Evaluation of Cell-Penetrating Peptides as Versatile, Effective Absorption Enhancers: Relation to Molecular Weight and Inherent Epithelial Drug Permeability.

PURPOSE: The poor permeability of new drug candidates across intestinal epithelial membranes complicates their development in oral form. This study investigated the potential of cell-penetrating peptides (CPPs) to improve the intestinal permeation and absorption of low-permeable low-molecular-weight (low-MW) drugs. METHODS: The in vitro epithelial permeation of six different drugs (metformin, risedronate, zanamivir, methotrexate [MTX], tacrolimus, and vincristine [VCR]) across Caco-2 cell monolayers was examined in the presence and absence of L- or D-penetratin, and the correlation between permeation enhancement efficiency and the properties of tested drugs was analyzed. In addition, a rat closed ileal loop absorption study was conducted to determine the in vivo effects of penetratin. RESULTS: MTX and VCR efficiently permeated Caco-2 monolayers in the presence of L- and D-penetratin, suggesting that CPPs enhanced the epithelial permeation of drugs with relatively high molecular weight and resultant limited intrinsic permeability. The in vivo rat closed ileal loop absorption study revealed the stimulatory effect of L- and D-penetratin on the intestinal absorption of MTX and VCR. CONCLUSIONS: CPPs are useful as oral absorption enhancers for low-permeable drugs.

Effects of intestinal luminal contents and the importance of microfold cells on the ability of cell-penetrating peptides to enhance epithelial permeation of insulin.

We previously reported that oral and intestinal absorption of insulin in rats and mice is significantly enhanced in vivo by coadministration with cell-penetrating peptides (CPPs). To evaluate the clinical use of CPPs as absorption enhancers, it is imperative to clarify the mechanisms associated with the permeation-stimulatory effect of CPPs in vitro. The confirmation experiment revealed a discrepancy between in vivo and in vitro effects of CPPs, such as D-octaarginine (D-R8) and L-penetratin, on epithelial permeation of insulin. The present study was designed to determine the factors that work in vivo but are deficient in an in vitro system consisting of Caco-2 cells. The effects of D-R8 and L-penetratin on permeation of insulin through the Caco-2 cell monolayer were partially boosted in fasted-state simulated intestinal fluid (FaSSIF). Contrary to expectation, the effects of CPPs on cellular uptake of insulin and the binding ratio of CPPs to insulin analyzed by surface plasmon resonance in normal buffer and FaSSIF were similar. Also, the effects of CPPs, especially D-R8, on cellular uptake of insulin, were stronger in Caco-2 cell monolayers with microfold cell (M cell)-like properties. These results suggested a key role of intestinal lipids and M cells in the stimulatory effect of CPPs on net epithelial permeation of insulin in vivo.

Hydrophobic Amino Acid Tryptophan Shows Promise as a Potential Absorption Enhancer for Oral Delivery of Biopharmaceuticals.

Cell-penetrating peptides (CPPs) have great potential to efficiently deliver drug cargos across cell membranes without cytotoxicity. Cationic arginine and hydrophobic tryptophan have been reported to be key component amino acids for cellular internalization of CPPs. We recently found that l-arginine could increase the oral delivery of insulin in its single amino acid form. Therefore, in the present study, we evaluated the ability of another key amino acid, tryptophan, to enhance the intestinal absorption of biopharmaceuticals. We demonstrated that co-administration with l-tryptophan significantly facilitated the oral and intestinal absorption of the peptide drug insulin administered to rats. Furthermore, l-tryptophan exhibited the ability to greatly enhance the intestinal absorption of other peptide drugs such as glucagon-like peptide-1 (GLP-1), its analog Exendin-4 and macromolecular hydrophilic dextrans with molecular weights ranging from 4000 to 70,000 g/mol. However, no intermolecular interaction between insulin and l-tryptophan was observed and no toxic alterations to epithelial cellular integrity-such as changes to cell membranes, cell viability, or paracellular tight junctions-were found. This suggests that yet to be discovered inherent biological mechanisms are involved in the stimulation of insulin absorption by co-administration with l-tryptophan. These results are the first to demonstrate the significant potential of using the single amino acid l-tryptophan as an effective and versatile bioavailability enhancer for the oral delivery of biopharmaceuticals.