In vitro and in vivo assessment of mu opioid receptor constitutive activity.

Constitutive (basal) signaling has been described and characterized for numerous G protein coupled receptors (GPCRs). The relevance of this activity to disease, drug discovery and development, and to clinical pharmacotherapy is just beginning to emerge. Opioid receptors were the first GPCR systems for which there was definitive evidence presented for constitutive activity, with numerous studies now published on the regulation of this activity (e.g., structure/activity of the receptor as it relates to basal activity, pharmacology of ligands that act as agonists, inverse agonists and "neutral antagonists," etc.). This chapter summarizes some of the methods used to characterize constitutive activity at the mu opioid receptor (MOR) in preclinical in vitro and in vivo model systems. This includes cell-based systems that are useful for higher throughput screening of novel ligands and for studying variables that can impact basal tone in a system. In vivo assays are also described in which constitutive activity is increased in response to acute or chronic opioid agonist exposure and where withdrawal is precipitated with antagonists that may function as inverse agonists or "neutral" antagonists. The methods described have inherent advantages and disadvantages that need to be considered in any drug discovery/development program. A brief discussion of progress toward understanding the clinical implications of MOR constitutive activity in the management of opioid addiction and chronic pain is also included in this chapter.

In vivo characterization of the opioid antagonist nalmefene in mice.

AIMS: The current study assessed the in vivo antagonist properties of nalmefene using procedures previously used to characterize the opioid antagonists naloxone, naltrexone, 6beta-naltrexol and nalbuphine. MAIN METHODS: ICR mice were used to generate antagonist dose-response curves with intraperitoneal (i.p.) nalmefene against fixed A(90) doses of morphine in models of morphine-stimulated hyperlocomotion and antinociception. Additional dose-response curves for antagonist precipitated opioid withdrawal were run in mice treated acutely (100mg/kg, s.c., -4h) or chronically (75mg pellet, s.c., -72h) with morphine. Comparisons were made between antagonist potency and degree of precipitated withdrawal. KEY FINDINGS: Nalmefene produced dose- and time-related antagonism of morphine-induced increases in locomotor activity with a calculated ID(50) (and 95% confidence interval) of 0.014 (0.007-0.027)mg/kg. Nalmefene produced rapid reversal of morphine-induced locomotor activity (5.1min for 50% reduction in morphine effect). A 0.32mg/kg dose of nalmefene produced blockade of morphine-induced antinociception in the 55 degrees C tail-flick test that lasted approximately 2h. Nalmefene was able to potently precipitate withdrawal in mice treated acutely or chronically with morphine. SIGNIFICANCE: These results demonstrate that nalmefene is similar to naloxone and naltrexone with respect to its in vivo pharmacology in mice. Specifically, nalmefene produces potent antagonism of morphine agonist effects while precipitating severe withdrawal. The compound has a slower onset and longer duration of action compared to naloxone and naltrexone. The data allows for a more complete preclinical comparison of nalmefene against other opioid antagonists including the putative opioid neutral antagonist 6beta-naltrexol.