Investigation of the Route of Absorption of Lipid and Sugar Modified Leu- Enkephalin Analogues and Their Enzymatic Stability Using the Caco-2 Cell Monolayer System

Abstract

It has been demonstrated that conjugation of lipoamino acids or glucose units to the endogenous opioid peptide, Leu-enkephalin can significantly improve the peptide's metabolic stability and absorption across biological barriers. The purpose of this study was to investigate the possible involvement of specific carrier proteins in the absorption of these peptide conjugates. A series of lipo- glycol- and liposaccharide peptide conjugates were synthesised and examined using the Caco-2 monolayer assay for evidence of interaction with the human H(+)-coupled oligopeptide transporter (hPepT1), glucose transporters and the multidrug resistance efflux pump, p-glycoprotein. The investigation involved determining the apparent permeability of each compound in the absence of any inhibitors and comparing this to the apparent permeabilities of each compound in the presence of glycylsarcosine, glucose or vinblastine, respective inhibitors of the above mentioned transporters. None of the peptide conjugates were found to be substrates for p-glycoprotein. Of the six peptide conjugates examined, only the C-terminus glucose conjugate of Leu-enkephalin (Enk-glu) showed evidence of transport by both glucose transporters and hPepT1. In contrast, N-terminus conjugation of both lipids and sugars appeared to provide the greatest protection against enzymatic degradation.

Converting Peptides into Drug Leads by Lipidation

“…Glycosylation compensates the low aqueous solubility of lipidated pepitdes and increases their membrane transportation through glucose transporters. Liposaccharide, the combination of LAA lipidation and glycosylation, is applied in the development of small-molecule drugs, peptides, vaccines and antibiotics [59,72,73,80,[150][151][152][153][154]. Hemolytic potency of liposaccharides was correlated with the chain length of LAA [72].…”

Tyr1-ψ[(Z)CF═CH]-Gly2 Fluorinated Peptidomimetic Improves Distribution and Metabolism Properties of Leu-Enkephalin

“…However, in these assays, minimal penetration in either the A → B or B → A directions discounted processes involving passive diffusion (see the Supporting Information for more details). 48,49 Further, considering that other enkephalin analogues are not substrates for P-gp, 49 we tentatively believe that 2 should also not be subject to efflux.…”

Peptides As Therapeutics with Enhanced Bioactivity

“…For example dermorphin (a natural opioid receptor agonist), bound by a cleavable disulfide bridge to OX26 (a monoclonal antibody that binds the transferrin receptor), was shown to produce analgesia, confirming that it passed the BBB [129]. Glycosylation of Leuenkephalin at the N-or C-termini enhanced transport profiles in a number of cases [102,146,147]. The permeation of the -anomer was 5-times greater than that of the -conformation [77].…”