Synthesis and characterization of an acylated di-peptide (Myr-Trp-Leu) with modified transmucosal transport properties.

In order to improve the buccal absorption of a dipeptide model compound, Tryptophan-Leucine (Trp-Leu), we have synthesised a lipophilic derivative by myristoylation of the N- terminal amino group of Trp-Leu. The acylated peptide (Myr-Trp-Leu) was characterized by HPTLC, purified and isolated by chromatography on a silica gel column. Its structure was confirmed by (13)C and (1)H NMR and mass spectroscopy. The increased lipophilicity of the Myr-Trp-Leu was compared to that of the native peptide by chromatography and by its partition coefficient between n-octanol and saline phosphate buffer. In addition, the sensitivity towards hydrolytic enzymes was studied. The interaction of Trp-Leu with liposomes as model membranes was also studied. The phase transition temperature of dipalmitoylphosphatidylcholine (DPPC) was lowered in the presence of Myr-Trp-Leu, while it was increased in the presence of native parent peptide. Permeation experiments performed in vitro with pig buccal mucosa showed that the Myr-Trp-Leu accumulated in the tissue at the various concentrations tested. In contrast, the native peptide was able to pass through the membrane at all concentrations used. Lipophilic modification of the peptide by acylation drastically changes its behaviour towards tissue systems.

Factors and strategies for improving buccal absorption of peptides.

Peptides and polypeptides have important pharmacological properties but only a limited number (e.g. insulin, oxytocin, vasopressin) have been exploited as therapeutics because of problems related to their delivery. The buccal mucosa offers an alternative route to conventional, parenteral administration. Peptides are generally not well absorbed through mucosae because of their molecular size, hydrophilicity and the low permeability of the membrane. Peptide transport across buccal mucosa occurs via passive diffusion and is often accompanied by varying degrees of metabolism. This review describes various approaches to improve the buccal absorption of peptides including the use of penetration enhancers to increase membrane permeability and/or the addition of enzyme inhibitors to increase their stability. Other strategies including molecular modification with bioreversible chemical groups or specific formulations such as bioadhesive delivery systems are also discussed.

Permeation of a myristoylated dipeptide across the buccal mucosa: topological distribution and evaluation of tissue integrity.

The ex vivo permeation of an acylated model dipeptide, Myristoyl-Tryptophan-Leucine (Myr-Trp-Leu) was studied using pig buccal mucosa. Myr-Trp-Leu, being lipophilic, did not readily penetrate across the membrane. Rather, it accumulated in the epithelial and connective tissue of the mucosal barrier. The topological distribution of Myr-Trp-Leu across the mucosa, following its application in ethanol/phosphate buffer (30/70 pH 7.4), was determinated by thin-sectioning of the tissue, extraction of the peptide, and high performance thin layer chromatography (HPTLC). The concentration profile depended, of course, on the duration of the experiment and appeared to be dependent upon the presence of sufficient ethanol in order that the peptide could be solubilized. This important role for ethanol then raised the question of the solvent's effect on tissue integrity. Light microscopic examination of the mucosa was, therefore, undertaken, under identical conditions to those used in the permeation experiments, to evaluate any perturbation induced by the ethanolic vehicle. No obvious effects were observed.