The m opioid receptor, MOR, displays spontaneous agonistindependent (basal) G protein coupling in vitro. To determine whether basal MOR signaling contributes to narcotic dependence, antagonists were tested for intrinsic effects on basal MOR signaling in vitro and in vivo, before and after morphine pretreatment. Intrinsic effects of MOR ligands were tested by measuring GTPgS binding to cell membranes and cAMP levels in intact cells. b-CNA, C-CAM, BNTX, and nalmefene were identi®ed as inverse agonists (suppressing basal MOR signaling). Naloxone and naltrexone were neutral antagonists (not affecting basal signaling) in untreated cells, whereas inverse agonistic effects became apparent only after morphine pretreatment. In contrast, 6a-and 6b-naltrexol and -naloxol, and 6b-naltrexamine were neutral antagonists regardless of morphine pretreatment. In an acute and chronic mouse model of morphine-induced dependence, 6b-naltrexol caused signi®cantly reduced withdrawal jumping compared to naloxone and naltrexone, at doses effective in blocking morphine antinociception. This supports the hypothesis that naloxoneinduced withdrawal symptoms result at least in part from suppression of basal signaling activity of MOR in morphinedependent animals. Neutral antagonists have promise in treatment of narcotic addiction.

In analgesic drug development, preclinical procedures are widely used to assess drug effects on pain-related behaviors. These procedures share two principal components: 1) a manipulation intended to produce a pain-like state in the experimental subject and 2) measurement of behaviors presumably indicative of that pain state. Drugs can then be evaluated for their ability to attenuate pain-related behaviors. In the simplest procedures, the pain state is produced by delivery of an acute noxious stimulus (e.g., a warm thermal stimulus), and the primary dependent measures focus on withdrawal responses or other nocifensive behaviors that increase in rate, frequency, or intensity in response to the noxious stimulus. This approach has been refined in two ways. First, new methods have been developed to induce more clinically relevant pain states. In particular, pain requiring clinical intervention is often associated with inflammation or neuropathy, and novel procedures have emerged to model these conditions and their ability to produce hypersensitive pain states, such as allodynia and hyperalgesia. Second, studies are incorporating a broader array of painrelated behaviors as dependent measures. For example, pain not only stimulates nocifensive behaviors but also suppresses many adaptive behaviors, such as feeding or locomotion. Measures of pain-suppressed behaviors can provide new insights into the behavioral consequences of pain and the effects of candidate analgesics. In addition, functional magnetic resonance imaging has emerged as a noninvasive tool for investigating changes in neural activity associated with pain and analgesia. Integration of these complementary approaches may improve the predictive validity of analgesic drug development.

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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

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Most peptides have not proved useful as neuroactive drugs because they are blocked by the blood-brain barrier and do not reach their receptors within the brain.Intraperitoneally administered L-serinyl JD-glucosde aalgues of [Metslenkephaln (glycopeptides) have been shown to be transported across the blood-brain barrier to bind with targeted p and &oplod receptors in the mouse brain. The opioid nature of the bing has been demonstrated with intracerebroventricularly a ered naloxone. Paradoxically, glucosylation decreases the ipophlt of the peptides while promoting transport across the phc endothe layer. It is suggested that glucose transporter GLUT-1 is responsible for the transport of the peptide message. Profound and long-lasting anesla has been observed in mice (tail-ck and hot-plate assays) with two of the glycopeptide a ues when administered intraperitoneally.

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).