Esterase-Sensitive Cyclic Prodrugs of Peptides: Evaluation of an Acyloxyalkoxy Promoiety in a Model Hexapeptide

Abstract

Purpose. To evaluate a cyclic acyloxyalkoxycarbamate prodrug of a model hexapeptide (H-Trp-Ala-Gly-Gly-Asp-Ala-OH) as a novel approach to enhance the membrane permeation of the peptide and stabilize it to metabolism.

Methods. Conversion to the linear hexapeptide was studied at 37°C in aqueous buffered solutions and in various biological milieus having measurable esterase activities. Transport and metabolism characteristics were assessed using the Caco-2 cell culture model.

Results. In buffered solutions the cyclic prodrug degraded chemically to the linear hexapeptide in stoichiometric amounts. Maximum stability was observed between pH 3–4. In 90% human plasma (t _1/2 = 100 ± 4 min) and in homogenates of the rat intestinal mucosa (t _- = 136 ± 4 min) and rat liver (t _- = 65 ± 3 min), the cyclic prodrug disappeared faster than in buffered solution, pH 7.4 (t _- = 206 ± 11 min). Pretreatment of these media with paraoxon significantly decreased the degradation rate of the prodrug. When applied to the apical side of Caco-2 cell monolayers, the cyclic prodrug (t _- = 282 ± 25 min) was significantly more stable than the hexapeptide (t _- = 14 min) and at least 76-fold more able to permeate (P _app = 1.30 ± 0.15 × 10⁻⁷ cm/ s) than the parent peptide (P _app ≤ 0.17 × 10⁻⁸ cm/s).

Conclusions. Preparation of a cyclic peptide using an acyloxyalkoxy promoiety reduced the lability of the peptide to peptidase metabolism and substantially increased its permeation through biological membranes. In various biological media the parent peptide was released from the prodrug by an apparent esterase-catalyzed reaction, sensitive to paraoxon inhibition.