A major problem in drug delivery is the oral delivery of protein drugs. It was formerly believed that the principal problem was due to denaturation of the proteins by gastric acid and digestion by gastrointestinal proteases. However, encapsulation methods have been developed that can delivery proteins intact to the intestines. This has led to the realization that the major, unsolved problem is that intact proteins cannot cross the intact intestinal epithelium. Physiologically, proteins are transported across epithelial cells by transcytosis. The major transcytotic process in adult human intestine is the transport of IgA from the basolateral to he apical surface by the polymeric immunoglobulin receptor (pIgR). This receptor binds its ligand, IgA, at the basolateral surface of the epithelial cell, and then is endocytosed, transported across the cell in vesicles, and exocytosed at the apical surface. At the apical surface, the extracellular, ligand- binding domain of the pIgR is proteolytically cleaved off and is released together with the IgA into external secretions. This cleaved fragment of the pIgR is called secretory component (SC). We have previously shown that ligands that bind to SC can bind to uncleaved pIgR on the apical surface, and can be endocytosed and transcytosed to the basolateral surface. However, such a ligand will also bind to me large amount of SC that has been cleaved and is present in the luminal contents, and this will effectively compete for binding of the ligand to intact pIgR on the apical surface. We have developed a new class of ligand that binds pIgR but does not bind SC. These ligands are efficiently endocytosed at the apical surface and transcytosed to the basolateral surface in vivo and in vitro, i.e. in the opposite of the normal direction. We have shown that these ligands can be conjugated to a 150 kDa cargo protein, which can be transported from intestinal lumen into blood in rats. Our goal now is to improve this technology for eventual use for protein drug delivery clinically.
In Specific Aim 1, we will analyze the exact route taken by molecules taking the apical to basolateral pathway, both in vivo and in vitro.
In Specific Aim 2 we will maximize transcytosis by optimizing our new class of ligands. We will vary off rate, pH sensitivity, and the exact region of the pIgR to which they bind.
| Zegers, Mirjam M P; O'Brien, Lucy E; Yu, Wei et al. (2003) Epithelial polarity and tubulogenesis in vitro. Trends Cell Biol 13:169-76 |
