Investigation On Novel Carrier Prodrug Structures Targeted To Oligopeptide Transporter1(PEPT1)

Oral administration is best accepted for its safty, convenience andless cost as well. But poorly oral bioavaibility is a critical obstacle in oralstrategies. Great efforts were involved in improving oral bioavaibility,among which, target carrier prodrug, especially targeting to PEPT1, was ahotspot in recent years.As a membrane protein, PEPT1is very hard to be purified,crystallized and homology modeling also. Since that, investigation onsubstrates structure based on in vitro or in vivo models was still the mostimportant part in carrier prodrug strategy targeted to PEPT1. Researchworks were mainly focusing on designing amino acid ester prodrugs ofparent compound via hydroxyl group, and dipeptide mono ester prodrugswere reported to have higher affinity for PEPT1than its amino acid monoester prodrug. But non hydroxyl group modification was hardly studied. Additionally, including peptide bond, α amino group and carboxyl groupwere considered as necessary structures for PEPT1recognition andtransport. But in the latter studies, except for α amino group, peptidebond and carboxyl group were proved to be not necessary. Therefore,substrate specificity of PEPT1is not fully understood.On analyzing the Peffof17cephalosporins on Caco-2monolayer, wefound that α amino group could be unnecessary for PEPT1recognitionand transport yet. According to the possible new trends in prodrug designtargeted to PEPT1and the hypothesis of α amino group to be unessential,in this study, a dipeptide modifided library was designed and synthesizedbased on click chemistry and solid phase peptide synthesis. Modificationon non hydroxyl group and non α amino group were two key structuralfeatures of the library and which was well characterized. Isolated purity,MS and~1H NMR were employed into the characterization of the wholelibrary, and30%compounds were confirmed with HR MS, additionally,15%compounds were fully characterized.Competitive inhibition assay was designed to evaluate the affinity toPEPT1of the library operated on transfected Hela cell model. SomeAZT-dipeptide conjugates can compete off over80%cell uptake ofcephalexin. In the further, cell uptake in vitro was conducted on Hela cellmodel and single-pass intestinal perfusion in vivo was operated on ratsrespectively. In this study, we found some dipeptide sequences with very highaffinity to PEPT1which were not reported before and could be used inprodrug strategy. Furthermore, results in vitro and in vivo affirmed thatPEPT1was involved in the uptake and transport of compound candidateswith two key structures, which was also not reported before. Moreover,our results also showed that the intestinal absorption of compoundcandidates with our selected dipeptide sequences was improvedsignificantly compared with parent structure.Findings in this study could enlarge parent drug selection andsignificantly expand the application of PEPT1in target carrier prodrugstrategy.