Received for publication April 9, 2004.
Revised May 16, 2004.
Accepted for publication May 17, 2004.

Antinociceptive Structure-Activity Studies with Enkephalin-Based Opioid Glycopeptides

Abstract

Development of opioid peptides as therapeutic agents has historically been limited due to pharmacokinetic issues including stability and blood-brain barrier (BBB) permeability. Glycosylation of opioid peptides can increase peptide serum stability and BBB penetration. To further define the requirements for optimizing in vivo antinociceptive potency following intravenous administration, we synthesized a series of enkephalin-based glycopeptides using solid phase Fmoc methods. The compounds differed in the 6th and subsequent amino acid residues (Ser or Thr), and in the attached carbohydrate moiety. In vitro binding and functional smooth muscle bioassays indicated that the addition of mono- or disaccharides did not significantly affect the opioid receptor affinity or agonist activity of the glycopeptides compared to their unglycosylated parent peptides. All of the glycopeptides tested produced potent antinociceptive effects in male ICR mice following intracerebroventricular injection in the 55°C tail-flick test. The calculated A₅₀ values for the Ser/Thr and monosaccharide combinations were all very similar, with values ranging from 0.02-0.09 nmol. Selected compounds were administered to mice intravenously and tested for antinociception to indirectly assess serum stability and BBB penetration. All compounds tested produced full antinociceptive effects with calculated A₅₀ values ranging from 2.2-46.4 µmol/kg, with the disaccharides having potencies that equaled or exceeded that of morphine on a µmol/kg basis. Substitution of a trisaccharide or bis- and tris-monosaccharides resulted in a falloff in antinociceptive potency. These results provide additional support for the utility of glycosylation to increase CNS bioavailability of small peptides and compliment our ongoing stability and blood-brain barrier penetration studies.

Key words: Antinociception, Delta opioid receptor, Enkephalin, Glycopeptide, Mu opioid receptor, Opioid

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