Development, Characterization And Application Of A Novel Mouse Line Humanized For The Intestinal Peptide Transporter PepT1

2015-10-01 13:14:43

The University of Michigan; 2015 October; Doctoral Thesis

Yongjun Hu


The proton-coupled oligopeptide transporter PepT1 is abundantly expressed on the apical side of small intestinal enterocytes and has major responsibility for the intestinal absorption of nutritional nitrogen, peptides and peptide-like drugs. However, there is growing evidence that a significant species difference exists in the affinity and capacity of substrates for PepT1. Therefore, a humanized PepT1 mouse model (huPepT1) was established by introducing hPepT1 genomic DNA into animals previously nulled for mouse PepT1. The mRNA and protein expression profiles indicated that huPepT1 mice had substantial but lower levels than wildtype animals in their expression of PepT1 in small intestine. However, colonic expression of PepT1 was greater in huPepT1 mice than wildtype mice, where the expression of PepT1 was quite low. In situ intestinal perfusion studies revealed that the permeability of glycylsarcosine (GlySar) and cefadroxil were similar, but lower, in the small intestine of huPepT1 mice as compared to wildtype animals. However, in colon, the permeability was greater in huPepT1 mice. Specificity studies, performed in the presence of potential inhibitors, demonstrated that GlySar and cefadroxil permeability was largely, if not solely, dependent upon PepT1 function in wildtype and humanized mice. However, a species difference was observed in the jejunal flux kinetics of GlySar and cefadroxil, where their Km values for PepT1 were 2-fold lower in humanized than wildtype mice. The in vivo studies indicated that the functional activity of intestinal PepT1 was fully restored for GlySar since nearly identical plasma concentration-time profiles and pharmacokinetic parameters were found following oral doses of GlySar in humanized and wildtype mice. After intravenous bolus doses of cefadroxil, virtually superimposable plasma concentration-time profiles were observed between wildtype and huPepT1 mice, and no differences were noted in clearance (CL), volume of distribution steady-state (Vss), and terminal half-life (T1/2) between these two genotypes. However, the Cmax, Tmax and AUC of humanized mice were 2-fold smaller than wildtype animals following oral dose escalation; T1/2 was unchanged. The slopes of partial cumulative AUC vs. time plots demonstrated that the absorption rate of cefadroxil was 2-fold greater in wildtype mice, and the AUC was dose-proportional in these animals. In contrast, a less than proportional increase was observed in AUC with increasing oral doses of cefadroxil in humanized mice. Finally, the AUC0-120 or Cmax of cefadroxil vs. dose profiles showed that humanized huPepT1 mice and humans (results obtained from the literature) were more similar visually than that of wildtype mice and humans. In concluding, this dissertation presents for the first time the generation and characterization of a mouse model humanized for the intestinal peptide transporter huPepT1. This animal model should provide a valuable tool in probing the role, relevance and regulation of PepT1,and in predicting the transport kinetics in humans. huPepT1 mice should also prove useful during the drug discovery process.

Chapter 1 : Research Objectives

Understanding the mechanisms of drug absorption, distribution, metabolism, excretion and transport (ADMET) is essential for improving the safety and efficacy of new chemical entities and therapeutic agents currently on the market. Using molecular biology techniques to clarify the role of proteins, particularly enzymes and transporters, in drug absorption and disposition is a priority of the pharmaceutical sciences. The first observation for active transport of the dipeptide glycylsarcosine (GlySar) in hamster jejunum (in vitro) lead to a new era of investigation for the cloning and functional characterization of transporters, and their relevance in drug kinetics and dynamics.

Among these well-defined transport proteins, peptide transporter 1 (PepT1), an integral membrane protein with 12 transmembrane domains, was brought to the forefront as a tempting delivery target for oral drugs and prodrugs. PepT1 transporter (SLC15A1) is one of four mammalian members in the solute carrier family 15 (SLC15). PepT1 protein is predominantly expressed on the apical side of microvilli cells in small intestine, along with potential expression in colon (more controversial). The PepT1 transporter is well studied and responsible for facilitating the uptake of di-/tri-peptides and peptide-like drugs across the cell membrane from intestinal lumen.

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