Alex W. Wilson

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.

Discovery of 2-[(2,4-Dichlorophenyl)amino]-N-[(tetrahydro- 2H-pyran-4-yl)methyl]-4-(trifluoromethyl)- 5-pyrimidinecarboxamide, a Selective CB2 Receptor Agonist for the Treatment of Inflammatory Pain

Selective CB2 receptor agonists are promising potential therapeutic agents for the treatment of inflammatory and neuropathic pain. A focused screen identified a pyrimidine ester as a partial agonist at the CB2 receptor with micromolar potency. Subsequent lead optimization identified 35, GW842166X, as the optimal compound in the series. 35 has an oral ED50 of 0.1 mg/kg in the rat FCA model of inflammatory pain and was selected as a clinical candidate for this indication.

Evaluation of the effects of lamotrigine, valproate and carbamazepine in a rodent model of mania.

Bipolar disorder is a psychiatric condition characterised by episodes of mania, depression, and underlying mood instability. Anticonvulsant drugs have an established place in the treatment of the disorder, but identifying novel drugs in this class is complicated by the absence of validated animal models. We have evaluated the efficacy of three anticonvulsant mood stabilising drugs (lamotrigine, valproate, and carbamazepine) in a model of mania, in which hyperactivity is induced by the combination of D-amphetamine and chlordiazepoxide. All three drugs were effective at preventing the hyperactivity. Lower doses of valproate and carbamazepine were required to prevent hyperactivity compared to doses required to block tonic-clonic seizures induced by pentylenetetrazole. Lamotrigine was equipotent in the two models. However, the complex pharmacology of the D-amphetamine/chlordiazepoxide model means that there may be several mechanisms by which hyperactivity can be reduced, and these may have more or less relevance to the treatment of bipolar disorder. To address this issue, we also evaluated effects of the three anticonvulsants on baseline locomotion, on activity in the presence of chlordiazepoxide alone, or on activity induced by D-amphetamine alone. Based on the results, we propose that hyperactivity induced by D-amphetamine/chlordiazepoxide may arise through dopaminergic drive coupled with disinhibition caused by low doses of the benzodiazepine. The efficacy of lamotrigine may then arise through a reduction in neuronal excitability or increased glutamate transmission, these latter a consequence of the disinhibition. Carbamazepine may also reduce excitability and glutamate release, but its broader pharmacology, manifested by sedation at higher doses complicates interpretation of its efficacy and reflects its poorer tolerability in the clinic. Valproate may be effective, at least in part, through an enhancement of GABAergic transmission. The predictive validity of the D-amphetamine/chlordiazepoxide model for efficacy in bipolar disorder remains to be established, and research with a wider range of clinically tested drugs is warranted to help validate the model further. In the meantime, the model may be useful for distinguishing novel anticonvulsant drugs with different mechanisms of action.

Activation of CB1 and CB2 receptors attenuates the induction and maintenance of inflammatory pain in the rat.

&NA; The aim of the present study was to investigate the effects of cannabinoid agonists on established inflammatory hyperalgesia. We have compared the effects of pre‐administration versus post‐administration of a potent non‐selective cannabinoid agonist HU210 and a selective CB2 receptor agonist JWH‐133 on hindpaw weight bearing and paw oedema in the carrageenan model of inflammatory hyperalgesia. For comparative purposes we also determined the effects of the μ‐opioid receptor agonist morphine and the COX2 inhibitor rofecoxib in this model. At 3 h following intraplantar injection of carrageenan (2%, 100 μl) there was a significant (P<0.001) reduction in weight bearing on the ipsilateral hindpaw, compared to vehicle treated rats and a concomitant increase in ipsilateral hindpaw volume (P<0.001), compared to vehicle treated rats. Systemic administration of HU210 (10 μg/kg) and JWH‐133 (10 mg/kg) at 3 h following injection of carrageenan, significantly attenuated decreases in ipsilateral hindpaw weight bearing (P<0.05 for both) and paw volume (P<0.001 for both). Pre‐administration of HU210 and JWH‐133 had similar effects on weight bearing in this model. Pre‐administered HU210 also significantly decreased carrageenan‐induced changes in paw volume (P<0.001), this was not the case for JWH‐133. Effects of post‐administered HU210 and JWH‐133 on ipsilateral hindpaw weight bearing and paw volume were comparable to the effect of systemic post‐administration of morphine and rofecoxib (3 mg/kg for both). In summary, both HU210 and JWH‐133 attenuated established inflammatory hypersensitivity and swelling, suggesting that cannabinoid‐based drugs have clinical potential for the treatment of established inflammatory pain responses.

An animal model of chronic inflammatory pain: Pharmacological and temporal differentiation from acute models

Clinically, inflammatory pain is far more persistent than that typically modelled pre‐clinically, with the majority of animal models focussing on short‐term effects of the inflammatory pain response. The large attrition rate of compounds in the clinic which show pre‐clinical efficacy suggests the need for novel models of, or approaches to, chronic inflammatory pain if novel mechanisms are to make it to the market. A model in which a more chronic inflammatory hypersensitivity phenotype is profiled may allow for a more clinically predictive tool. The aims of these studies were to characterise and validate a chronic model of inflammatory pain. We have shown that injection of a large volume of adjuvant to the intra‐articular space of the rat knee results in a prolonged inflammatory pain response, compared to the response in an acute adjuvant model. Additionally, this model also results in a hypersensitive state in the presence and absence of inflammation. A range of clinically effective analgesics demonstrate activity in this chronic model, including morphine (3 mg/kg, t.i.d.), dexamethasone (1 mg/kg, b.i.d.), ibuprofen (30 mg/kg, t.i.d.), etoricoxib (5 mg/kg, b.i.d.) and rofecoxib (0.3–10 mg/kg, b.i.d.). A further aim was to exemplify the utility of this chronic model over the more acute intra‐plantar adjuvant model using two novel therapeutic approaches; NR2B selective NMDA receptor antagonism and iNOS inhibition. Our data shows that different effects were observed with these therapies when comparing the acute model with the model of chronic inflammatory joint pain. These data suggest that the chronic model may be more relevant to identifying mechanisms for the treatment of chronic inflammatory pain states in the clinic.

Cyclooxygenase-1-Derived Prostaglandins in the Periaqueductal Gray Differentially Control C- versus A-Fiber-Evoked Spinal Nociception

Nonsteroidal anti-inflammatory drugs (NSAIDs) exert analgesic effects by inhibiting peripheral cyclooxygenases (COXs). It is now clear that these drugs also have central actions that include the modulation of descending control of spinal nociception from the midbrain periaqueductal gray (PAG). Descending control is a powerful determinant of the pain experience and is thus a potential target for analgesic drugs, including COX inhibitors. Noxious information from the periphery is conveyed to the spinal cord in A- and C-fiber nociceptors, which convey different qualities of the pain signal and have different roles in chronic pain. This in vivo study used different rates of skin heating to preferentially activate A- or C-heat nociceptors to further investigate the actions of COX inhibitors and prostaglandins in the PAG on spinal nociceptive processing. The results significantly advance our understanding of the central mechanisms underlying the actions of NSAIDs and prostaglandins by demonstrating that (1) in the PAG, it is COX-1 and not COX-2 that is responsible for acute antinociceptive effects of NSAIDs in vivo; (2) these effects are only evoked from the opioid-sensitive ventrolateral PAG; and (3) prostaglandins in the PAG exert tonic facilitatory control that targets C- rather than A-fiber-mediated spinal nociception. This selectivity of control is of particular significance given the distinct roles of A- and C-nociceptors in acute and chronic pain. Thus, effects of centrally acting prostaglandins are pivotal, we suggest, to both the understanding of nociceptive processing and the development of new analgesic drugs.

Chronic secondary hypersensitivity of dorsal horn neurones following inflammation of the knee joint

Abstract Intra‐articular injection of Freund’s complete adjuvant (FCA) into the rat knee joint produces a swelling of the joint and long lasting hypersensitivity. In this study we have used this model and in vivo electrophysiology to investigate the timecourse of spinal changes underlying chronic secondary hypersensitivity, by stimulating the ankle joint (an area outside the site of primary hypersensitivity), and have compared the results with behavioural data from the same population of animals at 4–8, 13–17 and 55–59 days following FCA injection. The magnitude of responses and the proportion of dorsal horn neurones receiving inputs from Aβ‐ Aδ‐ and C‐fibre afferents were monitored. At all timepoints, there was a significant increase in the ongoing activity of deep dorsal horn neurones when compared to naı¨ve rats, correlating well with the behavioural hypersensitivity. Both the magnitude of neuronal responses, and the proportion of neurones responding to electrical or mechanical stimulation in an area of secondary hypersensitivity, were significantly increased 4–8 and 13–17 days following FCA injection. However, while there was still behavioural hypersensitivity at 55–59 days there was a substantial decline in the responses to mechanical stimulation and A‐fibre responses to electrical stimulation, although the proportion of neurones responding in the C‐fibre latency remained elevated. These results suggest that the behavioural hypersensitivity is due to hyperexcitability at the level of the dorsal horn reflected as an increase of both C‐fibre responses and spontaneous activity.

Discovery of 1-[4-(3-Chlorophenylamino)-1-methyl-1H-pyrrolo[3,2-c]pyridin-7-yl]-1-morpholin-4-ylmethanone (GSK554418A), a Brain Penetrant 5-Azaindole CB2 Agonist for the Treatment of Chronic Pain

We report the synthesis and SAR of a series of novel azaindole CB(2) agonists. 6-Azaindole 18 showed activity in an acute pain model but was inactive in a chronic model. 18 is a Pgp substrate with low brain penetration. The template was redesigned, and the resulting 5-azaindole 36 was a potent CB(2) agonist with high CNS penetration. This compound was efficacious in the acute model and the chronic joint pain model.

Changes in dorsal root ganglion CGRP expression in a chronic inflammatory model of the rat knee joint: Differential modulation by rofecoxib and paracetamol

Neuropeptide‐expressing small diameter sensory neurones are thought to be vital in generating inflammatory hyperalgesic responses. Within the dorsal root ganglion (DRG), both the levels of the neuropeptide calcitonin gene‐related peptide (CGRP) and the numbers of CGRP‐immunoreactive (CGRP‐IR) DRG neurones have been shown to increase in a number of acute adjuvant‐induced inflammatory pain models. The aim of this study was to look specifically at changes in numbers of CGRP‐IR DRG neurones in a chronic model of inflammatory joint pain following complete Freunds adjuvant (CFA) injection into the rat knee. In this model, there were significant increases in the number of ipsilateral CGRP‐IR small DRG neurones at days 1, 16 and 35 following intra‐articular CFA, compared to saline‐injected sham animals. This correlated with the behavioural readouts of hypersensitivity and knee joint inflammation at the same time points. There was also a significant increase in the number of ipsilateral CGRP‐IR medium DRG neurones and contralateral CGRP‐IR small DRG neurones at day 1. Following dosing of CFA‐injected rats with rofecoxib (Vioxx™) or paracetamol, there was a significant decrease in the number of ipsilateral CGRP‐IR small and medium DRG neurones in rofecoxib‐ but not paracetamol‐treated rats. These data also correlated with behavioural readouts where hypersensitivity and knee joint inflammation were significantly reduced by rofecoxib but not paracetamol treatment. In conclusion, these data show that changes in ipsilateral CGRP expression within small DRG neurones are consistent with behavioural readouts in both time course, rofecoxib and paracetamol studies in this model of chronic inflammatory pain.

Discovery of GSK345931A: An EP(1) receptor antagonist with efficacy in preclinical models of inflammatory pain.

Herein we describe the medicinal chemistry programme to identify a potential back-up compound to the EP(1) receptor antagonist GW848687X. This work started with the lipophilic 1,2-biaryl benzene derivative 4 which displayed molecular weight of 414.9g/mol and poor in vivo metabolic stability in the rat and resulted in the identification of compound 7i (GSK345931A) which demonstrated good metabolic stability in the rat and lower molecular weight (381.9g/mol). In addition, 7i (GSK345931A) showed measurable CNS penetration in the mouse and rat and potent analgesic efficacy in acute and sub-chronic models of inflammatory pain.