Development of a new hazard scoring system in primary neuronal cell cultures for drug-induced acute neuronal toxicity identification in early drug discovery

Abstract

We investigated drug-induced acute neuronal electrophysiological changes using Micro-Electrode arrays (MEA) to rat primary neuronal cell cultures. Data based on 6-key MEA parameters were analyzed for plate-to-plate vehicle variability, effects of positive and negative controls, as well as data from over 100 reference drugs, mostly known to have pharmacological phenotypic and clinical outcomes. A Least Absolute Shrinkage and Selection Operator (LASSO) regression, coupled with expert evaluation helped to identify the 6-key parameters from many other MEA parameters to evaluate the drug-induced acute neuronal changes. Calculating the statistical tolerance intervals for negative-positive control effects on those 4-key parameters helped us to develop a new weighted hazard scoring system on drug-induced potential central nervous system (CNS) adverse effects (AEs). The weighted total score, integrating the effects of a drug candidate on the identified six-pivotal parameters, simply determines if the testing compound/concentration induces potential CNS AEs. Hereto, it uses four different categories of hazard scores: non-neuroactive, neuroactive, hazard, or high hazard categories. This new scoring system was successfully applied to differentiate the new compounds with or without CNS AEs, and the results were correlated with the outcome of in vivo studies in mice for one internal program. Furthermore, the Random Forest classification method was used to obtain the probability that the effect of a compound is either inhibitory or excitatory. In conclusion, this new neuronal scoring system on the cell assay is actively applied in the early de-risking of drug development and reduces the use of animals and associated costs.

Keywords: adverse effect (AE); hazard score system; micro-electrode array (MEA); neuronal cells; neuronal toxicity; seizures.

FIGURE 1

(A). Concept of Workflow on the identification of acute neuronal hazards. The compounds were tested in 48 well plates cultured with primary rat neurons using MEA. Effects (Data changes) in rat primary neurons were analyzed based on several measured parameters, measured in MEA, to have accumulated data points to define the range of scores. This strategy to determine a concentration-dependent hazard and to rank compound candidates is represented schematically. (B). Characteristic recordings of rodent primary cortical neurons. Electrophysiological spiking activity of primary cortical neurons in a single well in an MEA depicted in a time-sequenced raster plot. A vertical bar is drawn each time a neuron fires an action potential (black tick mark), and a burst of spikes is represented in blue on an MEA recording. The main parameters are depicted in (B) (16 electrodes) and (C) (single electrode).

FIGURE 2

Density plots show the TIs for vehicles and positive controls calculated for the six selected key parameters. Determination of Cutoffs using TIs to develop the scoring matrix. Examples of the TIs of compounds on AuCC (A), the burst duration (B), the weighted mean firing rate (C), and the median/mean Inter-Spike Interval (ISI) (D). The blue line corresponds to the upper and lower bound Tis for the vehicle, and the red line corresponds to the upper and lower bound Tis for the compounds. (E) The scoring matrix represents a point card where for each parameter a weighted score is given depending on the size and direction of the ΔΔ% effect. (F) Calculation of hazard scorings is done through a sum of points across all six parameters.

FIGURE 3

Effect of representative 106 reference drugs on the rat cortical neurons measured on the MEAs using the hazard score system defined by the electrophysiological changes per concentration. Concentrations were selected based on the therapeutic-free Cmax (shown in italics). n. a., not available. Green: no risk (non-neuronal active), yellow: neuronal active; Orange: hazard, and Red: high hazard.

FIGURE 4

Sensitivity, specificity, and accuracy were derived from the true positive, false negative, false positive, and true-negative classification of the 113 reference compounds on the outcome of the hazard scoring system.

FIGURE 5

Classification of 18 compounds into no effect (DMSO), (A) inhibitory, and (B) excitatory drugs by using Random Forest prediction. The results show the probability that the compounds will be classified into their categories at each concentration, allowing an addition to the hazard identification score system.

FIGURE 6

Assessment of the Hazard scoring of newly synthetized compounds on rat primary neurons measured by MEAs. (A). Pie charts showing the total distribution of different hazard levels for compounds regardless of concentrations (n = 79). (B). Pie charts showing the concentration-dependent distribution of different hazard scorings for compounds (n = 101). (C). Translational confirmation of the MEA-derived neuronal hazard scoring of 23 antisense oligonucleotides (ASOs) that were also evaluated in a phenotypic monitoring assay in vivo using adult C57Bl6 mice.

FIGURE 7

Translational confirmation of the MEA-derived neuronal hazard scoring of a reference compound (4-AP) that was also evaluated in a freely moving rat with EEG recordings. (A). Effects of 4-AP on the MEA in vitro with the hazard score for 0.3 and 10 µM 4-AP. (B). EEG recordings in a freely moving rat at baseline and vehicle and in the presence of 4-AP at different doses, 1 and 3 mg/kg.

FIGURE 8

Schematic showing standard paradigm for understanding the balance between excitation (+) and inhibition (−) in drug-induced seizures (A). (B) The normal balance between excitatory and inhibitory neuronal activity, receptor and ion channel function, neurotransmitters, and pathways. (C) Modulation of neuronal receptors or ion channels by drugs that decrease neuronal activity by increasing inhibitory neuronal activity or decreasing excitatory neuronal activity. (D) Modulation of neuronal receptors or ion channels by drugs that increase neuronal activity by increasing the excitatory neuronal activity or decreasing the inhibitory neuronal activity. Both (C, D) will result in an imbalance toward possible seizure occurrence.