In vitro stability and in vivo pharmacokinetic studies of a model opioid peptide, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE), and its cyclic prodrugs (acyloxyalkoxy-based cyclic prodrug of DADLE, coumarinic acid-based cyclic prodrug of DADLE, and oxymethyl-modified coumarinic acid-based cyclic prodrug of DADLE) were conducted. The enzymatic stability of DADLE and its prodrugs in various biological media was determined at 37°C in the presence and absence of paraoxon, a known esterase inhibitor. The prodrugs exhibited metabolic stability to exo-and endopeptidases, and esterase-catalyzed bioconversion of the prodrugs to DADLE was observed. For pharmacokinetic studies in rats, various biological samples (blood, bile, urine, and brain) were collected after i.v. administration of DADLE and its prodrugs. The samples were analyzed by highperformance liquid chromatography with tandem mass spectrometric detection, and the conversion from the prodrugs to intermediates to DADLE was monitored. The prodrugs exhibited similar pharmacokinetic properties and showed improved stability compared with DADLE in rat blood. This increased stability led to higher plasma concentrations of DADLE after i.v. administration of the prodrugs compared with i.v. administration of DADLE alone. In terms of elimination pathways, metabolism by endopeptidases was the major route for DADLE elimination, whereas rapid biliary excretion was the major route of elimination for the prodrugs. The rapid elimination of the prodrugs by the liver and the formation of stable intermediates after esterase hydrolysis limited the bioconversion efficiencies of the prodrugs to DADLE after i.v. administration. The substrate activity of the prodrugs for efflux transporters (e.g., Pglycoprotein) in the blood-brain barrier significantly restricted their access to the brain.The delivery of opioid peptides to the brain has presented significant challenges to pharmaceutical scientists due to the poor biopharmaceutical properties of the peptides. These include their lability to metabolism by exo-and endopeptidases and their low permeation across the blood-brain barrier (BBB) (Fricker and Drewe, 1996;Pauletti et al., 1996aPauletti et al., , 1997Prokai, 1998). The issue of metabolic lability has, for all practical purposes, been solved by medicinal chemists through the design of novel peptide bond bioisosteres to replace metabolically labile peptide bonds (Sawyer, 1995). Until recently, however, medicinal chemists have had less success in manipulating the structures of opioid peptides to achieve good BBB permeation while still retaining high affinity and selectivity for opioid receptors.Through prodrug strategies, some progress has been made recently in improving the BBB permeation characteristics of opioid peptides (Greene et al., 1996;Misicka et al., 1996;Patel et al., 1997;Prokai et al., 2000). For a prodrug strategy to be successful in delivering opioid peptides to the brain, the following criteria must be met: 1) the prodrug should have favorable physiochemical properties...
