This study investigated enteric nervous system (ENS) involvement in intestinal secretion induced by cyclic GMP-dependent secretagogues. The investigation was based upon the study of transepithelial ion transport in rat small and large intestine and in guinea pig caecum using voltage-clamped in vitro preparations mounted in Ussing chambers. ENS participation was established from the use of neural blocking agents (tetrodotoxin (TTX), bicuculline and capsaicin). The relative contribution of the myenteric plexus was assessed by selectively stripping tissues of the longitudinal muscle layer. All tissues, both unstripped and stripped, responded to <I>Escherichia coli </I>STa enterotoxin, guanylin and the nitric oxide (NO) donor sodium nitroprusside (SNP) with a dose-dependent increase in inward short circuit current (I_SC). Regarding STa/guanylin, TTX inhibited this I_SCin unstripped rat distal colon, ileum and guinea pig caecum, demonstrating that the ENS plays an important role in these tissues. In rat distal colon, TTX induced an abolition of the STa/guanylin response in both preparations, indicating submucous plexus involvement. In rat proximal colon there was no discernible TTX-sensitive component observed. The ileum displayed partial control from both the myenteric and submucous plexuses, whereas the caecum exhibited partial control from the myenteric plexus alone. Bicuculline inhibited STa action to a significant degree in the caecum while capsaicin inhibited secretion in the proximal colon. In rat small intestine, the SNP-induced I_SC was inhibited by TTX in both unstripped and stripped tissues. In contrast, inhibitory pathways were shown to exist in distal colon exposed to SNP, TTX revealing an enhancement of SNP-induced secretion in the stripped preparation. Thus, although there is clear involvement of the ENS in the actions of STa/guanylin and SNP, it is not possible to make a general statement regarding its contribution throughout the length of the alimentary canal due to the extent of inter segmental and inter species variations.

Protein therapeutics offer numerous advantages over small molecule drugs and are rapidly becoming one of the most prominent classes of therapeutics. Unfortunately, they are delivered almost exclusively by injection due to biological obstacles preventing high bioavailability via the oral route. In this work, numerous approaches to overcoming these barriers are explored. PH-Responsive poly(itaconic acid-co-N-vinylpyrrolidone) (P(IA-co-NVP)) hydrogels were synthesized, and the effects of monomer ratios, crosslinking density, microparticle size, protein size, and loading conditions were systematically evaluated using in vitro tests. P(IA-co-NVP) hydrogels demonstrated up to 69% greater equilibrium swelling at neutral conditions than previously-studied poly(methacrylic acid-co-N-vinylpyrrolidone) hydrogels and a 10-fold improvement in time-sensitive swelling experiments. Furthermore, P(IA-co-NVP) hydrogel microparticles demonstrated up to a 2.7-fold improvement in delivery of salmon calcitonin (sCT) compared to methacrylic acid-based systems, with a formulation comprised of a 1:2 ratio of itaconic acid to N-vinylpyrrolidone demonstrating the greatest delivery capability. Vast improvement in delivery capability was achieved using reduced ionic strength conditions during drug loading. Use of a 1.50 mM PBS buffer during loading yielded an 83-fold improvement in delivery of sCT compared to a standard 150 mM buffer. With this improvement, a daily dose of sCT could be provided using P(IA-co-NVP) microparticles in one standard-sized gel capsule. P(IA-co-NVP) was also tested with larger proteins urokinase and Rituxan. Crosslinking density provided a facile method for tuning hydrogels to accommodate a wide range of protein sizes. The effects of protein PEGylation were also explored. PEGylated sCT displayed lower release from P(IA-co-NVP) microparticles, but displayed increased apparent permeability across a Caco-2 monolayer by two orders of magnitude. Therefore, PEG-containing systems could yield high bioavailability of orally delivered proteins. Finally, a modified SELEX protocol for cellular selection of transcellular transport-initiating aptamers was developed and used to identify aptamer sequences showing enhanced intestinal perfusion. Over three selection cycles, the selected aptamer library showed significant increases in absorption, and from an initial library of 1.1 trillion sequences, 5-10 sequences were selected that demonstrated up to 10-fold amplification compared to the naïve library. These sequences could provide a means of overcoming the significant final barrier of intestinal absorption. / text