Pregnanolone-Based Prodrugs as a Strategy for Neuroprotective Drug-Like Compounds: Pregnanolone Pyroglutamate and Its Age‑dependent Anticonvulsant Effects in the 6-hz Seizure Model in Immature Rats.

Abstract

PURPOSE: Pediatric epilepsy remains a major therapeutic challenge due to the high prevalence of pharmacoresistant seizures, and the lack of drugs tailored to immature brain physiology. This study tested a pregnanolone‑based prodrug strategy in which the metabolically labile C‑3 hydroxyl group is transiently masked with a polar moiety designed to regenerate pregnanolone in vivo and thereby preserve or enhance its anticonvulsant efficacy. METHODS: Four C‑3‑conjugated pregnanolone analogues (compounds-) were synthesized and characterized as positive modulators of γ‑aminobutyric acid type A receptors (GABARs) in vitro. Their in vivo activity was evaluated in the 6-Hz transcorneal stimulation model in 15‑ and 25‑day‑old Wistar rats, with detailed quantification of seizure duration and severity, and direct comparison to the endogenous neurosteroid allopregnanolone and the approved neuroactive steroid zuranolone. RESULTS: All four prodrugs showed nanomolar GABAreceptor modulation, consistent with the expected main neurosteroid mechanism of action. Compound(pregnanolone pyroglutamate), designed to be enzymatically cleaved to release pregnanolone, displayed the most consistent and robust in vivo efficacy, producing marked, age‑dependent reductions in seizure duration and severity across multiple parameters in both age groups. Additionally, compoundsanddisplayed enhanced efficacy in younger animals. Plasma stability experiments demonstrated rapid biotransformation of compoundto pregnanolone, supporting the hypothesis that its anticonvulsant effect is mediated by in vivo regeneration of the parent neurosteroid. CONCLUSION: These findings validate a pregnanolone‑prodrug approach in which C‑3 masking groups are used primarily to deliver and release endogenous pregnanolone in vivo, and they identify compoundas a lead candidate for further optimization as an anticonvulsant for pediatric epilepsy. The work provides a translationally relevant framework for developing neurosteroid‑based prodrugs with improved drug‑like properties for pharmacoresistant seizures in the developing brain.