The high attrition rate in pharmaceutical development is recognized as the biggest contributing factor to the astronomical cost of bringing a drug to market, which is now estimated at around $2.6 billion. Identifying and addressing the causes of attrition is therefore of paramount importance. One of the major reasons for drug attrition is central nervous system (CNS) toxicity, often resulting from off-target effects on neuronal ion channels that ultimately lead to seizures. However, these neurotoxic effects are often discovered relatively late in development, at a stage when significant time and resource has already been invested
Brett Spangler is project manager of Lonza’s Bioscience Solutions division
An effective way of addressing this problem is to perform comprehensive CNS safety pharmacology testing as early as possible to identify potential seizurogenic effects of candidate drugs. One option is to use in-vivo studies, which have historically been the gold standard in neurotoxicity testing. However, these can be costly and time-consuming, and are therefore not appropriate for high-throughput screening in drug discovery. Moreover, with pharmaceutical companies looking to reduce dependence on animal testing, alternative models that can achieve high throughput and good reliability are urgently required.
To address this need, in-vitro models employing both intracellular and extracellular recording techniques are available. While intracellular techniques are more well-established, they have several limitations for industrial toxicology screening. In this article, we review the major challenges in neurotoxicity testing, and discuss how recent advances in extracellular recording techniques are now enabling reliable and consistent CNS safety pharmacology testing.
The limitations of intracellular recording techniques
Intracellular recording techniques are powerful tools for measuring neuronal electrophysiological activity. Of these, the most widely adopted approach is patch clamping, a technique in which an open-tipped pipette is applied to neuronal cell membranes to enable the recording of currents through single ion channels. Though this method can provide detailed information on how drugs affect electrical excitability, it is notoriously labor-intensive and tedious to perform, and throughput has historically been a significant limitation.
While recent technological advances have enabled the development of automated systems with higher throughput, these have several limitations. Some platforms, for example, are restricted to specific cell lines, while others use low-resistance patches that sacrifice data quality to generate higher data volumes. High-throughput patch clamping techniques have therefore not been widely adopted in CNS safety pharmacology testing.
A further limitation of patch clamping is that it is restricted to single-channel or single-cell recordings. Though it is now possible to employ advanced robotics to record from multiple simultaneously patch-clamped neurons, these techniques are still essentially limited to studying a few neurons at once. As a result, patch clamping cannot be used to assess coordinated electrical activity across a population of cells. Since seizure-like activity is such an important factor in CNS toxicity, this is a major limitation.
Given these drawbacks of intracellular recording systems, developers are moving towards extracellular recording techniques, which are better equipped to measure seizure-like activity and achieve higher throughput. Of the extracellular techniques available, one of the most promising strategies is the use of microelectrode arrays (MEAs).
MEAs: A robust solution for in-vitro toxicology testing
In MEA models, in-vitro cultures of neuronal networks are grown on chips with an embedded array of microelectrodes. These electrodes provide multiple recording points to detect extracellular electrophysiological activity, thus allowing scientists to non-invasively assess seizure-like activity across a whole population of neurons. MEAs therefore hold great promise for neurotoxicity testing, as they allow researchers to measure how the electrophysiological activity of the network is affected by neuroactive test substances. Furthermore, these models can be used to assess chronic as well as acute neurotoxicity. Sustaining the neuronal culture for several days, for example, enables scientists to investigate whether the epileptiform activity induced by a test compound changes over time.
A further advantage of MEA models is that they can be used on an industrial scale for routine screening. Indeed, advances in high-throughput MEA technologies mean it is now possible to have electrode arrays embedded in every well of a multiwell plate, such that 200 compounds can be assessed per day. In addition to high throughput, MEA models also demonstrate exceptional consistency: a multi-site study showed good intra- and inter-lab reproducibility in over 100 experiments, providing encouraging evidence for the robustness of this technique.
The latest MEA systems are therefore able to overcome the challenges of in-vitro neurotoxicity testing in terms of modelling seizure-like activity and achieving high throughput. This makes them a useful tool for CNS safety pharmacology assessments, and indeed, some benchtop MEA systems are now validated to measure proconvulsant activity. For example, a recent study showed that network electrophysiological responses could reliably distinguish proconvulsant compounds from other classes (excitatory compounds, inhibitory compounds and anti-epileptic drugs). This study used Lonza’s rat cortical neurons cultured on the Axion BioSystems Maestro MEA platform, and employed advanced software to analyze network electrophysiology. Analysis revealed that proconvulsant compounds could not be distinguished purely based on the firing rate of the network as a whole, but that excellent discrimination could be achieved by assessing the synchronicity of activity. This importance of network synchrony highlights the value of extracellular recording systems in assessing whole populations of neurons.
With such high-throughput and reliable models, it is now possible to generate in-vitro neurotoxicity data that are likely to be representative of the situation in vivo. Moreover, with continuing advances in cell culture systems, MEA models will be refined to further increase their physiological relevance.
The growing potential of MEA models
While MEA assays using primary cells currently provide a reliable and powerful tool for toxicological screening in industry, research is now exploring how to improve these models further. These new advances have yet to translate into widespread use, but reflect the exciting future potential in this field.
Of particular interest are human induced pluripotent stem cells (hiPSCs), which could be used to generate models more representative of human neurotoxicity. Indeed, with their greater physiological relevance, these cells have been described as “the future of in-vitro toxicology.” However, it is difficult to differentiate hiPSCs into neurons with adequate functional activity, and it may require several months for cells to show the required characteristics.12 As such, primary rodent cell models, the current industry standard for neurotoxicity testing, will likely continue to be required as a control in iPSC studies. Ultimately, there is still substantial work needed to characterize the functional properties of hiPSC-derived neuronal networks, and it is not yet possible to use these cells to produce the robust, consistent and reliable performance needed for industrial use.
Another progressive approach is to use more complex cultures on MEAs. This could prove a very valuable strategy, as standard neuronal cultures do not fully reflect the complex 3D neuronal circuitry or include interactions with non-neuronal cells, which play a vital role in some mechanisms of neurotoxicity. To address this issue, high-throughput MEA systems have been adapted for mixed organotypic cell cultures, acute brain slices and three-dimensional organoid cultures (commonly referred to as “mini-brains”).15 Again, these advances bode well for increasing the physiological relevance of MEA models, but they have not yet translated to widespread industrial use.
Harnessing the benefits of MEA models
MEA models using primary rodent cell cultures provide high-throughput, consistent and reliable systems to assess neuronal electrophysiological activity. The key advantage of MEA models over intracellular recording techniques is their ability to measure activity across a population of neurons, as this enables the generation of results more reflective of seizure toxicity in vivo. It is likely that physiological relevance will improve even further in the future as models are refined to incorporate hiPSCs or more complex 3D cultures.
Advancing in-vitro testing for neurotoxicity will allow more effective screening of candidate drugs earlier on, reducing the time invested in unsuccessful projects and limiting the overall cost of drug development. Ultimately, improved neurotoxicity testing will provide a real benefit to patients, by enabling valuable new therapies to be developed more efficiently.
Brett Spangler is project manager of Lonza’s Bioscience Solutions division
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