lontophoretic Delivery of a Series of Tripeptides Across the Skin in Vitro

The iontophoresis of eight tripeptides, of the general structure alanine–X–alanine, has been measured across hairless mouse skin in vitro. The peptides were blocked (a) at the carboxyl terminus using the mixed anhydride reaction with t-butylamine and (b) at the amino terminus by acetylation with ¹⁴C-acetic anhydride. The nature of the central residue (X) was varied by selecting one of five neutral amino acids, two negatively chargeable moieties (aspartic and glutamic acids), and a positively chargeable species (histidine). Constant current iontophoresis at 0.36 mA/cm², using Ag/AgCl electrodes, was performed for 24 hr in diffusion cells, which allowed both anode and cathode to be situated on the same (epidermal) side of a single piece of skin. Due to a combination of osmotic and electroosmotic forces, the anodal iontophoretic flux of neutral peptides was significantly greater than passive transport. Steady-state fluxes were not achieved, however, suggesting that time-dependent changes in the properties of the skin barrier may be occurring. Limited, further experiments confirmed that, on a 24-hr time scale, these changes were not fully reversible. The cathodal delivery of anionic permeants was well controlled at a steady and highly enhanced rate by the current flow. This behavior closely paralleled earlier work using simple negatively charged amino acids and N-acetylated amino acid derivatives. It appears that the normalized iontophoretic flux of these anionic species is independent of lipophilicity but may be inversely related to molecular weight. The positively charged peptide, Ac–Ala–His–Ala–NH(Bu^t), showed greater anodal iontophoretic enhancement when delivered from a donor solution at pH 4.0 than from a solution at pH 7.4. This was consistent with (a) the corresponding behavior of histidine alone and (b) the existence of a pK_a for these compounds at 6. Steady-state delivery was not achieved, although the levels of enhancement, especially at pH 4, were the largest observed. A preliminary investigation of tripeptide stability to either (i) electrolysis in the donor compartment or (ii) cutaneous metabolism revealed very little degradation under the conditions of the experiment. Overall, this research supports the principle of enhanced peptide delivery across the skin by iontophoresis and indicates a number of areas (e.g., mechanism and extent of current-induced changes in skin barrier function, molecular size dependence, pathways of current flow) on which further work should be focused.