Structural requirements of penetratin absorption enhancement efficiency for insulin delivery

Penetratin, a 16-residue peptide, is used widely as a highly efficient delivery carrier for a wide range of poorly permeable therapeutic cargoes. The crucial structural features of penetratin remain unclear, as demonstrated by the difficulties encountered in designing new molecules. The efficiency in enhancing nasal insulin absorption was compared between l-penetratin and 20 of its analogues in rats. We also measured lactate dehydrogenase (LDH) leakage as an indicator of cytotoxicity and scored the histopathological irritation. Substitution of a cationic residue (Arg or Lys) with Leu or addition of tetra-arginine to the C- or N-terminus of penetratin caused considerable reduction in the enhancing efficiency properties of the modified analogues. Mutual exchanging of Arg and Lys in corresponding analogues produced nearly inactive analogues, although changing Arg to Lys in the same analogue produced similar penetratin activity. In addition, activity was impaired markedly upon modification of penetratin within amphiphilic (Trp) or hydrophobic (Ile and Phe) residues. Chain size-modified analogues lacked the ability to induce nasal insulin absorption. In contrast, rearrangement of the modified analogues by C,N-half-exchange and reverse analogues produced activity similar to that of the original penetratin. The enhancing activity was inhibited almost completely upon sequence arrangement of the resulting analogues. Surprisingly, a shuffle (Arg, Lys fix) 2 analogue increased insulin absorption significantly, reaching a relative bioavailability value 1.85-times that of original penetratin. This analogue caused negligible release of LDH in nasal lavage fluid and maintained the integrity of the nasal respiratory epithelium. In conclusion, modulation of amino acid sequences by fixing the cationic residue positions can augment penetratin-enhanced nasal absorption and may lead to improvements in nasal insulin absorption.