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Closing the Gap: Developing an in vitro Neurotoxicity Assay

The regulatory authorities still rely heavily on animal in vivo testing for adult and developmental neurotoxicity testing. Several in vitro tests have been developed to support the in vivo data, however, many have limitations for their use.

One in vitro approach commonly used for electrophysiological assessment is the brain slice assay. Despite its frequent use, this assay has several drawbacks in that it’s low throughput, difficult and expensive to run and doesn’t always offer a useful level of predictivity.

Neurite outgrowth assays provide an in vitro alternative and can assess whether a compound has a toxic impact on neuron growth. This is often considered a neural development assay as it measures the ability of neurons to put out neurites during differentiation. This assay fails to determine the mechanism(s) of toxicity and it doesn’t report endpoints that reflect electrophysiologically-based neuronal function. However, this assay is considerably less expensive to run than brain slice assays, and it can be configured for high throughput analysis.

Multielectrode Arrays (MEAs) are emerging as a powerful tool to understand functional neurotoxic response. Because neuronal function is based on electrical impulses (action potentials), MEA-based assays can measure subcytotoxic responses and examine the effects that compounds have not just on how neurites grow, but how they function.

We developed eCiphr®Neuro using MEA technology in an effort to provide a platform that quantified seizurogenic responses following exposure to compounds which is a major cause of attrition in CNS drug development. Seizures can be the result of synchronous firing of neurons. While drug-induced causes of synchronous firing may be assessed by a target-based approach, often this isn’t practical because the underlying cause could lie within hundreds of different channels, receptors and/or contributing proteins. By utilizing an MEA platform, we are able to examine and record functional phenotypic endpoints, independent of mechanistic or morphological changes.

It’s an assay that we’ve continued to develop because, despite the fact that MEAs have been used to explore functionality of electrically excitable cells for many years, the technology is now starting to see novel, highly predictive value in preclinical development. In a recent experiment we tested brucine because it is chemically related to strychnine and is known as a glycine receptor antagonist, which elicits a seizure-like response. Jenifer Bradley, a member of the toxicology team at Cyprotex US, said, “Our goal in testing brucine was to quantitate seizure-like responses using the 11 endpoints reported by the assay. When we saw that it acted as a GABAA antagonist at low doses and the expected glycine receptor antagonist at high doses, we were surprised and really excited. We hadn’t expected to see that kind of sensitivity. Conclusively demonstrating that MEAs can differentiate compound classes and specific toxicity pathways is a significant step in further validating the technology for in vitro neurotoxicity assessment.”

Cyprotex and Axion Biosystems are co-hosting a workshop on 15th May 2015 to present on how MEAs are being used to evaluate neurotoxic and cardiotoxic risk in vitro. Learn more: www.cyprotex.com/mea-workshop

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