L-type amino acid transporter 1 utilizing prodrugs of ferulic acid revealed structural features supporting the design of prodrugs for brain delivery

Abstract

Ferulic acid (FA), a natural antioxidant, has displayed some potential benefits against Alzheimer's disease. However, due to its poor blood-brain barrier (BBB) permeation and low bioavailability, the clinical use of FA for the treatment of Alzheimer's disease has been limited. In the present study, we applied an L-type amino acid transporter (LAT1) – mediated prodrug approach to deliver FA into the mouse brain and synthetized three novel LAT1-utilizing prodrugs of FA. We used a previously proposed methodology for the development of transporter-utilizing prodrugs and investigated their cellular uptake via LAT1 in vitro in ARPE-19 cells, BBB permeation using in situ perfusion in mice and pharmacokinetics after a single i.p. injection in mice; and compared the findings to our previous structure-pharmacokinetics relationship analysis of LAT1-utilizing prodrugs. In addition, we evaluated interspecies differences in the bioconversion rate of the ester-based prodrug in mouse and human plasma and liver S9 subcellular fraction. It was found that amide-based prodrugs with an aromatic ring in the promoiety were effectively bound to LAT1 and utilized the transporter for cellular uptake in vitro and crossed the BBB after in situ perfusion in mice. In addition, the amide prodrug with the promoiety directly conjugated in the meta-position to FA was bioconverted to the parent drug in mouse brain. Importantly, the study showed that the analogous ester-based prodrug did bind to LAT1 but did not utilize the transporter for cellular uptake in ARPE-19 cells. However, the presence of an ester linker between the prodrug and the parent drug promoted favorable bioconversion properties in human in comparison to mouse tissues in vitro i.e. the ester prodrug showed higher stability in human plasma (75% of intact prodrug in 5 h) and liver S9 subcellular fraction (181 min) in comparison to mouse plasma (t½ 2.6 min) and liver S9 fraction (t½ 23.3 min), suggesting that ester-based prodrugs may offer potential benefits in humans. In conclusion, switching from an amide to ester linker between the promoiety and the parent drug can affect the bioconversion rate of prodrugs in different species as well as influencing their cellular uptake mechanism. Furthermore, the results demonstrated the effective application of structure-pharmacokinetic relationships and screening methodology for developing LAT1-utilizing prodrugs and highlighted the importance of evaluating the biotransformation of parent drug and prodrugs in different species.