Expression and functional analysis of proton -dependent oligopeptide transporter members

This proposal is aimed at utilizing fundamental molecular and cell biological principles to study the influence of proton-dependent oligopeptide (POT) transporters on the absorption, distribution, metabolism and excretion (ADME) of di- and tripeptides and peptide-based drug therapeutics. Ultimately, the research in the dissertation may help to aid in the rational lead candidate selection of therapeutics that possess optimal biopharmaceutical properties related to a compound\u27s “developability.” Integration of this information into dosage form design and development is the long term goal of the project that will hopefully lead to increased bioavailability of related agents that serve as POT substrates.^ This dissertation is divided into several separate Chapters. Chapter 1 provides an introduction to the material discussed in other Chapters and provides a general overview of drug transporters that have been linked to the absorption of peptide-related substrates across the gastrointestinal tract. Chapter 2 details the initial studies conducted in our laboratory illustrating preliminary differences in the expression of specific POT transporters in several patients that varied in age, gender and ethnicity. Particular emphasis was placed on transporter expression within the gastrointestinal tract (GIT), the site of oral absorption of therapeutic agents. Specifically, several members of the Proton-Dependent Oligopeptide Transporter (POT) superfamily were identified within the GIT. These include the well characterized human Peptide Transporter 1 (hPepT1) and Peptide Transporter 2 (hPepT2), and the more recently characterized human Peptide/Histidine Transporter 1 (hPHT1) and 2 (hPHT2) orthologues. The hypothesis driving these studies was that by establishing a broader understanding of the specific transporters expressed within the GIT, we will ultimately enable rational, regiospecific drug dosage form design for targeting for the drug release in “absorption windows” where the optimal bioavailability of peptide-based drugs can be achieved.^ Chapters 3 and 4 of this proposal are primarily focused with elucidating some of the functional characteristics of hPHT1. Chapter 3 focuses on the functional evaluation of hPHT1 in a stably transfected COS-7 cell line of simian origin to contrast the results with the studies performed previously in our laboratory utilizing hPHT1 transiently transfected COS-7 cells. Chapter 4 illustrates our evaluation of hPHT1 function utilizing shRNA technology to knockdown expression levels in the recently been developed hCMEC/D3 cell line. The hCMEC/D3 cell line was derived from a primary human blood-brain barrier (BBB) culture that was immortalized and selected through cloning to yield a proliferative population with properties that mimic the in vivo brain microvessel endothelial cells. These studies compliment those performed with the wild-type and hPHT1 stably transfected hCMEC/D3 by Dr. Stephen Carl in our laboratory, further confirming the functional characteristics of the transporter in the brain.^ Furthermore, Chapter 5 discusses the effect of media supplementation on the differentiation-dependent changes in drug transporter expression and function in the HT-29 human adenocarcinoma cell line. Our laboratory was particularly focused on utilizing this cell line to transfect hPHT1 and hPepT1 transcripts for over-expression and functional assessment primarily because of their low endogenous transporter expression that leads to minimal system “noise”. By identifying these differences, we hope to eventually develop a standardized cell culturing protocol that will better enable comparative drug screening studies between laboratories utilizing cells from the same progenitor line. Finally, Chapter 6 will give brief concluding remarks summarizing the previous results of this dissertation and also gives future perspectives on possible avenues to take this project down the road.