Characterization of the physicochemical interactions between exenatide and two intestinal permeation enhancers: Sodium caprate (C10) and salcaprozate sodium (SNAC).

A common approach to tackle the poor intestinal membrane permeability of peptides after oral administration is to formulate them with a permeation enhancer (PE). Increased oral bioavailability for oral peptide candidates has been reported from clinical trials when either salcaprozate sodium (SNAC) or sodium caprate (C10) is incorporated in the formulation. However, little is known about how they physically interact with peptides in solution. Our objective was to compare the biophysical interactions between the GLP-1 analogue exenatide (Byetta®, Lilly), and C10 or SNAC using a variety of advanced analytical techniques. First, critical micelle concentration was measured in different buffers for both PEs. Dynamic light scattering (DLS) measurements revealed specific supramolecular structures arising from exenatide-PE association. Surface plasmon resonance (SPR) indicated the formation of exenatide-PE complexes with a high contribution from non-specific interactions and rapid binding kinetics, resulting in overall low affinities. DLS and isothermal titration calorimetry (ITC) were used to examine the supramolecular organization of the PEs, and revealed thermodynamic signatures characterized by unfavourable enthalpic contributions compensated by favourable entropic ones, but with low-affinity estimates in water (KD in the 10-100 µM range). With affinity capillary electrophoresis (ACE), weak interactions between exenatide and SNAC or C10 were confirmed in saline, with a dissociation constant around 10 µM and 30 µM respectively. In biorelevant intestinal media, the bile salts in FaSSIF and FeSSIF further reduced the binding of both agents to exenatide (KD ≈ 100 µM), indicating that the interaction between the PEs and exenatide might be inhibited by bile salts in the GI lumen. This study suggests that the interactions of both PEs with exenatide follow a similar non-covalent mechanism and are of low affinity.

Comparison of the effects of the intestinal permeation enhancers, SNAC and sodium caprate (C10): Isolated rat intestinal mucosae and sacs.

SNAC and C10 are intestinal permeation enhancers (PEs) used in formulations of peptides for oral delivery in clinical trials. Our aims were to compare their: (i) mechanism of action in isolated rat intestinal mucosae mounted in Ussing chambers and in non-everted gut sacs, (ii) effects on mucosa integrity in those models and also in in situ intra-jejunal instillations and (iii) interactions with intestinal mucus. SNAC increased the apparent permeability coefficient (Papp) of the paracellular marker, FITC-dextran 4000 (FD4), across isolated rat gastric mucosae in concentration-dependent fashion, whereas C10 did not, while both reduced the transepithelial electrical resistance (TEER). In isolated jejunal and colonic mucosae, both agents increased the Papp of [14C]-mannitol and FD4 whereas C10 but not SNAC reduced TEER. 20 mM SNAC was required to achieve the efficacy of 10 mM C10 in jejunal and colonic mucosae. In isolated non-everted jejunal and colonics sacs, FD4 flux increases were observed in the presence of both PEs. Histology of mucosae revealed that both PEs induced minor epithelial damage to the mucosa at concentrations that increased fluxes. Jejunal tissue withstood epithelial damage in the following order: intra jejunal in situ instillations > jejunal sacs > isolated jejunal mucosae. Both PEs modulated viscoelastic properties of porcine jejunal mucus without altering rheological properties. In conclusion, SNAC and C10 are reasonably efficacious PEs in rat intestinal tissue with common overall mechanistic features. Their potency and toxic potential are low, in agreement with clinical trial data.

A head-to-head Caco-2 assay comparison of the mechanisms of action of the intestinal permeation enhancers: SNAC and sodium caprate (C10).

Salcaprozate sodium (SNAC) and sodium caprate (C10) are the two leading intestinal permeation enhancers (PEs) in oral peptide formulations in clinical trials. There is debate over their mechanism of action on intestinal epithelia. The aims were: (i) to compare their effects on the barrier function by measuring transepithelial electrical resistance (TEER), permeability of FITC-4000 (FD4) across Caco-2 monolayers, and on immunohistochemistry of tight junction (TJ)-associated proteins; and (ii) to compare cellular parameters using conventional end-point cytotoxicity assays and quantitative high content analysis (HCA) of multiple sub-lethal parameters in Caco-2 cells. C10 (8.5 mM) reversibly reduced TEER and increased FD4 permeability across monolayers, whereas SNAC had no effects on either parameter except at cytotoxic concentrations. C10 exposure induced reorganization of three TJ proteins, whereas SNAC only affected claudin-5 localization. High concentrations of C10 and SNAC were required to cause end-point toxicology changes in vitro. SNAC was less potent than C10 at inducing lysosomal and nuclear changes and plasma membrane perturbation. In parallel, HCA revealed that both agents displayed detergent-like features that reflect initial membrane fluidization followed by changes in intracellular parameters. In conclusion, FD4 permeability increases in monolayers in response to C10 were in the range of concentrations that altered end-point cytotoxicity and HCA parameters. For SNAC, while HCA parameters were also altered in a similar overall pattern as C10, they did not lead to increased paracellular flux. These assays show that both agents are primarily surfactants, but C10 has additional TJ-opening effects. While these in vitro assays illucidate their epithelial mechanism of action, clinical experience suggests that they over-estimate their toxicology in the dynamic intestinal environment.

The UCD nanosafety workshop (03 December 2018): towards developing a consensus on safe handling of nanomaterials within the Irish university labs and beyond - a report.

Careful handling of the nanomaterials (NMs) in research labs is crucial to ensure a safe working environment. As the largest university in Ireland, University College Dublin (UCD) has invested significant resources to update researchers working with NMs. Due to sizes often <100 nm, the NMs including nanoparticles, harbor unprecedented materialistic properties, for example, enhanced reactivity, conductivity, fluorescence, etc. which albeit conferring the NMs an edge over bulk materials regarding the applied aspects; depending on the dose, also render them to be toxic. Thus, a set of regulatory guidelines have emerged regarding safe handling of the NMs within occupational set-ups. Unfortunately, the current regulations based on the toxic chemicals and carcinogens are often confusing, lack clarity, and difficult to apply for the NMs. As a research-intensive university, a diverse range of research activities occur within the UCD labs, and it is difficult, at times impossible, for the UCD Safety, Insurance, Operational Risk & Compliance (SIRC) office to develop a set of common guidelines and cater throughout all its labs conducting research with the NMs. Hence, a necessity for dialog and exchange of ideas was felt across the UCD which encouraged the researchers including early stage researchers (e.g. PhDs, Postdocs) from multiple schools to participate in a workshop held on the 03 December 2018. The workshop tried to follow a pragmatic approach, where apart from discussing both the in vitro and in vivo scenarios, practical cases simulating situations faced frequently in the labs were discussed. This report summarizes the findings made during the workshop by this emerging critical mass in UCD.

Intestinal Permeation Enhancers for Oral Delivery of Macromolecules: A Comparison between Salcaprozate Sodium (SNAC) and Sodium Caprate (C10).

Salcaprozate sodium (SNAC) and sodium caprate (C10) are two of the most advanced intestinal permeation enhancers (PEs) that have been tested in clinical trials for oral delivery of macromolecules. Their effects on intestinal epithelia were studied for over 30 years, yet there is still debate over their mechanisms of action. C10 acts via openings of epithelial tight junctions and/or membrane perturbation, while for decades SNAC was thought to increase passive transcellular permeation across small intestinal epithelia based on increased lipophilicity arising from non-covalent macromolecule complexation. More recently, an additional mechanism for SNAC associated with a pH-elevating, monomer-inducing, and pepsin-inhibiting effect in the stomach for oral delivery of semaglutide was advocated. Comparing the two surfactants, we found equivocal evidence for discrete mechanisms at the level of epithelial interactions in the small intestine, especially at the high doses used in vivo. Evidence that one agent is more efficacious compared to the other is not convincing, with tablets containing these PEs inducing single-digit highly variable increases in oral bioavailability of payloads in human trials, although this may be adequate for potent macromolecules. Regarding safety, SNAC has generally regarded as safe (GRAS) status and is Food and Drug Administration (FDA)-approved as a medical food (Eligen®-Vitamin B12, Emisphere, Roseland, NJ, USA), whereas C10 has a long history of use in man, and has food additive status. Evidence for co-absorption of microorganisms in the presence of either SNAC or C10 has not emerged from clinical trials to date, and long-term effects from repeat dosing beyond six months have yet to be assessed. Since there are no obvious scientific reasons to prefer SNAC over C10 in orally delivering a poorly permeable macromolecule, then formulation, manufacturing, and commercial considerations are the key drivers in decision-making.