ABSTRACT
Endomorphin 1 (Endo-1=Tyr-Pro-Trp-Phe-NH(2)), an endogenous opioid with high affinity and selectivity for mu-opioid receptors, mediates acute and neuropathic pain in rodents. To overcome metabolic instability and poor membrane permeability, the N- and C-termini of Endo-1 were modified by lipoamino acids (Laa) and/or sugars, and 2',6'-dimethyltyrosine (Dmt) replacement of Tyr. Analogues were assessed for mu-opioid receptor affinity, inhibition of cAMP accumulation, enzymatic stability, and permeability across Caco-2 cell monolayers. C-terminus modification decreased receptor affinity, while N-terminus C8-Laa improved stability and permeability with slight change in receptor affinity. Dmt provided a promising lead compound: [C8Laa-Dmt[1]]-Endo-1 is nine times more stable (t(1/2)=43.5min), >8-fold more permeable in Caco-2 cell monolayers, and exhibits 140-fold greater mu-opioid receptor affinity (K(imu)=0.08nM).
Subject(s)
Oligopeptides/chemical synthesis , Oligopeptides/metabolism , Peptide Library , Animals , Biological Availability , Caco-2 Cells , Caprylates/chemical synthesis , Cell Line, Tumor , Cell Membrane Permeability , Glycosylation , Humans , Hydroxylation , Lauric Acids/chemical synthesis , Lipid Metabolism , Peptide Fragments/metabolism , RatsABSTRACT
Caco-2 cell permeability and stability assays were used as an in vitro model to study the intestinal epithelial transport and stability of two analogues of thyrotropin-releasing hormone (TRH; Pyr-His-Pro-NH2). Peptide 1 (Pyr-His-Pro-D-glucopyranuronamide) was more permeable across the Caco-2 cell monolayer compared with the permeability of the parent TRH peptide (Papp=5.10+/-1.89x10(-6) cm/s c.f. Papp=0.147+/-0.0474x10(-6) cm/s respectively). The permeability of peptide 1 was improved threefold by attaching a 2-aminooctanoic acid moiety to the N-terminus to form peptide 2 (2-aminooctanoic acid-Gln-His-Pro-D-glucopyranuronamide) (Papp=16.3+/-2.47x10(-6) cm/s). The half-life for both peptide 1 and peptide 2 was approximately 20 min in a homogenate of Caco-2 cells compared with the half-life of TRH which is approximately 3 min. It was concluded that the permeability of peptides 1 and 2 was enhanced because of their increased stability, while the higher permeability of peptide 2 compared with peptide 1 may be attributed to its increased lipophilicity which results in enhanced passive diffusion.
