Intact penetratin metabolite permeates across Caco-2 monolayers

TY - CONF

T1 - Intact penetratin metabolite permeates across Caco-2 monolayers

AU - Birch, Ditlev

AU - Christensen, Malene Vinther

AU - Stærk, Dan

AU - Franzyk, Henrik

AU - Nielsen, Hanne Mørck

PY - 2015

Y1 - 2015

N2 - Purpose Peptide and protein-based therapeutics often show high potency and selectivity towards a target when compared to small-molecule therapeutics. However, due to the hydrophilic nature and size of these drugs, the passage across the mucosal membranes of the body is often highly restricted. Previous studies have demonstrated that cell-penetrating peptides (CPPs) may be used as carriers in order to improve the bioavailability of a therapeutic cargo like insulin after oral administration. Penetratin, a commonly used CPP, has been shown to increase the uptake of insulin across Caco-2 cell monolayers. The carrier peptide itself is, however, degraded by peptidases in the brush border as well as inside the cells and so far not much attention have been given to the properties of the metabolites of penetratin and their effects on the biological membrane and the delivery of the cargo. Therefore, the aim of the present study was to investigate penetratin metabolites with respect to effects on cellular viability, their epithelial permeation and cell uptake. Methods Extracellular and intracellular degradation of penetratin was assessed by incubation of the carrier peptide on the apical side of Caco-2 cells cultured on permeable filter inserts and in cell lysates, respectively. The epithelial permeation of penetratin and the formed metabolites was assessed by using Caco-2 monolayers cultured on permeable filter inserts. Results Preliminary data revealed that at least one specific metabolite is formed upon both intracellular and extracellular degradation of penetratin (figure 1A). Following incubation with epithelium for 4 hours, the metabolite permeated the Caco-2 monolayer and the concentration increased approximately 10-fold when compared to a sample collected following 15 minutes of incubation (figure 1B). Figure 1: A) Formation of the penetratin metabolite following extracellular (open circles) and intracellular (closed circles) degradation of 10 µM penetratin. B) Permeation of the penetratin metabolite across Caco-2 monolayers during 4 hours after exposure to 10 µM penetratin (open circles) or 100 µM penetratin (closed circles). Mean ± SEM, n = 3-6. Conclusion At least one specific metabolite formed following extracellular and intracellular degradation of penetratin permeates across Caco-2 monolayers with a rate depending on the formation of the metabolite. The formation of the metabolite occurs more rapidly intracellularly than extracellularly. Further studies on the structure in relation to cytotoxicity, permeation and uptake of the metabolite will reveal the importance of these findings and how they may be applied for drug delivery purposes.

AB - Purpose Peptide and protein-based therapeutics often show high potency and selectivity towards a target when compared to small-molecule therapeutics. However, due to the hydrophilic nature and size of these drugs, the passage across the mucosal membranes of the body is often highly restricted. Previous studies have demonstrated that cell-penetrating peptides (CPPs) may be used as carriers in order to improve the bioavailability of a therapeutic cargo like insulin after oral administration. Penetratin, a commonly used CPP, has been shown to increase the uptake of insulin across Caco-2 cell monolayers. The carrier peptide itself is, however, degraded by peptidases in the brush border as well as inside the cells and so far not much attention have been given to the properties of the metabolites of penetratin and their effects on the biological membrane and the delivery of the cargo. Therefore, the aim of the present study was to investigate penetratin metabolites with respect to effects on cellular viability, their epithelial permeation and cell uptake. Methods Extracellular and intracellular degradation of penetratin was assessed by incubation of the carrier peptide on the apical side of Caco-2 cells cultured on permeable filter inserts and in cell lysates, respectively. The epithelial permeation of penetratin and the formed metabolites was assessed by using Caco-2 monolayers cultured on permeable filter inserts. Results Preliminary data revealed that at least one specific metabolite is formed upon both intracellular and extracellular degradation of penetratin (figure 1A). Following incubation with epithelium for 4 hours, the metabolite permeated the Caco-2 monolayer and the concentration increased approximately 10-fold when compared to a sample collected following 15 minutes of incubation (figure 1B). Figure 1: A) Formation of the penetratin metabolite following extracellular (open circles) and intracellular (closed circles) degradation of 10 µM penetratin. B) Permeation of the penetratin metabolite across Caco-2 monolayers during 4 hours after exposure to 10 µM penetratin (open circles) or 100 µM penetratin (closed circles). Mean ± SEM, n = 3-6. Conclusion At least one specific metabolite formed following extracellular and intracellular degradation of penetratin permeates across Caco-2 monolayers with a rate depending on the formation of the metabolite. The formation of the metabolite occurs more rapidly intracellularly than extracellularly. Further studies on the structure in relation to cytotoxicity, permeation and uptake of the metabolite will reveal the importance of these findings and how they may be applied for drug delivery purposes.

M3 - Poster

T2 - Annual Meeting of the American Association of Pharmaceutical Sciences

Y2 - 25 October 2015 through 29 October 2015

ER -