Colon-Targeted Cell-Permeable NFκB Inhibitory Peptide Is Orally Active against Experimental Colitis

Abstract

For the purpose of development of orally active peptide therapeutics targeting NFκB for treatment of inflammatory bowel disease (IBD), two major barriers in oral delivery of therapeutic peptides, metabolic lability and tissue impermeability, were circumvented by introduction of a colon-targeted delivery system and cell permeable peptides (CPP) to NFκB inhibitory peptides (NIP). Suppression of NFκB activation was compared following treatment with various CPP conjugated NIPs (CPP-NIP). The most potent CPP-NIP was loaded in a capsule coated with a colon specific polymer, which was administered orally to colitic rats. The anti-inflammatory activity of the colon-targeted CPP-NIP was evaluated by measuring inflammatory indices in the inflamed colonic tissue. For confirmation of the local action of the CPP-NIP, the same experiment was done after rectal administration. Tissue permeability of the CPP-NIP was examined microscopically and spectrophotometrically using FITC-labeled CPP-NIP (CPP-NIP-FITC). NEMO binding domain peptide (NBD, TALDWSWLQTE) fused with a cell permeable peptide CTP (YGRRARRRARR), CTP-NBD, was most potent in inhibiting NFκB activity in cells. Colon-targeted CTP-NBD, but not colon-targeted NBD and CTP-NBD in an enteric capsule, ameliorated the colonic injury, which was in parallel with decrease in MPO activity and the levels of inflammatory mediators. Intracolonic treatment with CTP-NBD alleviated rat colitis and improved all the inflammatory indicators. CTP-NBD-FITC was detected at much greater level in the inflamed tissue than was NBD-FITC. Taken together, introduction of cell permeability and colon targetability to NIP may be a feasible strategy for an orally active peptide therapy for treatment of IBD.

Enhanced therapeutic efficacy of budesonide in experimental colitis with enzyme/pH dual-sensitive polymeric nanoparticles

“…MPO activity was measured according to an established method. 11 Briefly, distal colon specimens were minced with a sharp scissor in 1 mL of hexadecyltrimethylammonium bromide (HTAB) buffer (0.5% in 50 mM phosphate buffer, pH 6.0) on ice and homogenized. HTAB was added to adjust the pooled homogenate concentration to 100 mg of tissue per milliliter, followed by sonication for 10 seconds, three freeze-thaw cycles, and centrifugation at 14,000 rpm at 4°C for 3 minutes.…”

“…10,11 Therefore, an ideal colon-specific delivery system for UC therapeutics should prevent premature drug release before reaching the colon and deliver sufficient amounts of the drug to the inflamed area of the distal colon. Fine tuning of the formulation may be necessary to account for longer transit times in the gut, as well as slightly higher pH values (around 7.3) in the distal small intestine.…”

Effects of the properties of short peptides conjugated with cell-penetrating peptides on their internalization into cells

“…However, this conventional method does have some problems: (1) purification of the target protein and construction of the assay system for evaluation of protein function are difficult; (2) peptide activity and/or the internalization ability of CPPs often change when conjugated to each other 19 ; (3) even if the function of the peptide is confirmed in vitro , it does not guarantee similar functioning in cells because of possible interference factors, including other intracellular molecules. To address these problems, it is important that a CPP-binding peptide library, assuring internalization into cells, is constructed before screening.…”

Colon-targeted delivery of piceatannol enhances anti-colitic effects of the natural product: potential molecular mechanisms for therapeutic enhancement

“… 25 To assess recovery of colonic injury, colonic tissue sections were subjected to hematoxylin-eosin (H&E) staining as described previously. 26 …”