RESUMO
A prodrug approach is a powerful method to temporarily change the physicochemical and thus, pharmacokinetic properties of drugs. However, in site-selective targeted prodrug delivery, tissue or cell-specific bioconverting enzyme is needed to be utilized to release the active parent drug at a particular location. Unfortunately, ubiquitously expressed enzymes, such as phosphatases and carboxylesterases are well used in phosphate and ester prodrug applications, but less is known about enzymes selectively expressed, e.g., in the brain and enzymes that can hydrolyze more stable prodrug bonds, such as amides and carbamates. In the present study, L-type amino acid transporter 1 (LAT1)-utilizing amide prodrugs bioconverting enzyme was identified by gradually exploring the environment and possible determinants, such as pH and metal ions, that affect amide prodrug hydrolysis. Based on inducement by cobalt ions and slightly elevated pH (8.5) as well as localization in plasma, liver, and particularly in the brain, aminopeptidase B was proposed to be responsible for the bioconversion of the majority of the studied amino acid amide prodrugs. However, this enzyme hydrolyzed only those prodrugs that contained an aromatic promoiety (L-Phe), while leaving the aliphatic promoeities (L-Lys) and the smallest prodrug (with L-Phe promoiety) intact. Moreover, the parent drugs' structure (flexibility and the number of aromatic rings) largely affected the bioconversion rate. It was also noticed in this study, that there were species differences in the bioconversion rate by aminopeptidase B (rodents > human), although the in vitro-in vivo correlation of the studied prodrugs was relatively accurate.
RESUMO
Non-steroidal anti-inflammatory drugs (NSAIDs) can have protective effects in the brain by inhibition of cyclooxygenases (COX). However, the delivery into the brain across the blood-brain barrier (BBB) and particularly into the brain parenchymal cells is hindered. Therefore, in the present study, we developed four l-type amino acid transporter 1 (LAT1)-utilizing prodrugs of flurbiprofen, ibuprofen, naproxen, and ketoprofen, since LAT1 is expressed on both, the BBB endothelial cells as well as parenchymal cells. The cellular uptake and utilization of LAT1 by novel prodrugs were studied in mouse cortical primary astrocytes and immortalized microglia (BV2), and the release of the parent NSAID in several tissue and cell homogenates. Finally, the effects of the studied prodrugs on prostaglandin E2 (PGE2) production and cell viability were explored. The gained results showed that all four prodrugs were carried into their target cells via LAT1. They also released their parent NSAIDs via carboxylesterases (CES) and most likely also other un-identified enzymes, which need to be carefully considered when administrating these compounds orally or intravenously. Most importantly, all the studied prodrugs reduced the PGE2 production in astrocytes and microglia after lipopolysaccharide (LPS)-induced inflammation by 29-94% and without affecting the cell viability with the studied concentration (20 µM).
