In Vitro Membrane Permeation Studies and in Vivo Antinociception of Glycosylated Dmt¹-DALDA Analogues

Abstract

In this study the μ opioid receptor (MOR) ligands DALDA (Tyr-d-Arg-Phe-Lys-NH₂) and Dmt¹-DALDA (Dmt-d-Arg-Phe-Lys-NH₂, Dmt = 2',6'-dimethyltyrosine) were glycosylated at the N- or C-terminus. Subsequently, the modified peptides were subjected to in vitro and in vivo evaluation. In contrast to the N-terminally modified peptide (<b>3</b>), all peptide analogues derivatized at the C-terminus (<b>4</b>-<b>7</b>) proved to possess high affinity and agonist potency at both MOR and DOR (δ opioid receptor). Results of the Caco-2 monolayer permeation, as well as in vitro blood-brain barrier model experiments, showed that, in the case of compound <b>4</b>, the glycosylation only slightly diminished the lumen-to-blood and blood-to-lumen transport. Altogether, these experiments were indicative of transcellular transport but not active transport. In vivo assays demonstrated that the peptides were capable of (i) crossing the blood-brain barrier (BBB) and (ii) activating both the spinal ascending as well as the descending opioid pathways, as determined by the tail-flick and hot-plate assays, respectively. In contrast to the highly selective MOR agonist Dmt¹-DALDA <b>1</b>, compounds <b>4</b>-<b>7</b> are mixed MOR/DOR agonists, expected to produce reduced opioid-related side effects.