In vivo receptor occupancy of mGlu5 receptor antagonists using the novel radioligand [3H]3-methoxy-5-(pyridin-2-ylethynyl)pyridine)

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Abstract

In vivo receptor occupancy of mGlu5 receptor antagonists was quantified in rat and mouse brain using the mGlu5 receptor selective antagonist [3H]3-methoxy-5-(pyridin-2-ylethynyl)pyridine) ([3H]methoxy-PEPy). Administration of [3H]methoxy-PEPy (50 μCi/kg i.v.) to mGlu5 receptor-deficient mice revealed binding at background levels in forebrain, whereas wild-type mice exhibited 14-fold higher binding in forebrain relative to cerebellum. Systemic administration of the mGlu5 receptor antagonists 2-methyl-6-(phenylethynyl)pyridine (MPEP) and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) reduced the binding of [3H]methoxy-PEPy in rats and mice, reflecting mGlu5 receptor occupancy by these compounds. MPEP (10 mg/kg i.p.) and MTEP (3 mg/kg i.p.) maintained >75% receptor occupancy for 2 h in rats, while in mice MPEP and MTEP achieved >75% occupancy for only 30 and 15 min, respectively. Compound levels in plasma were substantially lower in mice suggesting species differences in receptor occupancy result from differences in absorption or metabolism of the compounds. These findings demonstrate that [3H]methoxy-PEPy is useful for determining the occupancy of mGlu5 receptors in the brain.

Introduction

Metabotropic glutamate (mGlu) receptors are G-protein coupled receptors which play an important role in mediating glutamate neurotransmission in the brain (Conn and Pin, 1997). There are presently eight subtypes of mGlu receptors, which are subdivided into three groups based on sequence homology, signaling pathways, and agonist selectivity (Pin and Duvoisin, 1995). The mGlu5 receptor subtype is a member of the Group I family of mGlu receptors which initiate cellular responses through Gq/11 protein coupling to phospholipase C and stimulation of phosphoinositide hydrolysis.

mGlu5 receptors are present in high densities in many forebrain regions (e.g. striatum, hippocampus, and frontal cortex) and are present along key points of the pain neuraxis (e.g. thalamus, the dorsal surface of the spinal cord, and dorsal root ganglion). mGlu5 receptor agonists and antagonists have a broad range of neurobiological effects, and antagonists have been considered for several therapeutic indications. In vivo stimulation of mGlu5 receptors by intracerebral or intrathecal administration of the mGlu1/5 receptor agonist 3,5-dihydroxyphenylglycine (DHPG) or the mGlu5 receptor agonist 2-chloro-5-hydroxyphenylglycine (CHPG) can lead to increases in locomotor activity and seizures Camon et al., 1998, Chapman et al., 2000, neurotoxicity (Camon et al., 1998), and behaviors consistent with increased nociception Fisher and Coderre, 1996, Karim et al., 2001. In contrast, mGlu5 receptor antagonists have shown activity in animal models of anxiety Brodkin et al., 2002b, Spooren et al., 2000, Tatarczynska et al., 2001, depression (Tatarczynska et al., 2001), Parkinson's disease (Breysse et al., 2002), pain Bordi and Ugolini, 2000, Walker et al., 2001, and seizures (Chapman et al., 2000).

The behavioral effects of mGlu5 receptor antagonists have been principally demonstrated in rats and, to a lesser extent, in mice. It is not known, however, if there are inherent differences between these two species with respect to activities of mGlu5 receptor antagonists. In addition, with the availability of mGlu5 receptor knockout mice, as well as the development of other relevant transgenic and knockout mice that may be utilized in mGlu5 receptor research, it becomes important to have a clear understanding of the actions of mGlu5 receptor antagonists in mice.

We have recently shown that [3H]methoxymethyl-3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine ([3H]methoxymethyl-MTEP), a potent and selective mGlu5 receptor antagonist, is useful for labeling mGlu5 receptors both in vitro and in vivo (Anderson et al., 2002). Similarly, the structurally related mGlu5 receptor antagonists, [3H]3-methoxy-5-(pyridin-2-ylethynyl)pyridine ([3H]methoxy-PEPy) (Cosford et al., 2003) and [3H]2-[(3-methoxyphenyl)ethynyl]-6-methylpyridine ([3H]M-MPEP) (Gasparini et al., 2002), have both been shown to selectively label mGlu5 receptors in vitro. Here we demonstrate that [3H]methoxy-PEPy is also useful for labeling mGlu5 receptors in vivo and for determining functional brain penetration and receptor occupancy of unlabeled mGlu5 receptor antagonists following systemic administration. Our results demonstrate key differences in the time course of receptor occupancy between rats and mice that may provide useful guidelines to follow for selecting doses and time lines for behavioral assessments of mGlu5 receptor activities in these two species.

Section snippets

Animals and compounds

Male Sprague–Dawley rats (175–225 g) were purchased from Harlan (San Diego, CA) while mGlu5 receptor knockout mice (20–25 g) (Lu et al., 1997) and C57Bl/6 mice were purchased from The Jackson Laboratory (Bar Harbor, Maine). Wild-type litter mates served as controls for the knockout mice. All animals were group housed on a 12-h light/dark cycle with free access to food and water. All procedures were approved by the Institutional Animal Care and Use Committee in accordance with The Guide for the

Time course of in vivo binding of [3H]methoxy-PEPy

The binding of [3H]methoxy-PEPy in both Sprague–Dawley rats and C57Bl/6 mice was maximal 1 min post-tail vein dosing (Fig. 1), producing 4435±245 dpm in rats (n=4) and 2755±401 dpm (n=3) in mice. In addition, the level of binding in hippocampus relative to cerebellum was approximately 10:1 for rats, and the level of binding in forebrain relative to cerebellum in mice was approximately 8:1. Because maximal binding in hippocampus or forebrain was observed at 1 min, in all subsequent experiments, [

Discussion

An in vivo receptor occupancy assay for determining the functional brain penetration and receptor binding of mGlu5 receptor antagonists has importance for developing these compounds for neurological and psychiatric disorders. Receptor occupancy assays rely on the selective labeling of a receptor by a specific radioligand and a reduction in radioligand binding results from occupancy of the receptor by the unlabeled compound. Systemic injections of unlabeled mGlu5 receptor antagonists MPEP and

Acknowledgments

The authors wish to acknowledge the excellent work of Ashok Chaudary in the synthesis of [3H]methoxy-PEPy.

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