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

Abstract

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.

Keywords: Excipient cytotoxicity; Intestinal permeation enhancers; Oral peptide delivery; SNAC; Sodium caprate; Tight junctions.