Sue D. Collins

Prostanoid receptor EP1 and Cox-2 in injured human nerves and a rat model of nerve injury: a time-course study

BackgroundRecent studies show that inflammatory processes may contribute to neuropathic pain. Cyclooxygenase-2 (Cox-2) is an inducible enzyme responsible for production of prostanoids, which may sensitise sensory neurones via the EP1 receptor. We have recently reported that while macrophages infiltrate injured nerves within days of injury, they express increased Cox-2-immunoreactivity (Cox-2-IR) from 2 to 3 weeks after injury. We have now investigated the time course of EP1 and Cox-2 changes in injured human nerves and dorsal root ganglia (DRG), and the chronic constriction nerve injury (CCI) model in the rat.MethodsTissue sections were immunostained with specific antibodies to EP1, Cox-2, CD68 (human macrophage marker) or OX42 (rat microglial marker), and neurofilaments (NF), prior to image analysis, from the following: human brachial plexus nerves (21 to 196 days post-injury), painful neuromas (9 days to 12 years post-injury), avulsion injured DRG, control nerves and DRG, and rat CCI model tissues. EP1 and NF-immunoreactive nerve fibres were quantified by image analysis.ResultsEP1:NF ratio was significantly increased in human brachial plexus nerve fibres, both proximal and distal to injury, in comparison with uninjured nerves. Sensory neurones in injured human DRG showed a significant acute increase of EP1-IR intensity. While there was a rapid increase in EP1-fibres and CD-68 positive macrophages, Cox-2 increase was apparent later, but was persistent in human painful neuromas for years. A similar time-course of changes was found in the rat CCI model with the above markers, both in the injured nerves and ipsilateral dorsal spinal cord.ConclusionDifferent stages of infiltration and activation of macrophages may be observed in the peripheral and central nervous system following peripheral nerve injury. EP1 receptor level increase in sensory neurones, and macrophage infiltration, appears to precede increased Cox-2 expression by macrophages. However, other methods for detecting Cox-2 levels and activity are required. EP1 antagonists may show therapeutic effects in acute and chronic neuropathic pain, in addition to inflammatory pain.

GW274150, a novel and highly selective inhibitor of the inducible isoform of nitric oxide synthase (iNOS), shows analgesic effects in rat models of inflammatory and neuropathic pain.

Abstract Nitric oxide (NO), synthesised by different isoforms of nitric oxide synthase (NOS), has been linked with the development and maintenance of nociception. We studied the role of the inducible isoform, iNOS, in two different rat pain models with an inflammatory component. iNOS was immunohistochemically detected locally in the paw 6 h after Freunds Complete Adjuvant (FCA) injection, showing a plateau at 24–72 h and falling slowly in the following weeks. This correlated with the late phase of the hypersensitivity to pain revealed in the behavioural tests. A highly selective iNOS inhibitor GW274150 (1–30 mg/kg orally, 24 h after FCA) suppressed the accumulation of nitrite in the inflamed paw indicating substantial iNOS inhibition. At the same time it partially reversed FCA‐induced hypersensitivity to pain and edema in a dose‐dependent manner. After Chronic Constriction Injury (CCI) surgery to the sciatic nerve, iNOS presence was only detected locally in the region of the nerve (inflammatory cells). GW274150 (3–30 mg/kg orally, 21 days after surgery) also reversed significantly the CCI‐associated hypersensitivity to pain. No iNOS was detectable in dorsal root ganglia, spinal cord or brain in either model. This study demonstrates a role for peripherally‐expressed iNOS in pain conditions with an inflammatory component and the potential value of iNOS inhibitors in such conditions.

Novel histamine H3 receptor antagonists GSK189254 and GSK334429 are efficacious in surgically-induced and virally-induced rat models of neuropathic pain.

&NA; Several studies have implicated a potential role for histamine H3 receptors in pain processing, although the data are somewhat conflicting. In the present study we investigated the effects of the novel potent and highly selective H3 receptor antagonists GSK189254 (6‐[(3‐cyclobutyl‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepin‐7‐yl)oxy]‐N‐methyl‐3‐pyridinecarboxamide hydrochloride) and GSK334429 (1‐(1‐methylethyl)‐4‐({1‐[6‐(trifluoromethyl)‐3‐pyridinyl]‐4‐piperidinyl}carbonyl)hexahydro‐1H‐1,4‐diazepine) in two rat models of neuropathic pain, namely the chronic constriction injury (CCI) model and the varicella‐zoster virus (VZV) model. Both GSK189254 (0.3, 3 and/or 10 mg/kg p.o.) and GSK334429 (1, 3 and 10 mg/kg p.o.) significantly reversed the CCI‐induced decrease in paw withdrawal threshold (PWT) measured using an analgesymeter and/or von Frey hairs. In addition, GSK189254 (3 mg/kg p.o.) and GSK334429 (10 mg/kg p.o.) both reversed the VZV‐induced decrease in PWT using von Frey hairs. We also investigated the potential site of action of this analgesic effect of H3 antagonists using autoradiography. Specific binding to H3 receptors was demonstrated with [3H]‐GSK189254 in the dorsal horn of the human and rat spinal cord, and in human dorsal root ganglion (DRG), consistent with the potential involvement of H3 receptors in pain processing. In conclusion, we have shown for the first time that chronic oral administration of selective H3 antagonists is effective in reversing neuropathic hypersensitivity in disease‐related models, and that specific H3 receptor binding sites are present in the human DRG and dorsal horn of the spinal cord. These data suggest that H3 antagonists such as GSK189254 and GSK334429 may be useful for the treatment of neuropathic pain.

Investigation into the role of P2X3/P2X2/3 receptors in neuropathic pain following chronic constriction injury in the rat: an electrophysiological study

1 Two P2X3/P2X2/3 receptor antagonists with different potencies were profiled electrophysiologically in a rat model of nerve injury. 2 A‐317491 has poor CNS penetrance (blood : brain, 1 : <0.05), and was therefore administered intravenously in chronic constriction injury (CCI)‐ and sham‐operated rats to study the involvement of P2X3 subunit‐containing receptors in the periphery in neuropathic pain. A‐317491 and Compound A were administered topically to the spinal cord to investigate the central contribution. 3 There were no significant inhibitory effects of A‐317491 intravenous (i.v.) seen in sham‐operated animals compared to vehicle controls. In CCI‐operated animals, there were significant inhibitory effects of 3 mg kg−1 A‐317491 i.v. on C fibre‐evoked responses, and with 10 mg kg−1 A‐317491 i.v. on Aδ and C fibre‐evoked responses. No significant effects of A‐317491 were observed after topical application to the spinal cord. In contrast, when Compound A was administered spinally in CCI animals, there was a decrease in Aδ and C fibre‐evoked responses, and wind up. 4 These changes indicate that A‐317491 has a selective effect on neuronal responses in CCI animals compared to sham, demonstrating an increased involvement of P2X3/P2X2/3 receptors in sensory signalling following nerve injury. In addition, the more potent antagonist Compound A was effective spinally, unmasking a potential central role of P2X3/P2X2/3 receptors at this site post nerve injury. These data support a role for P2X3/P2X2/3 antagonists in the modulation of neuropathic pain.

Discovery and structure-activity relationships of a series of pyroglutamic acid amide antagonists of the P2X7 receptor.

A computational lead-hopping exercise identified compound 4 as a structurally distinct P2X(7) receptor antagonist. Structure-activity relationships (SAR) of a series of pyroglutamic acid amide analogues of 4 were investigated and compound 31 was identified as a potent P2X(7) antagonist with excellent in vivo activity in animal models of pain, and a profile suitable for progression to clinical studies.

Localisation and modulation of prostanoid receptors EP1 and EP4 in the rat chronic constriction injury model of neuropathic pain

Immunohistochemistry was used to examine the expression of prostaglandin E2 receptors EP1 and EP4 in sciatic nerves from the rat chronic constriction injury (CCI) model of neuropathic pain. At 21 days post‐surgery the CCI rats had developed mechanical hyperalgesia on the operated side, and quantitative image analysis showed a highly significant doubling of the area occupied by EP1‐ and EP4‐positive pixels in sections from CCI nerves when compared to sham‐operated controls. Co‐localisation studies with the marker ED1 revealed that 73% of the EP1‐positive cells and 54% of the EP4‐positive cells in the injured nerves represented infiltrating macrophages. Cells negative for ED1 and positive for either EP1 or EP4 were characterised as Schwann cells from their morphology and expression of myelin basic protein and S100 antigens. Similar EP1‐ and EP4‐positive Schwann cell profiles were observed in sections of uninjured control nerves. Low levels of EP receptor expression were found in neurofilament‐immunostained axons, but no consistent differences were observed in the levels of axonal EP staining between CCI and control tissue. These data provide further evidence of the importance of prostaglandins in the pathogenesis of neuropathic pain, and suggest that not only infiltrating macrophages but also Schwann cells may be involved in the modulation of these mediators in response to nerve injury.

Identification of [4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)-2-pyrimidinyl] amines and ethers as potent and selective cyclooxygenase-2 inhibitors.

A novel series of [4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)-2-pyrimidine-based cyclooxygenase-2 (COX-2) inhibitors, which have a different arrangement of substituents compared to the more common 1,2-diarylheterocycle based molecules, have been discovered. For example, 2-(butyloxy)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)pyrimidine (47), a member of the 2-pyrimidinyl ether series, has been shown to be a potent and selective inhibitor with a favourable pharmacokinetic profile, high brain penetration and good efficacy in rat models of hypersensitivity.