title: Enhanced paracellular transport of insulin can be achieved via transient induction of myosin light chain phosphorylation.
abstract: 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 4kDa fluorescent dextran but not 70kDa 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 70kDa 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.
authors.affiliation: Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK.
authors.forename: Alistair
authors.initials: A
authors.lastname: Taverner
authors.affiliation: Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK.
authors.forename: Ruggero
authors.initials: R
authors.lastname: Dondi
authors.affiliation: Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK.
authors.forename: Khaled
authors.initials: K
authors.lastname: Almansour
authors.affiliation: Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK.
authors.forename: Floriane
authors.initials: F
authors.lastname: Laurent
authors.affiliation: Welsh School of Pharmacy, Cardiff University, Cardiff, CF10 3XF, UK.
authors.forename: Siân-Eleri
authors.initials: SE
authors.lastname: Owens
authors.affiliation: Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK.
authors.forename: Ian M
authors.initials: IM
authors.lastname: Eggleston
authors.affiliation: Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK.
authors.forename: Nikoletta
authors.initials: N
authors.lastname: Fotaki
authors.affiliation: Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK. Electronic address: Rjm37@bath.ac.uk.
authors.forename: Randall J
authors.initials: RJ
authors.lastname: Mrsny
mesh_terms: D000818:Animals; D001692:Biological Transport; D001786:Blood Glucose / Q000032:analysis; D018938:Caco-2 Cells; D006801:Humans; D007328:Insulin / Q000008:administration & dosage* / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D009124:Muscle Proteins; D018994:Myosin Light Chains / Q000378:metabolism*; D043263:Myosin-Light-Chain Phosphatase; D009842:Oligopeptides / Q000008:administration & dosage* / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases; D010766:Phosphorylation; D017208:Rats, Wistar
chemical_list: D001786:Blood Glucose; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D018994:Myosin Light Chains; D009842:Oligopeptides; C107507:PPP1R14A protein, human; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase; C490372:PPP1R12A protein, human
keywords: Cell penetrating peptide; Insulin delivery; Myosin light chain phosphatase; Paracellular transport; Protein–protein interactions; Tight junction function