In order to efficiently combat neuroinflammation, it is essential to deliver the anti-inflammatory drugs to their target sites in the brain. Pro-drugs utilizing the L-type amino acid transporter 1 (LAT1) can be transported across the blood-brain barrier (BBB) and the cellular barriers of the brain's parenchymal cells. In this study, we evaluated, for the first time, the efficacy of LAT1-utilizing prodrugs of ketoprofen (KPF) on cyclooxygenase (COX) enzymes in vitro and prostaglandin E2 production in vivo by using an enzymatic assay and liquid chromatography- tandem mass spectrometry method, respectively. Aliphatic amino acid-conjugated pro-drugs inhibited the peroxidase activity of COX in vitro in their intact form (85% inhibition, IC50 ≈ 1.1 µM and 79%, IC50 ≈ 2.3 µM), which was comparable to KPF (90%, IC50 ≈ 0.9). Thus, these compounds acted more as KPF derivatives rather than pro-drugs. In turn, aromatic amino acid-conjugated pro-drugs behaved differently. The ester pro-drug inhibited the COX peroxidase activity in vitro (90%, IC50 ≈ 0.6 µM) due to its bioconversion to KPF, whereas the amide pro-drug was inactive toward COX enzymes in vitro. However, the amide pro-drug released KPF in the mouse brain in sufficient and effective amounts measured as reduced PGE2 levels.
Keywords: COX peroxidase assay; LAT1-utilizing pro-drugs; blood-brain barrier; membrane transport; neuro-inflammation; prostaglandin E2; targeted brain delivery; transporters.