A tremendous amount of knowledge has been acquired in recent years regarding the proteins that make up and regulate the intercellular junctional complex. The claudin family of transmembrane proteins is now understood to be the key transmembrane protein that provides selectivity to ions that move through the paracellular pathway. As has been shown in the kidney
nephron, it now seems clear that different expression levels of claudin family members can result in differential permselectivity to ions. Less is known about how the expression levels of different claudins might control the passage of solutes, and this will be a key piece of understanding that will impact the successful implementation of PPE-induced TJ modulation. The nature of the paracellular pores (or channels) that are formed by the extracellular domains of TJ proteins continues to emerge. The recent application of a sieving model to describe the flux behavior of PEG oligomers of differing size has revealed the possibility of two distinct populations of pores, a relatively abundant restrictive pore that dominates the solute selectivity of smaller molecules, and a less abundant, non-restrictive pore that allows passage of larger molecules79,213. This model is particularly intriguing in light of the recent discovery that the composition and barrier at tricellular TJ is leakier compared to that of bicellular TJ4. The use of more structurally rigid hydrophilic markers will be helpful to confirm the two pore model of the paracellular pathway and determine how these pores change in the presence of PPEs. Gauging the size limitations that exist in opened TJ pores will be particularly important to determine whether PPE technology can successfully be applied to improve the intestinal absorption of larger hydrophilic peptide or protein therapeutic agents.
The structural class of PPEs that can loosely be categorized as amphiphilic, which contains many endogenous molecules and food components, was the first group of PPEs to be investigated and still is an active area of research. Despite the demonstration of increased permeability of hydrophilic solutes in vitro and in vivo in animal models, the registration of these agents for use in humans has not yet been accomplished. Many of these have failed perhaps due to high concentrations required in vivo and the narrow window of selectivity between TJ opening and cytotoxicity. Furthermore, the in vivo potency is not well-predicted based on in vitro data,
suggesting that there has been over-reliance on current in vitro intestinal models to predict in vivo efficacy of PPEs. Modification of the existing in vitro models to better mimic the in vivo setting could improve the in vivo predictivity of the in vitro models, and could also lead to PPE structural modifications that may result in improvement in their in vivo potency.
Non-amphiphilic PPEs demonstrate much less membrane perturbation and typically lower cytotoxicity, and can also offer additional advantages. For example, the cysteine- containing mucoadhesive polymers or thiomers demonstrate not only paracellular permeability enhancing activity, but also have mucoadhesive and protease inhibiting properties which may all contribute to higher absorption of hydrophilic peptide and protein molecules. Non-amphiphilic PPEs that act via cell surface receptors, such as Zot, or peptide fragments that interact with extracellular loops of TJ proteins show greater promise to be TJ-selective. Recent work has demonstrated that lipophilic prodrugs moieties of TJ peptides have resulted in PPEs that are more stable to peptidases, and can rapidly open TJs after apical application to cell monolayers14. While this represents a vast improvement over non-modified TJ peptides, further studies will be needed to demonstrate the in vivo efficacy of these PPEs. Also showing promise with regard to greater TJ selectivity are the non-amphiphilic PPEs that inhibit PLC -dependent pathways (e.g., U-73122). An intriguing implication of this finding is that since PLC activity is regulated by cell surface receptors such as P2y2, it is conceivable that an endogenous ligand acts to maintain TJ tone via a cell surface receptor—an interaction that could be selectively modulated with extracellular antagonists resulting in a selective opening of the TJs.
While the etiology of inflammatory bowel disease is likely to be multifactorial, at least one possible contributing factor is pre-existing chronic leakiness of the intestinal epithelium, which results in passage of luminal antigens, or in turn leads to inflammation of intestinal
tissues. This, as well as the heightened leakiness of traumatized intestinal epithelium that is known to result in passage of bacterial fragments such as LPS, with life-threatening consequences due to septic shock, raise concerns that breaching the intestinal barrier by a PPE may result in absorption of unwanted toxins into the body. However, it is important to note that these concerns are not founded on published studies wherein PPEs have been shown to cause systemic toxicity. Furthermore, it is important to balance these concerns based on the purported role of TJ leakiness in various intestinal pathologies against the knowledge that TJ leakiness occurs due to normal physiological processes. For example, intestinal TJs are opened to facilitate the rapid absorption of glucose and amino acids, to allow paracellular passage of neutrophils or to allow rapid repair of wounded epithelium. Future studies will need to address whether the perceived danger of increasing absorption of luminal toxins after transient modification of paracellular permeability through the action of a PPE actually results in adverse events.