Morphine, and its congeners are the most widely prescribed drugs for moderate to severe pain. These “narcotics” produce an array of side effects, including pruritus, miosis, euphoria, sedation, constipation, and respiratory depression. Since their discovery in 1975, much effort has been expended to convert endogenous opioid peptides Met/Leu-enkephalin into analgesics with the hope that they would provide pain relief with reduced side effects. To date, we are aware of only one report of an enkephalin-based peptide being tested as an analgesic in humans. The compound produced potent and fully efficacious analgesia in severe pain states, but required intrathecal administration.1
Many peptide analogs have been synthesized having greatly improved resistance to enzyme degradation and corresponding increases in plasma stability and serum half-life.2,3
The majority of these metabolically stable analogs fail to cross blood-brain barrier (BBB) in appreciable amounts. The BBB is both a physical and metabolic barrier between the CNS and the systemic circulation, which serves to regulate and protect the microenvironment of the brain.4
The BBB includes tight junctions between the endothelial cells of brain capillaries that behave as a continuous lipid bilayer.5
Glycosylation is a post-translational protein modification, which has been shown to increase metabolic stability,6
and has been shown to promote BBB penetration of enkephalins to produce CNS activity after peripheral administration in rodents.7,8
In some cases these glycosides have been shown to produce antinociceptive effects with greatly reduced side effects compared to traditional small molecules.9,10
Circular dichroism (CD) and nuclear Overhauser studies (NOESY) of these glycosylated enkephalins revealed that they adopt restricted and relatively well-defined conformational ensembles in sodium dodecylsulfate (SDS) micelles, whereas they form predominantly random coil conformations in H2
In a related study it was shown that glycosylation of the highly MOR selective peptide Tyr-(D)Ala-NMePhe-Gly(OH) (“DAMGO”) improved bioavailability and in vivo
These MOR selective analogs showed greater potencies than morphine. Theoretically calculated amphipathicities of these glyco-DAMGO analogs correlated with antinociceptive potency (i.v.
administration), presumably reflecting enhanced transport properties.
In this study we report on phosphorylation as an alternative strategy to enhance peptide stability and BBB penetration. Dass and Mahalaksmi showed that phosphorylated enkephalin was stable to proteolysis.13
Further, phosphorylated opioid peptides have been isolated from bovine adrenal medulla, providing further impetus to investigate the effects of phosphorylation on peptide pharmacology.14
The highly selective MOR agonist DAMGO15
and the mixed MOR/DOR agonist YtGFL-amide16,17
were chosen as the opioid “messages” for this study, which have previously been shown to produce centrally mediated effects upon glycosylation. The chemical structures of the lead peptides and their phosphate derivatives studied are depicted in .
Chemical structures of the compounds tested in this study.