Chemical and enzymatic stability as well as transport properties of a Leu-enkephalin analogue and ester prodrugs thereof

Abstract The Leu-enkephalin analogue (Tyr- d -Ala-Gly-Phe-Leu-NH 2 ) was synthesized together with three esters prodrugs hereof. The prodrugs synthesized were the O-acetyl , O-propionyl and O-pivaloyl esters of the tyrosine phenolic group. The compounds were isolated in good purity (HPLC purity >99%) and in good yields (60–75%). The chemical and enzymatic stability of the prodrugs has been investigated in detail. The prodrugs studied are quite chemically stable and the degradation of the prodrugs follows the pattern previously shown for similar esters (U-shaped pH-profile; maximal stability at pH 4–5). The prodrugs are degraded quantitatively in plasma to the parent peptide with half-lives in the range 2.9 min–2.6 h. Type B esterases were shown to be involved in the degradation as the half-lives increased in the presence of paraoxon. No significant stabilization was seen in 10% porcine gut homogenate. Half-lives in the same order were seen for the analogue and the prodrugs in pure Leucine aminopeptidase solution. The analogue was stable in Carboxypeptidase A solution whereas a faster degradation of the prodrugs was seen in this media. Furthermore the transport properties of the compounds has been studied. A P app value of 0.284×10 −6 cm/s for the analogue was obtained for the transport across Caco-2 cell monolayers in the BL-AP direction. The P app values were increased by a factor of 2, 7 and 18 for the acetyl-, propionyl- and pivaloylprodrug. The increase could be explained by higher lipofilicities of the prodrugs compared to the analogue.