Elsevier

Neuropharmacology

Volume 38, Issue 5, 15 May 1999, Pages 611-623
Neuropharmacology

Selective NMDA NR2B antagonists induce antinociception without motor dysfunction: correlation with restricted localisation of NR2B subunit in dorsal horn

https://doi.org/10.1016/S0028-3908(98)00218-4Get rights and content

Abstract

The present study investigated the regional distribution of the N-methyl-d-aspartate (NMDA) receptor containing the NR2B subunit protein in rat lumbar spinal cord and examined whether selective NR2B antagonists would exhibit antinociception with reduced side-effect liability than subtype non-selective NMDA antagonists and anticonvulsants. Immunocytochemical studies showed the NR2B subunit had a restricted distribution, with moderate labelling of fibres in laminas I and II of the dorsal horn suggesting a presynaptic location on primary afferent fibres and possible involvement in pain transmission. In the in vivo studies, the NMDA/glycine antagonists (MK-801, 0.02–1 mg/kg i.p., L-687,414 10–300 mg/kg i.p., and L-701,324 1–10 mg/kg i.p.) and the anticonvulsant, gabapentin (10–500 mg/kg p.o.), induced rotarod deficits at antinociceptive doses. In contrast, the selective NR2B antagonists, (±)-CP-101,606 (1–100 mg/kg p.o.) and (±)-Ro 25-6981 (3–100 mg/kg i.p.) showed a significant dose window. (±)-CP-101,606 caused no motor impairment or stimulation in rats at doses up to 100 mg/kg p.o., which is far in excess of those inhibiting allodynia in neuropathic rats (ID50 4.1 mg/kg, p.o.). (±)-Ro 25-6981 also showed a significant separation (ID50 allodynia 3.8 mg/kg, i.p.), however, some disruption of rotarod performance was observed at 100 mg/kg. The anticonvulsant lamotrigine (3–500 mg/kg p.o.) also showed a good dose window. These findings demonstrate that NR2B antagonists may have clinical utility for the treatment of neuropathic and other pain conditions in man with a reduced side-effect profile than existing NMDA antagonists.

Introduction

Neuropathic pain in man can occur following injury to the peripheral or central nervous system arising from multiple causes including chemotherapy, traumatic injury and herpes zoster infection. These neuropathies may be persistent and are particularly problematic because they are often poorly managed by conventional opioid analgesics (Arner and Meyerson, 1988) and non-steroidal anti-inflammatory drugs (Max et al., 1988). Recent attention has focused on N-methyl-d-aspartate (NMDA) receptor antagonists for the treatment of neuropathic pain. Ketamine, dextromethorphan and CPP (3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid) have been reported to produce symptomatic relief in a number of neuropathies including postherpetic neuralgia, central pain caused by spinal cord injury, and phantom limb pain (Kristensen et al., 1992, Eide et al., 1995, Knox et al., 1995, Max et al., 1995). However, these agents also induce unacceptable side effects at analgesic doses including hallucinations, dysphoria, and disturbances of cognitive and motor function which prohibit their widespread use. Therefore, in order to exploit NMDA receptor antagonists as possible treatments for neuropathic pain it is necessary to develop new agents with a reduced side-effect liability.

The NMDA receptor complex comprises of multiple protein subunits, of which there is at least one NMDAR1 (NR1) subunit (with up to eight splice variants) and at least one of a family of NR2 subunits (NR2A-NR2D) (for review, see Whiting and Priestley, 1996). In situ hybridisation and immunohistochemical studies have shown the NMDA subunits to be differentially distributed in the brain (Ishii et al., 1993, Wenzel et al., 1995, Laurie et al., 1997); for example, NR1 and NR2A mRNA and protein are abundantly expressed throughout the rat brain, whereas NR2B mRNA and protein is restricted to the forebrain. The more discrete distribution of NR2B subunit in the CNS may support a reduced side-effect profile of agents that act selectively at this site. For example, lack of expression in the cerebellum may suggest a reduced propensity for causing ataxia compared with existing NMDA receptor antagonists such as competitive antagonists which act at the glutamate recognition site (e.g. CPP), non-competitive antagonists that block the associated ion channel (e.g. dizocilpine [MK-801]) and competitive antagonists of the glycine co-agonist site (e.g. L-687,414 [R(+)-cis-β-methyl-3-amino-1-hydroxypyrrolid-2-one maleate], and L-701,324 [7-chloro-4-hydroxy-3-(3-phenoxy) phenyl-2(H)-quinolone]) which show little (<5-fold) or no selectivity for NR2B-containing over NR2A-containing receptor subtypes (Priestley et al., 1995, Grimwood et al., 1996). At present, the literature does not establish whether the NR2B subunit is expressed in the rat spinal cord. Luque et al. (1994) found weak to moderate expression of mRNA for NR2B in laminae II and IX, but Tölle et al. (1993) were unable to detect mRNA for NR2B in the spinal cord. NR2B protein was found in laminae I and II in rat dorsal horn (Yung, 1998) but there are no reports showing the distribution within the dorsal and ventral horn. A primary objective of the present study was therefore to define the distribution of the NR2B subunit protein within the rat spinal cord.

The NR2B subunit is particularly interesting because selective antagonists to receptors containing this subunit have recently become available. For example, functional selectivity for NR2B subunit has been shown with CP-101,606 [(1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propranol; Chenard et al., 1995] and Ro 25-6981 [R-(R*,S*)-α-(4-hydroxyphenyl)-β-methyl-4-(phenyl-methyl)-1-piperidine propranol; Trube et al., 1996] and their parent compound, ifenprodil (Williams, 1993, Grimwood et al., 1996). Moreover, both (±)-CP-101,606 and ifenprodil have been reported to possess antinociceptive activity in animal assays of inflammatory hyperalgesia (Taniguchi et al., 1997, Sakurada et al., 1998). A second objective of the present study was to examine whether selective NMDA NR2B containing receptor antagonists also possess antinociceptive activity in an animal assay of neuropathic pain and determine whether they have a reduced liability to induce motor impairment or psychomotor effects than non-competitive NMDA and glycine site antagonists, and some anticonvulsants which have been used to treat neuropathic pain in man. Experimental peripheral neuropathy has been investigated in rats using a chronic sciatic nerve constriction injury (Bennett and Xie, 1988). These animals display many of the characteristics seen in patients including mechanical allodynia (nociceptive response to innocuous stimulation) and heat hyperalgesia (exaggerated responses to noxious thermal stimulation). This assay is able to detect antinociceptive effects of drugs which are effective in alleviating neuropathic pain in man, such as the anticonvulsants, gabapentin and lamotrigine (Hunter et al., 1997) and the NMDA antagonists, ketamine and dextrorphan (an active metabolite of dextromethorphan) (Mao et al., 1993). We have also assessed these compounds using an assay of inflammatory hyperalgesia and on spinal nociceptive reflexes to define the spectrum of analgesic activity.

Section snippets

Preparation of antisera

Peptide sequences (20 amino acids) corresponding to amino acid residues 1298-1316 for the NR2A subunit and 1149-1168 for the NR2B subunit of the NMDA receptor complex were selected from the C terminus as described by Ishii et al. (1993) to minimise homology with other known proteins. Peptides were synthesised by Affiniti Research Products (UK) by solid-phase methodology utilising 9-fluorenylmethyloxycarbonyl (Fmoc) for Naamino and t-butyl based groups for side chain temporary protection. Upon

Distribution of NR2A and NR2B subunits in rat spinal cord

Immunostaining with the antibody to NR2A was widespread and observed in cell bodies throughout the dorsal and ventral horn of the rat lumbar spinal cord and on motor neurones (Fig. 2a, c). In contrast, staining with the NR2B antibody showed a more restricted distribution, with moderate staining of fibres in laminae I-II of the dorsal horn suggesting a presynaptic location on primary afferent fibres (Fig. 2b, d). Some staining of cell bodies and fibres was also seen around the central canal

Discussion

The aim of the present studies was to establish the distribution of receptors containing the NR2B subunit in the spinal cord and to determine whether selective NR2B antagonists possess antinociceptive effects with a reduced side-effect profile compared with a non-competitive NMDA antagonist, with glycine site antagonists and anticonvulsants.

Using specific antibodies to the NR2A or NR2B subunit, distinct staining patterns were observed in rat lumbar spinal cord. The NR2B antibody produced

Acknowledgements

We are grateful to our colleagues J. Elliott, J. Kulagowski and M. Rowley for the synthesis of L-687,414, L-701,324, (±)-Ro 25-6981 and (±)-CP-101,606 and to J. Attack for the preparation and purification of the antibodies for the NR2A and NR2B subunits. We also wish to thank to P. Hutson and S. Grimwood for helpful discussions during the preparation of this manuscript.

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