitro model that reflects the 3D architecture of cardiac tissue with mature cell phenotypes.

3Rs benefits

• ​Assessment of developmental drug pre-candidates on cardiac contractility relies exclusively on the use of animals prior to clinical trials. A typical study uses 12 to 24 dogs.
• A physiologically relevant 3D in vitro model will replace the use of animals in these studies and reduce the number of animals needed to assess the mechanism of action of any drug-induced effect.
• An in vitro system where load can be adjusted to model the pathophysiology of disease tissue could also be used to reduce animal use in efficacy studies. A typical efficacy study uses approximately 24 mice.
• Once validated, an in vitro platform could be used to screen compounds and provide earlier go/no-go decisions hence reducing the number of animals used to test compounds that would ultimately fail in development.

Phase 1 winners

Project teams led by:

• Professor Chris Denning, University of Nottingham, £100,000.
• Professor Cesare Terraciano, Imperial College London, £99,958.
• Professor Wolfram Zimmerman, University Medical Center Göttingen, £100,000.

Phase 2 winner

Project team led by:

• Professor Chris Denning, University of Nottingham, £999,915.

Full Challenge information

Through the InPulse Challenge, the team have developed a suite of assays to assess cardiac liabilities of drugs using hiPSC cardiac myocytes (CMs).

The team compared different lines of hiPSC-CMs, including PluriCyte cells from Ncardia, in combination with different platform technologies, each tested through the same set of compounds provided by the Sponsor to establish the optimum configuration for assessment of the effects of drugs on cardiac toxicity with a focus on contractility.

Two hiPSC-CMs configurations were selected to provide a comprehensive final assay set: a 2D monolayer cell platform to provide high throughput readouts and a 3D engineered heart tissue system (EHT Technologies) that is lower throughput but can replicate better the cardiac microenvironment to produce higher levels of cell maturity and provide more detailed mechanistic insights (Figure 1).

Figure 1. Engineered Heart Tissue from EHT Technologies.

Using the 2D monolayers of hiPSC-CMs, two technology platforms were utilised to provide assay read outs:

• The Triple Transient Measurement System (TTM) developed by Leiden University that can continually measure contractility, Ca2+ handling and electrophysiology within a repeating 3 msec window (Figure 2).

Figure 2. TTM system developed by Leiden University

• The CellOPTIQ system developed by Clyde Biosciences in Glasgow that can measure contractility, Ca2+ handling and electrophysiology from the same region of the well.

Assessment of the effects of drugs on the contraction of hiPSC-CMs EHTs is performed via calculation of absolute force from the extent of deflection of two silicon posts, whilst calcium handling is reported using a lentiviral genetically encoded calcium indicator.

The team also developed MUSCLEMOTION- a versatile open software tool to quantify cardiomyocyte and cardiac muscle contraction in vitro and in vivo. This single software platform can be used to quantify contractility in:

• Single adult rabbit cardiomyocytes.
• hiPSC-CM as single cells, monolayers, cardio spheres and EHTs with or without drug or disease challenge.
• In vivo healthy or diseased zebrafish hearts.
• Human MRI scans from patients who were healthy or with deficiency in heart function.

The InPulse Challenge has led to greater validation of the hiPSC-CM model in cardiovascular safety testing and produced a wealth of knowledge and data to inform the optimal choice of cardiac cell platform to assess contractility.

For further information please contact Professor Chris Denning in the first instance.