Building a robust CNS screening strategy: Integrating in vivo and in vitro assays to better predict CNS risk

Abstract

Attrition within clinical development due to central nervous system (CNS)-related adverse events continues to highlight the need to refine CNS screening strategies. The primary preclinical method to assess CNS risk is the Irwin/Functional Observation Battery (FOB). However, these tests are significantly limited by subjective assessment and temporal resolution. Automated neurobehavioral assessment, such as the Home Cage Analyzer (HCA), can overcome these issues. In addition, in vitro methods, such as microelectrode array (MEA), provide greater throughput and improved translation when using human iPSCs. Ideally, the combination of neurobehavioral and electrophysiological endpoints would converge to provide a clearer picture of CNS perturbance. Critically, a robust CNS screening strategy hinges upon understanding the limitations of such methods and identifying potential gaps in sensitivity between them. This study aimed to validate the Home Cage Analyzer (HCA) and test for convergence or difference in sensitivity between in vivo and in vitro screening using potent CNS active compounds from two distinct drug classes: psychostimulants and sedatives. For HCA experiments, eight male Wistar Han rats (Charles River) were implanted with temperature sensitive RFID transponders (Biomark USA) and pair housed in individually ventilated cages (Tecniplast). Rats were maintained under a 12-h light/dark cycle and habituated to the testing room and HCA rack for 1 week prior to initiation of dosing. Using a crossover Latin-square design, each rat received a single dose of d-amphetamine (0.25, 1, or 3 mg/kg), diazepam (0.5, 1.5, 5 mg/kg), or saline/vehicle. For MEA experiments, hiPSC-derived cortical neuron/astrocyte network (Fujifilm CDI) or rat cortex (E18.5; QBM Biosciences) was cultured in a Maestro Multi-electrode Array System (Axion BioSystems; 48-well plate). l-amphetamine in saline (0.12, 0.37, 1.11, 3.33, 10, 30 μM) or diazepam in DMSO (0.1, 0.3, 1, 3, 10 μM). As expected, we found a dose-dependent increase in activity after treatment with amphetamine in the HCA. Conversely, rats treated with diazepam showed decreased activity levels within the HCA. In line with neurobehavioral effects, diazepam caused a significant decrease in spiking and burst frequency in the MEA, resulting in a well-defined sedative phenotype. Surprisingly, amphetamine application in iPSCs exhibited no phenotypic response within the MEA, including no change in burst rate. Importantly, this lack of an effect in the MEA could be interpreted as a “clean” result, thus representing a missed hazard. In contrast, diazepam demonstrated consistent effects in the HCA and MEA. Together, these results indicate complementarity between in vitro MEA assay and in vivo automated HCA when screening sedatives while revealing a gap between these assays for amphetamine. Thus, a multi-modal approach is critical to a robust CNS screening strategy.