Pharmacokinetic Evaluation of Neutral Sphinghomyelinase2 (nSMase2) Inhibitor Prodrugs in Mice and Dogs

<b>Background</b>: Extracellular vesicles (EVs) can carry pathological cargo, contributing to disease progression. The enzyme neutral sphingomyelinase 2 (nSMase2) plays a critical role in EV biogenesis, making it a promising therapeutic target. Our lab previously identified a potent and selective inhibitor of nSMase2, named DPTIP (IC₅₀ = 30 nM). Although promising, DPTIP exhibits poor pharmacokinetics (PKs) with a low oral bioavailability (%F < 5), and a short half-life (t_1/2 ≤ 0.5 h). To address these limitations, we previously developed DPTIP prodrugs by masking its phenolic hydroxyl group, demonstrating improved plasma exposure in mice. Recognizing that species-specific metabolic differences can influence prodrug PK, we expanded our studies to evaluate selected prodrugs in both mice and dogs. <b>Methods:</b> The scaleup of selected prodrugs was completed and two additional valine- ester based prodrugs were synthesized. Mice were dosed prodrugs via peroral route (10 mg/kg equivalent). For dog studies DPTIP was dosed via intravenous (1 mg/kg) or peroral route (2 mg/kg) and prodrugs were given peroral at a dose 2 mg/kg DPTIP equivalent. Plasma samples were collected at predetermined points and analyzed using developed LC/MS-MS methods. <b>Results:</b> In mice, several of the tested prodrugs showed similar or improved plasma exposures compared to DPTIP. However, in dog studies, the double valine ester prodrug <b>9</b>, showed significant improvement with an almost two-fold increase in DPTIP plasma exposure (AUC_0-t = 1352 vs. 701 pmol·h/mL), enhancing oral bioavailability from 8.9% to 17.3%. <b>Conclusions:</b> These findings identify prodrug <b>9</b> as a promising candidate for further evaluation and underscore the critical role of species-specific differences in prodrug PKs.