Narcotic analgesics cause addiction by poorly understood mechanisms, involving opioid receptor (MOR). Previous cell culture studies have demonstrated significant basal, spontaneous MOR signaling activity, but its relevance to narcotic addiction remained unclear. In this study, we tested basal MORsignaling activity in brain tissue from untreated and morphinepretreated mice, in comparison to antagonist-induced withdrawal in morphine-dependent mice. Using guanosine 5Ј- O-(3-[35 S]thio)triphosphate ([ 35 S]GTP␥S) binding and adenylyl cyclase activity assay in brain homogenates, we demonstrated that morphine pretreatment of mice enhanced basal MOR signaling in mouse brain homogenates and, moreover, caused persistent changes in the effects of naloxone and naltrexone, antagonists that elicit severe withdrawal in dependent subjects. Naloxone and naltrexone suppressed basal [35 S]GTP␥S binding (acting as "inverse agonists") only after morphine pretreatment, but not in drug-naive animals. Moreover, naloxone and naltrexone stimulated adenylyl cyclase activity in striatum homogenates only after morphine pretreatment, by reversing the inhibitory effects of basal MOR activity. After cessation of morphine treatment, the time course of inverse naloxone effects on basal MOR signaling was similar to the time course of naltrexone-stimulated narcotic withdrawal over several days. The neutral antagonist 6␤-naltrexol blocked MOR activation without affecting basal signaling (G protein coupling and adenylyl cyclase regulation) and also elicited substantially less severe withdrawal. These results demonstrate long-lasting regulation of basal MOR signaling as a potential factor in narcotic dependence.

The -opioid receptor displays basal signaling activity, which seems to be enhanced by exposure to opioid agonists. This study assesses the in vivo pharmacology of the putative "neutral" antagonist 6␤-naltrexol in comparison to other ligands with varying efficacy, such as naloxone, an inverse agonist in the opioid-dependent state. ICR mice were used to generate full antagonist dose-response curves for naloxone, naltrexone, nalbuphine, and 6␤-naltrexol in blocking acute antinociceptive effects of morphine and precipitating opioid withdrawal in models of physical dependence. 6␤-Naltrexol was roughly equipotent to naloxone and between 4.5-and 10-fold less potent than naltrexone in blocking morphine-induced antinociception and locomotor activity, showing that 6␤-naltrexol enters the central nervous system. In contrast to naloxone and naltrexone, 6␤-naltrexol precipitated only minimal withdrawal at high doses in an acute dependence model and was ϳ77-and 30-fold less potent than naltrexone and naloxone, respectively, in precipitating withdrawal in a chronic dependence model. 6␤-Naltrexol reduced the inverse agonist effects of naloxone in vitro and in vivo, as expected for a neutral antagonist. Therefore, the pharmacological effects of 6␤-naltrexol differ markedly from those of naloxone and naltrexone in the opioid-dependent state. A reduction of withdrawal effects associated with neutral -opioid receptor antagonists may offer advantages in treating opioid overdose and addiction.Basal signaling/constitutive activity of G-protein coupled receptors is now firmly established largely on the basis of in vitro results (Kenakin, 2003(Kenakin, , 2004a, with receptor ligands displaying a range of efficacies from full agonists to full inverse agonists. However, for receptor systems that display constitutive activity, many questions remain to be resolved. 1) Do the in vitro observations translate into changes that can be measured in vivo? 2) Are there clinical applications for inverse agonists versus neutral antagonists? 3) How is basal receptor activity regulated, and how does disease and/or chronic drug exposure alter levels of basal signaling and ligand efficacy? Studies on ␤-adrenergic receptors, for example, suggest that these issues may contribute to patient outcomes in diseases such as congestive heart failure and asthma (Maack et al., 2000;Callaerts-Vegh et al., 2004).We and others have established that the -and ␦-opioid receptors display basal signaling, which is altered by exposure to opioid agonists (Costa and

We have previously reported the chemistry and antinociceptive properties of a series of glycosylated enkephalin analogs (glycopeptides) exhibiting approximately equal affinity and efficacy at ␦ opioid receptors (DORs) and opioid receptors (MORs).

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 9-fluorenylmethyloxy carbamate methods. The compounds differed in the sixth 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 with 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 A 50 values for the Ser/Thr and monosaccharide combinations were all very similar with values ranging from 0.02 to 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 A 50 values ranging from 2.2 to 46.4 mol/kg with the disaccharides having potencies that equaled or exceeded that of morphine on a micromoles per kilogram basis. Substitution of a trisaccharide or bis-and tris-monosaccharides resulted in a decrease in antinociceptive potency. These results provide additional support for the utility of glycosylation to increase central nervous system bioavailability of small peptides and compliment our ongoing stability and blood-brain barrier penetration studies.