Elsevier

Neuropharmacology

Volume 48, Issue 5, April 2005, Pages 658-672
Neuropharmacology

Pharmacological and pharmacokinetic characterization of the cannabinoid receptor 2 agonist, GW405833, utilizing rodent models of acute and chronic pain, anxiety, ataxia and catalepsy

https://doi.org/10.1016/j.neuropharm.2004.12.008Get rights and content

Abstract

To date, two cannabinoid receptors have been identified, CB1 and CB2. Activation of these receptors with non-selective cannabinoid receptor agonists reduces pain sensitivity in animals and humans. However, activation of CB1 receptors is also associated with central side effects, including ataxia and catalepsy. More recently, a role for selective CB2 agonists in pain modification has been demonstrated. GW405833, a selective CB2 agonist, was recently reported to partially reverse the inflammation and hyperalgesia in a rat model of acute inflammation. In the current report, we extend the characterization and therapeutic potential of this compound. For the first time, we show that GW405833 selectively binds both rat and human CB2 receptors with high affinity, where it acts as a partial agonist (∼50% reduction of forskolin-mediated cAMP production compared to the full cannabinoid agonist, CP55,940). We also report for the first time that intraperitoneal administration of GW405833 (0.3–100 mg/kg) to rats shows linear, dose-dependent increases in plasma levels and substantial penetration into the central nervous system. In addition, GW405833 (up to 30 mg/kg) elicits potent and efficacious antihyperalgesic effects in rodent models of neuropathic, incisional and chronic inflammatory pain, the first description of this compound in these models. In contrast, analgesia, sedation and catalepsy were not observed in this dose range, but were apparent at 100 mg/kg. Additionally, GW405833 was not antihyperalgesic against chronic inflammatory pain in CB2 knockout mice. These data support the tenet that selective CB2 receptor agonists have the potential to treat pain without eliciting the centrally-mediated side effects associated with non-selective cannabinoid agonists, and highlight the utility of GW405833 for the investigation of CB2 physiology.

Introduction

Cannabinoid receptors mediate many of the effects of Δ9-tetrahydrocannabinol (THC), the psychotropic agent found in marijuana. These receptors belong to the superfamily of G protein-coupled receptors, and, to date, two have been cloned and characterized: CB1 and CB2, which share 48% identity at the amino acid level (Matsuda et al., 1990, Munro et al., 1993). Both signal through activation of pertussis toxin-sensitive G proteins to inhibit adenylate cyclase, and both are positively coupled to the activation of mitogen activated protein kinases (for review, see Howlett et al., 2002).

In addition to THC, numerous other natural products, endogenous ligands and synthetic small molecules can modify the activity of the cannabinoid receptors (Howlett et al., 2002). Anandamide was the first endocannabinoid identified, and to date, remains the most widely studied (Devane et al., 1992). Subsequently, numerous other putative endocannabinoids and endogenous fatty acid amides have been proposed as native ligands for the cannabinoid receptors (for review, see Chapman and Finn, 2003). In vitro and in vivo pharmacological evidence suggests that most of these ligands elicit their effects through either the CB1 receptor or both CB1 and CB2 receptors.

Cannabinoids and cannabinoid receptors have been implicated in pain transduction and perception (for review, see Chapman and Finn, 2003) as well as neuroinflammation (for review, see Walter and Stella, 2004). The expression patterns for these receptors support a role in these processes. The expression of CB1 is primarily restricted to neurons of the central and peripheral nervous systems, including primary afferent nociceptive neurons (Galiegue et al., 1995, Ross et al., 2001). In contrast, the expression of CB2 is primarily localized to cells of the immune system (Galiegue et al., 1995). However, recent data suggest the expression of CB2 receptors on peripheral primary afferent nociceptive neurons as well (Ross et al., 2001).

A number of publications have demonstrated the analgesic, antihyperalgesic and anti-inflammatory actions of selective CB1 agonists and non-selective CB1/CB2 agonists (for review, see Chapman and Finn, 2003). As such, cannabinoids represent a potential therapeutic approach for treating chronic pain. However, CB1 agonists that penetrate the central nervous system result in catalepsy, sedation and undesirable psychotropic effects, which have also limited the therapeutic utility of non-selective, brain-permeable cannabinoid agonists.

It is becoming increasingly apparent that selective agonism of CB2 receptors may also constitute a novel strategy for treating chronic pain. For example, the non-selective cannabinoid agonist, HU210, reversed carrageenan-induced edema through CB2, as the CB2-selective antagonist SR144528 blocked this effect (Clayton et al., 2002). In addition, compounds that exhibit selectivity for CB2 over CB1 have proven efficacious in a number of pain models. Here, the CB2-selective THC derivative, HU-308, reduced both the inflammation associated with arachidonic acid-induced ear swelling and late phase formalin-induced pain behavior in mice, again in an SR144528-sensitive manner (Hanuš et al., 1999). In addition, the CB2-selective compound, AM1241, has been shown to be anti-inflammatory, analgesic and efficacious against inflammatory and neuropathic pain when administered either locally or systemically (for review, see Malan et al., 2003). The effects of AM1241 were reversible with selective CB2 antagonists, not reversible with selective CB1 antagonists, and remained in animals that were null mutants for the CB1 receptor (Ibrahim et al., 2003). GW405833 is an alternative CB2-selective agonist that has been previously shown to both reduce edema formation and inhibit the hypersensitivity associated with intraplantar injection of carrageenan (Clayton et al., 2002). These effects were inhibited by SR144528, providing evidence that the effect of GW405833 is mediated by CB2 receptors (Clayton et al., 2002).

In the current study, we have extended the characterization and therapeutic potential of GW405833. We describe the affinity of this compound for human and rat CB1 and CB2 receptors in vitro, as well as its functional activity in CHO-K1 cells expressing human CB2 receptors. In rats, we have performed an in vivo pharmacokinetic profile, including assessment of its penetration into the central nervous system. We also describe the effect of GW405833 in assays of anxiety and acute nociception as well as models of inflammatory-, neuropathic- and incision-induced pain. Additionally, we have performed a limited side-effect profile of GW405833, including assays for catalepsy and motor deficits. Finally, we describe the effect of the compound in a mouse model of chronic inflammatory pain utilizing mice that do not express the CB2 receptor.

Section snippets

Materials

Radioisotopes were purchased from PerkinElmer Life Sciences (Boston, MA). Human CB1 receptors expressed in CHO-K1 cells and a human CB2 cDNA expression construct (hCB2-D1 in pcDNA3) were purchased from EuroScreen (Brussels, Belgium). (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN55,212-2) and (−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55,940) were purchased from

GW405833 is a selective CB2 agonist

The affinities of GW405833 for the human cannabinoid CB1 and CB2 receptors were measured using competition radioligand binding assays on CHO-K1 cell membranes expressing the recombinant receptors (Fig. 2). GW405833 showed high binding affinity to the human CB2 receptor with a concentration-dependent displacement of [3H]-CP55,940 (Fig. 2A). The affinity of GW405833 for this receptor (3.92 ± 1.58 nM; n = 3) was comparable to that of the non-selective cannabinoid agonists CP55,940 and WIN55,212-2 (2.33 ±

Discussion

Selective CB2 cannabinoid receptor agonists are beginning to show promise as therapeutic agents. Given the lack of CB2 receptors on neurons of the central nervous system, this approach may offer an opportunity to treat these conditions without the central side effects commonly associated with brain-permeable CB1-selective or CB1/CB2 non-selective agonists. In this paper, we present the in vitro and in vivo pharmacological and in vivo pharmacokinetic properties of GW405833, a selective CB2

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