Esterase-Sensitive Cyclic Prodrugs of Peptides: Evaluation of a Phenylpropionic Acid Promoiety in a Model Hexapeptide

Abstract

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

Methods. Conversion to the linear hexapeptide was studied at 37°C in HBSS, pH 7.4, and in various biological milieus having measurable esterase activities. Transport and metabolism characteristics were assessed using the Caco-2 cell culture model.

Results. In aqueous buffered solution, pH 7.4, the cyclic prodrug degraded quantitatively (t _1/2 = 1795 ± 289 min) to the linear hexapeptide and the lactone. Substantially faster degradation of the cyclic prodrug was observed in 90% human plasma (t _1/2 = 508 ± 24 min), and in homogenates of Caco-2 cells (t _1/2 = 940 ± 13 min), the rat intestinal mucosa (t _1/2 = 1286 ± 32 min), and rat liver (t _1/2 = 840 ± 42 min). Pretreatment of these biological 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 was significantly more stable than the hexapeptide and at least 71-fold more able to permeate (P_app = 1.21 ± 0.12 × 10⁻⁷ cm/s) than was the parent peptide (P_app ≤ 0.17 × 10⁻⁸ cm/s). In the presence of 0.1 mM palmitoyl-DL-carnitine, the transport rate of the cyclic prodrug (P_app = 2.19 × 10⁻⁶ cm/s) was 1250-fold greater than that of the linear hexapeptide.

Conclusions. Preparation of a cyclic peptide using a phenylpropionic acid 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.