Cholix (Chx) is expressed by the intestinal pathogen Vibrio cholerae as a single chain of 634 amino acids (~70.7 kDa protein) that folds into three distinct domains, with elements of the second and third domains being involved in accessing the cytoplasm of nonpolarized cells and inciting cell death via ADP-ribosylation of elongation factor 2, respectively. In order to reach nonpolarized cells within the intestinal lamina propria, however, Chx must cross the polarized epithelial barrier in an intact form. Here, we provide in vitro and in vivo demonstrations that a nontoxic Chx transports across intestinal epithelium via a vesicular trafficking pathway that rapidly achieves vesicular apical to basal (A→B) transcytosis and avoids routing to lysosomes. Specifically, Chx traffics in apical endocytic Rab7 + vesicles and in basal exocytic Rab11 + vesicles with a transition between these domains occurring in the ER-Golgi intermediate compartment (ERGIC) through interactions with the lectin mannose-binding protein 1 (LMAN1) protein that undergoes an intracellular redistribution that coincides with the reorganization of COPI + and COPII + vesicular structures. Truncation studies demonstrated that domain I of Chx alone was sufficient to efficiently complete A→B transcytosis and capable of ferrying genetically conjoined human growth hormone (hGH). These studies provide evidence for a pathophysiological strategy where native Chx exotoxin secreted in the intestinal lumen by nonpandemic V. cholerae can reach nonpolarized cells within the lamina propria in an intact form by using a nondestructive pathway to cross in the intestinal epithelial that appears useful for oral delivery of biopharmaceuticals. One-Sentence Summary: Elements within the first domain of the Cholix exotoxin protein are essential and sufficient for the apical to basal transcytosis of this Vibrio cholerae-derived virulence factor across polarized intestinal epithelial cells.

The intestinal epithelium functions to effectively restrict the causal uptake of luminal contents but has been demonstrated to transiently increase paracellular permeability properties to provide an additional entry route for dietary macromolecules. We have examined a method to emulate this endogenous mechanism as a means of enhancing the oral uptake of insulin. Two sets of stable Permeant Inhibitor of Phosphatase (PIP) peptides were rationally designed to stimulate phosphorylation of intracellular epithelial myosin light chain (MLC) and screened using Caco-2 monolayers in vitro. Apical application of PIP peptide 640, designed to disrupt protein–protein interactions between protein phosphatase 1 (PP1) and its regulator CPI-17, resulted in a reversible and non-toxic transient reduction in Caco-2 monolayer trans-epithelial electric resistance (TEER) and opening of the paracellular route to 4 kDa fluorescent dextran but not 70 kDa dextran in vitro. Apical application of PIP peptide 250, designed to impede MYPT1-mediated regulation of PP1, also decreased TEER in a reversible and non-toxic manner but transiently opened the paracellular route to both 4 and 70 kDa fluorescent dextrans. Direct injection of PIP peptides 640 or 250 with human insulin into the lumen of rat jejunum caused a decrease in blood glucose levels that was PIP peptide and insulin dose-dependent and correlated with increased pMLC levels. Systemic levels of insulin suggested approximately 3–4% of the dose injected into the intestinal lumen was absorbed, relative to a subcutaneous injection. Measurement of insulin levels in the portal vein showed a time window of absorption that was consistent with systemic concentration-time profiles and approximately 50% first-pass clearance by the liver. Monitoring the uptake of a fluorescent form of insulin suggested its uptake occurred via the paracellular route. Together, these studies add validation to the presence of an endogenous mechanism used by the intestinal epithelium to dynamically regulate its paracellular permeability properties and better define the potential to enhance the oral delivery of biopharmaceuticals via a transient regulation of an endogenous mechanism controlling the intestinal paracellular barrier.

Please cite only the published version using the reference above.See http://opus.bath.ac.uk/ for usage policies.Please scroll down to view the document. In the conditioned place preference extinction and reinstatement method, a combination of 0.3 mg/kg buprenorphine and 1.0 mg/kg naltrexone completely blocked drug-primed reinstatement in cocaine-conditioned rats (conditioned with 3 mg/kg cocaine, drug prime was 3 mg/kg cocaine), and attenuated drug-primed reinstatement in morphine-conditioned rats (conditioned with 5 mg/kg morphine, drug prime was 1.25 mg/kg morphine). These data add to the growing evidence that a buprenorphine/naltrexone combination may be protective against relapse in a polydrug abuse situation.

IL-10 is a potent anti-inflammatory cytokine capable of suppressing a number of proinflammatory signals associated with intestinal inflammatory diseases, such as ulcerative colitis and Crohn's disease. Clinical use of human IL-10 (hIL-10) has been limited by anemia and thrombocytopenia following systemic injection, side effects that might be eliminated by a gut-restricted distribution. We have identified a transcytosis pathway used by cholix, an exotoxin secreted by nonpandemic forms of the intestinal pathogen Vibrio cholerae. A nontoxic fragment of the first 386 aa of cholix was genetically fused to hIL-10 to produce recombinant AMT-101. In vitro and in vivo characterization of AMT-101 showed it to efficiently cross healthy human intestinal epithelium (SMI-100) by a vesicular transcytosis process, activate hIL-10 receptors in an engineered U2OS osteosarcoma cell line, and increase cellular phospho-STAT3 levels in J774.2 mouse macrophage cells. AMT-101 was taken up by inflamed intestinal mucosa and activated pSTAT3 in the lamina propria with limited systemic distribution. AMT-101 administered to healthy mice by oral gavage or to cynomolgus monkeys (nonhuman primates) by colonic spray increased circulating levels of IL-1R antagonist (IL-1Ra). Oral gavage of AMT-101 in two mouse models of induced colitis prevented associated pathological events and plasma cytokine changes. Overall, these studies suggest that AMT-101 can efficiently overcome the epithelial barrier to focus biologically active IL-10 to the intestinal lamina propria.

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