Pregnanolone Glutamate: A Dual-Fate Delivery System for Neuroactive Steroids in Perinatal Focal Cerebral Ischemia

Pregnanolone glutamate (PG) is a synthetic neurosteroid analog showing promise for treating ischemic brain injury, yet its blood–brain barrier (BBB) transport and metabolic fate remain unclear. We investigated the pharmacokinetics of PG in postnatal day 12 rats of both sexes subjected to ET‑1–induced focal hippocampal ischemia. Animals received PG (1 mg/kg i.p.) or vehicle; serum and hippocampal steroidomes were profiled 60 minutes post-administration using GC-MS/MS (hippocampus: n=16 PG+, n=27 PG-; multi-tissue subset: n=6 PG+, n=21 PG-). Our data reveal a "dual-fate" mechanism: PG undergoes rapid systemic hydrolysis as a prodrug but also crosses the BBB intact, with significant parent conjugate accumulation in hippocampus (42.3 pmol/g). The brain functioned as a "metabolic sink," passively accumulating peripherally generated metabolites—such as 17-hydroxypregnanolone—despite local absence of synthesizing enzymes (e.g., CYP17A1). Crucially, PG induced "metabolic segregation" within the CNS: the pharmacological 5β-pathway was saturated (~170-fold pregnanolone increase), while endogenous neuroprotective 5α-pathway (allopregnanolone) homeostasis remained preserved, contrasting with peripheral metabolic saturation. Preferential hippocampal accumulation of 3-oxo and 3β-isomers suggests autonomous regulatory buffering via oxidative HSD17B enzymes, protecting against excessive GABAergic inhibition. This unique pharmacokinetic profile—combining metabolic segregation with active central buffering—defines PG as a dual-mechanism delivery system that generates central neuroactive metabolites without disrupting endogenous neurosteroidogenesis, positioning it as a promising neurotherapeutic candidate minimizing physiological steroid homeostasis disruption.