A staged zebrafish-mouse screening strategy for antiseizure compound prioritization

Epilepsy affects more than 50 million people worldwide, and the identification of new antiseizure medications (ASMs) with adequate blood-brain barrier (BBB) penetration remains a major challenge. The pentylenetetrazol (PTZ)-induced zebrafish model is widely used for early ASM screening, but most studies use larvae at 7 days post-fertilization (dpf) or earlier, before later stages of BBB maturation are fully assessed. Here, we evaluated whether staged testing in 7-, 14-, and 21-dpf zebrafish could improve prioritization of CNS-active compounds before mammalian testing. Thirty-one Vitamin K (VK) analogs were screened in 7-dpf zebrafish using PTZ-induced seizure-like hyperlocomotor activity as a phenotypic endpoint. Seventeen compounds significantly reduced PTZ-induced hyperlocomotor activity at 7 dpf. Based on quantitative selection criteria, three compounds were advanced to dose-response studies in older zebrafish, and two compounds retained activity at 14 and 21 dpf. These compounds were then evaluated in the mouse 6 Hz seizure model. Both compounds showed protection at 32 mA, whereas only compound 3d remained active in the higher-intensity 44 mA model. Brain-to-plasma analysis confirmed CNS exposure for 3d and 3l, with ratios of 2.64 and 0.853, respectively. These findings support later-stage zebrafish screening as a practical phenotypic prioritization step for selecting CNS-active compounds for mammalian seizure models, while highlighting the need for direct mammalian validation and further PK/PD characterization.