Selective NMDA NR2B antagonists induce antinociception without motor dysfunction: correlation with restricted localisation of NR2B subunit in dorsal horn
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.
References (43)
- et al.
Lack of analgesic effects of opioids on neuropathic and idiopathic forms of pain
Pain
(1988) - et al.
The effects of ifenprodil and eliprodil on voltage-dependent Ca2+ channels and in gerbil cerebral ischaemia
Eur. J. Pharmacol.
(1996) - et al.
A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man
Pain
(1988) - et al.
L-745,337: a selective inhibitor of cyclooxygenase-2 elicits antinociception but not gastric ulceration in rats
Neuropharmacology
(1994) - et al.
Central and peripheral antialgesic action of aspirin-like drugs
Eur. J. Pharmacol.
(1978) - et al.
The effect of novel anti-epileptic drugs in rat experimental models of acute and chronic pain
Eur. J. Pharmacol.
(1997) - et al.
Regional, developmental and interspecies expression of the four NMDAR2 subunits, examined using monoclonal antibodies
Brain Res. Mol. Brain Res.
(1997) - et al.
Alternatively spliced isoforms of the N-methyl-d-aspartate receptor subunit 1 are differentially distributed within the rat spinal cord
Neuroscience
(1994) - et al.
Intrathecal treatment with dextrorphan or ketamine potently reduces pain-related behaviours in a rat model of peripheral mononeuropathy
Brain Res.
(1993) - et al.
CP-101, 606, a potent neuroprotectant selective for forebrain neurons
Eur. J. Pharmacol.
(1997)
The N-methyl-d-aspartate receptor antagonist dextromethorphan selectively reduces temporal summation of second pain in man
Pain
Involvement of spinal NMDA receptors in capsaicin-induced nociception
Pharmacol. Biochem. Behav.
Peripheral and central mechanisms of cutaneous hyperalgesia
Prog. Neurobiol.
Ethical guidelines for investigations of experimental pain in conscious animals
Pain
Anticonvulsant and behavioural profile of L-701,324, a potent, orally active antagonist at the glycine modulatory site on the N-methyl-d-aspartate receptor complex
J. Pharmacol. Exp. Ther.
Lamotrigine control of idiopathic trigeminal neuralgia
J. Neurol. Neurosurg. Psychiatry
(1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propranol: a potent new neuroprotectant which blocks N-methyl-d-aspartate responses
J. Med. Chem.
Subunit composition of N-methyl-d-aspartate receptors in the central nervous system that contain the NR2D subunit
Mol. Pharmacol.
Continuous subcutaneous administration of the N-methyl-d-aspartate (NMDA) receptor antagonist ketamine in the treatment of postherpetic neuralgia
Pain
Modulation of 45Ca2+ influx into cells stably expressing recombinant human NMDA receptors by ligands acting at distinct recognition sites
J. Neurochem.
Cited by (322)
Presynaptic glutamate receptors in nociception
2023, Pharmacology and TherapeuticsLong-term reversal of chronic pain behavior in rodents through elevation of spinal agmatine
2023, Molecular TherapyCaMKII binding to GluN2B at S1303 has no role in acute or inflammatory pain
2021, Brain ResearchA comprehensive description of GluN2B-selective N-methyl-D-aspartate (NMDA) receptor antagonists
2020, European Journal of Medicinal ChemistryCitation Excerpt :Intracellular Ca2+ overaccumulation induces a series of Ca2+-dependent enzymes which may cause neuronal damage and death (excitotoxicity) [34]. The selective antagonism of GluN2B subunit-containing NMDA receptors has emerged as an effective strategy to curb the pathological roles of excitotoxicity in progression of various diseases, such as Alzheimer’s Disease, ischemic stroke, neuropathic pain, Parkinson’s Disease, and depression [35,36]. Alzheimer’s disease (AD) is a progressive, chronic neurodegenerative disorder that influences memory and cognitive function in the aged population [37].
Therapeutic potential of N-methyl-D-aspartate receptor modulators in psychiatry
2024, Neuropsychopharmacology