Nick M. Clayton

CB1 and CB2 cannabinoid receptors are implicated in inflammatory pain.

&NA; The cannabinoid agonist, HU210 has been evaluated in vivo in nociceptive and inflammatory pain models in the rat. The ED50 for the anti‐nociceptive (increasing mechanical withdrawal threshold) effect was 0.1 mg/kg−1 i.p., and for anti‐hypersensitivity and anti‐inflammatory activity was 5 &mgr;g/kg−1 i.p. (in the carrageenan model). The selective CB1 antagonist, AM281 (0.5 mg/kg−1 i.p.) reversed effects of HU210 (10 and 30 &mgr;g/kg−1 i.p.) in both nociceptive and inflammatory models of hypersensitivity. The selective CB2 antagonist, SR144528 (1 mg/kg−1 i.p.) antagonised effects of HU210 (30 &mgr;g/kg−1 i.p.) in the carrageenan induced inflammatory hypersensitivity. The CB2 agonist, 1‐(2,3‐Dichlorobenzoyl)‐5‐methoxy‐2‐methyl‐(2‐(morpholin‐4‐yl)ethyl)−1H‐indole (GW405833) inhibited the hypersensitivity and was anti‐inflammatory in vivo. These effects were blocked by SR144528. These findings suggest that CB1 receptors are involved in nociceptive pain and that both CB1 and CB2 receptors are involved in inflammatory hypersensitivity. Future studies will investigate effects on identified inflammatory cells within the inflamed tissue to further elucidate the role of cannabinoid receptors.

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.

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.

Review article: the therapeutic potential of 5‐HT3 receptor antagonists in the treatment of irritable bowel syndrome

There is evidence from studies, in both animals and humans, that 5‐HT3 receptor blockade has potential value in the treatment of irritable bowel syndrome, particularly in those patients with diarrhoea‐predominant bowel habits. New findings suggest that 5‐HT3 receptors exist on gut afferent neurones and that their activation by locally released 5‐HT leads to visceral nociceptive stimulation, in addition to increased neuronally‐mediated motor and secretory activity. If this concept is validated, it will provide a rationale for the use of 5‐HT3 receptor antagonists in patients with increased gut motility, reduced fluid absorption and low nociceptive thresholds leading to abdominal pain.

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.

Changes in vanilloid receptor 1 (TRPV1) expression following lingual nerve injury.

We have investigated a possible role for vanilloid receptor 1 (TRPV1), a transducer of noxious stimuli, in the development of neuropathic pain following injury to a peripheral branch of the trigeminal nerve. In nine adult ferrets the left lingual nerve was sectioned and recovery permitted for 3 days, 3 weeks or 3 months (3 ferrets per group). A retrograde tracer, fluorogold, was injected into the damaged nerve to identify associated cell bodies in the trigeminal ganglion. Three further ferrets, receiving only tracer injection, served as uninjured controls. Indirect immunofluorescence for TRPV1 and image analysis was used to quantify the percentage area of staining (PAS) of TRPV1 in the left and right lingual nerves. Additionally, the proportion of fluorogold positive and fluorogold negative cells expressing TRPV1 in the ganglion was determined. TRPV1 expression increased significantly at the injury site of damaged nerves 3 days after injury and this was matched by a reduction in the proportion of fluorogold positive cells expressing TRPV1 in the ganglion. At 3 weeks TRPV1 expression at the injury site was still high, while in the ganglion was significantly greater than in the controls. In the 3‐month recovery group TRPV1 expression in both nerve fibres and ganglion cells, was not significantly different from controls and there were no changes in expression in the fluorogold negative cells in the ganglion at any time point studied. These data suggest that after injury there is an increase in the axonal transport of TRPV1 from the cell bodies to the damaged axons and this is followed by an increase in synthesis in the ganglion. These changes in expression may be involved in development of sensory disturbances or dysaesthesia after injury.

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.

Activation of the α7-Nicotinic Acetylcholine Receptor Reverses Complete Freund Adjuvant–Induced Mechanical Hyperalgesia in the Rat Via a Central Site of Action

UNLABELLED The role of specific nicotinic receptor (nAChR) subtypes in antinociception has not been fully elucidated because of the lack, until recently, of selective tool compounds. (R)-N-(1-azabicyclo[2.2.2]oct-3-yl)(5-(2-pyridyl)thiopene-2-carboxamide) (compound B) is reported to be an agonist selective for the alpha(7)nAChR and in the present study was found to be efficacious in inflammatory pain models in 2 species. Compound B reversed complete Freund adjuvant-induced reductions in paw withdrawal thresholds in rat and mouse in a dose-related manner, producing maximum reversals of 65% +/- 4% at 10 mg/kg and 87% +/- 15% at 20 mg/kg. When rats and mice were predosed with the centrally penetrant, broad-spectrum nicotinic receptor antagonist mecamylamine, the efficacy of the agonist was significantly inhibited, producing reversals of only 11% +/- 5% at 10 mg/kg and 5% +/- 13% at 20 mg/kg, confirming activity via nicotinic receptors. Rats were also predosed systemically with the selective low-brain penetrant alpha(7)-antagonist methyllycaconitine, which had no effect on agonist activity (90% +/- 18% at 10 mg/kg), suggesting a central involvement. This hypothesis was further established with methyllycaconitine completely inhibited the agonist effect when dosed intrathecally (1% +/- 7%). PERSPECTIVE These studies provide good rationale for the utility of selective, central nervous system penetrant agonists at the alpha(7)-nicotinic receptor for the treatment of inflammatory pain.

Changes in sodium channel expression following trigeminal nerve injury.

We have investigated the expression of TTX-sensitive (TTXs) and TTX-resistant (TTXr) sodium channel subtypes following injury to the inferior alveolar nerve (IAN), in order to determine their potential role in the development of trigeminal neuropathic pain. In seven anaesthetised ferrets, fluorogold (2%) was injected into the left IAN to identify cell bodies with axons in this nerve. In four animals, the nerve was sectioned distal to the injection site and the remaining three served as controls. After 3 days, the animals were perfused with 4% paraformaldehyde. The left and right IANs and trigeminal ganglia were processed using indirect immunofluorescence with specific primary antibodies to TTXs subtypes Na(v)1.3 and Na(v)1.7 and TTXr subtypes Na(v)1.8 and Na(v)1.9. Image analysis was used to quantify the percentage area of staining (PAS) in the nerves. In the ganglia, counts were made of positively labelled cells in the fluorogold population. PAS for Na(v)1.8 and Na(v)1.9 was significantly greater in injured nerves than in either contralateral or control nerves. After injury, significantly fewer cells in the ganglia expressed Na(v)1.3 (controls 36.9%; injured 13.1%), Na(v)1.7 (controls 17.0%; injured 8.1%) and Na(v)1.9 (controls 60.3%; injured 29.0%) (p<0.05, unpaired t test). These changes are different from those previously reported in the dorsal root ganglion following damage to peripheral nerves of spinal origin. As they occur at a time of known high abnormal neural discharge, it seems likely that changes in sodium channel expression may play a role in nerve injury-induced trigeminal pain.

Pyridine-3-carboxamides as novel CB 2 agonists for analgesia

We describe herein the medicinal chemistry approach which led to the discovery of a novel pyridine-3-carboxamide series of CB(2) receptor agonists. The SAR of this new template was evaluated and culminated in the identification of analogue 14a which demonstrated efficacy in an in vivo model of inflammatory pain.