Human induced pluripotent stem cell-derived neural platforms rapidly assess toxicity of potential anti-viral therapeutics
Stephen Dea, Ileana Slavin, Priyanka Arunkumar, Janet Seelke, Mary Anne Lofstrom, Blake Anson, Fabian Zanella, and Cassiano Carromeu
Given the speed with which viral threats can rise to pandemic levels, there is an urgent need for antiviral therapies to combat them. Repurposing existing medications is a way to reduce both the time and cost of drug development. However, because these infections are non-discriminatory and medications may be applied to new population segments, sophisticated screening systems are needed to ensure safety across new patient demographics, including for example testing for potential developmental neurotoxicity in pregnant women. Here, we present human iPSC-derived cell-based neural screening platforms to investigate the toxicological profile of potential therapeutic compounds affecting the Central Nervous System (CNS) at multiple stages from developing to mature. Using our platform, we tested the safety profiles of 29 compounds described as potential anti-viral medications. Interestingly, many compounds displayed high toxicity on early stage neural tissues but not on later stages. Compounds were further evaluated for functional assessment in high-throughput calcium flux and lower-throughput multi-electrode array assays. We found that in general 3D neurospheroids were more sensitive than 2D cultures. Of the 29 drugs tested, only two displayed acceptable neurotoxicity profiles with no adverse functional effects at the dosages tested. These results highlight the importance of employing human neural cultures at different stages of neural development to fully understand the neurotoxicity profile of potential therapeutics across normal ontogeny.
11:15 am - 1:00 pm EST
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Human iPSC-Derived Neurospheroids Reduce Late-Stage Attrition by Providing a Front-Line Assay for Early Identification of Safe and Effective Compounds
P. Negraes, N. Sodhi, S. Romero, K. Fichter, D. Phan, C. Turori, B. Anson, O. Guicherit, B. Van Hese, and C. Carromeu
Compound safety and efficacy are necessary and complementary components of the drug discovery pipeline, and tools for accurate and rapid screening of both endpoints are vital. iPSC-derived neurospheroids offer a promising in vitro cellular model for early-stage, human-first studies to identify the safest and most effective compounds for pipeline progression and decreased late-stage attrition. Here we deployed healthy and patient iPSC-derived neurospheroids in a high-throughput screen to identify compounds that rescue the disease phenotype without inducing neurotoxicity. Neurospheroids in 384-well plates underwent chronic treatment with a library of 296 well-annotated compounds followed by simultaneous multiplexed functional, structural, and viability evaluation for phenotypic rescue and/or neurotoxicity. Functional assessment employed FLIPR-based readouts of neural activity where over 70 individual endpoints were quantified and used to rank-order rescue. Structural and viability assessments were taken with microscopic and ATP-based evaluations that, along with the FLIPR readout, were used to ascertain the level and concentration dependence of any toxicity. Example results include 1) compounds that showed toxic responses without any phenotypic rescue; 2) compounds that showed various levels of rescue with evidence of toxicity at higher doses; and 3) 27 compounds that showed good to excellent rescue without any functional, structural, or viability-based toxicity. Compounds eliciting phenotypic recovery spanned several cellular pathways including (but not limited to) the serotonergic, dopaminergic, cholinergic, and GABAergic pathways. Further dissection of cholinergic-based rescue revealed that the primary effect was due to inhibiting the action of acetylcholinesterase. Our results demonstrate a highly specific platform for simultaneously uncovering potential neurotoxicity while identifying the most effective compounds capable of restoring neural function from both healthy and patient-derived material. This “human-first” platform brings relevant biology for both safety and efficacy endpoints into early drug discovery, enables toxicity testing on patient derived material, and holds the potential to reduce costs and accelerate development by ensuring that only the most effective and safest compounds are progressed along the development pipeline.
11:15 am - 1:00 pm EST
Human iPSC-Derived Neural Spheroids Provide a High-Throughput Platform for Early Neurotoxicity Detection
B. Van Hese, K. Prum, A. LaCroix, N. Coungeris, B. Anson, O. Guicherit, P. Yeh, B. Jones, G. J. Smits3 and C. Carromeu
The current gold standard for pre-clinical CNS testing is the rodent functional observational battery (FOB) which offers safety assessments and dose-setting information for clinical trials. However, serious adverse events uncovered at this point cause a significant loss of time and money. Furthermore, as the FOB depends on visual observations of gross neuromotor abnormalities and early stage in vitro testing often relies on non-functional, viability-based endpoints, neurotoxicity is still one of the primary reasons for toxicity-based withdrawal of marketed compounds. Therefore, in order to progress the most promising and safest compounds to the clinic, we developed and present a high-throughput human based neural platform coupled with high-volume data analysis to identify CNS liability early in the drug discovery process. Human iPSC neural spheroids showing spontaneous, synchronous activity in 384-well plates were exposed to negative control, seizurogenic, peripheral neuropathy-inducing, and withdrawn compounds. The functional impact of acute (1-6 hrs) and chronic (7-day) compound exposure was assessed via kinetic Ca2+ imaging and quantified by extracting over 70 waveform parameters. Holistic analysis showed minimal impact of the negative controls and significant perturbation with 5/5, 12/16, and 13/16 of the peripheral neuropathy, seizurogenic, and withdrawn compounds, respectively. Notably, the peripheral neuropathy compounds in general required chronic exposure to show significant effects and the fingerprint of activity pattern was similar across this class of compounds including increased waveform heterogeneity and variability in peak height and peak rise time. Several interesting observations were also apparent from the withdrawn compound group including 1) The novel finding of pre-clinical toxicity with Minaprine, Carmofur, and Mepazine, 2) The need for chronic exposure to elict Carmofur-based toxicity in both in vitro and clinical settings, 3) Insights on the potential advantages of human based high-throughput screening and FOB results for difficult to detect chronic dosing effects. Together these results suggest that human iPSC-derived neural spheroids and a human-first testing paradigm provide a specific high throughput platform for early detection of neurotoxicity across, but not limited to, seizurogenesis and several forms of neuropathy.