Andy Billinton

The putative cannabinoid receptor GPR55 plays a role in mechanical hyperalgesia associated with inflammatory and neuropathic pain

Abstract It has been postulated that the G protein‐coupled receptor, GPR55, is a third cannabinoid receptor. Given that the ligands at the CB1 and CB2 receptors are effective analgesic and anti‐inflammatory agents, the role of GPR55 in hyperalgesia associated with inflammatory and neuropathic pain has been investigated. As there are no well‐validated GPR55 tool compounds, a GPR55 knockout (GPR55−/−) mouse line was generated and fully backcrossed onto the C57BL/6 strain. General phenotypic analysis of GPR55−/− mice revealed no obvious primary differences, compared with wild‐type (GPR55+/+) littermates. GPR55−/− mice were then tested in the models of adjuvant‐induced inflammation and partial nerve ligation. Following intraplantar administration of Freund’s complete adjuvant (FCA), inflammatory mechanical hyperalgesia was completely absent in GPR55−/− mice up to 14 days post‐injection. Cytokine profiling experiments showed that at 14 days post‐FCA injection there were increased levels of IL‐4, IL‐10, IFNγ and GM‐CSF in paws from the FCA‐injected GPR55−/− mice when compared with the FCA‐injected GPR55+/+ mice. This suggests that GPR55 signalling can influence the regulation of certain cytokines and this may contribute to the lack of inflammatory mechanical hyperalgesia in the GPR55−/− mice. In the model of neuropathic hypersensitivity, GPR55−/− mice also failed to develop mechanical hyperalgesia up to 28 days post‐ligation. These data clearly suggest that the manipulation of GPR55 may have therapeutic potential in the treatment of both inflammatory and neuropathic pain.

Comparative cellular distribution of GABAA and GABAB receptors in the human basal ganglia: Immunohistochemical colocalization of the α1 subunit of the GABAA receptor, and the GABABR1 and GABABR2 receptor subunits

The GABAB receptor is a G‐protein linked metabotropic receptor that is comprised of two major subunits, GABABR1 and GABABR2. In this study, the cellular distribution of the GABABR1 and GABABR2 subunits was investigated in the normal human basal ganglia using single and double immunohistochemical labeling techniques on fixed human brain tissue. The results showed that the GABAB receptor subunits GABABR1 and GABABR2 were both found on the same neurons and followed the same distribution patterns. In the striatum, these subunits were found on the five major types of interneurons based on morphology and neurochemical labeling (types 1, 2, 3, 5, 6) and showed weak labeling on the projection neurons (type 4). In the globus pallidus, intense GABABR1 and GABABR2 subunit labeling was found in large pallidal neurons, and in the substantia nigra, both pars compacta and pars reticulata neurons were labeled for both receptor subunits. Studies investigating the colocalization of the GABAA α1 subunit and GABAB receptor subunits showed that the GABAA receptor α1 subunit and the GABABR1 subunit were found together on GABAergic striatal interneurons (type 1 parvalbumin, type 2 calretinin, and type 3 GAD neurons) and on neurons in the globus pallidus and substantia nigra pars reticulata. GABABR1 and GABABR2 were found on substantia nigra pars compacta neurons but the GABAA receptor α1 subunit was absent from these neurons. The results of this study provide the morphological basis for GABAergic transmission within the human basal ganglia and provides evidence that GABA acts through both GABAA and GABAB receptors. That is, GABA acts through GABAB receptors, which are located on most of the cell types of the striatum, globus pallidus, and substantia nigra. GABA also acts through GABAA receptors containing the α1 subunit on specific striatal GABAergic interneurons and on output neurons of the globus pallidus and substantia nigra pars reticulata. J. Comp. Neurol. 470:339–356, 2004.

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.

Non-acidic pyrazole EP1 receptor antagonists with in vivo analgesic efficacy

Replacement of the carboxylic acid group in a series of previously described methylene-linked pyrazole EP(1) receptor antagonists led to the discovery of amide, reversed amide and carbamate derivatives. Two compounds, 10a and 10b, were identified as brain penetrant compounds and both demonstrated efficacy in the CFA model of inflammatory pain.

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.

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.

Discovery of a novel indole series of EP1 receptor antagonists by scaffold hopping

We describe the medicinal chemistry approach that generated a novel indole series of EP(1) receptor antagonists. The SAR of this new template was evaluated and culminated in the identification of compound 12g which demonstrated in vivo efficacy in a preclinical model of inflammatory pain.

Identification of 2-oxo-N-(phenylmethyl)-4-imidazolidinecarboxamide antagonists of the P2X(7) receptor.

A backup molecule to compound 2 was sought by targeting the most likely metabolically vulnerable site in this molecule. Compound 18 was subsequently identified as a potent P2X(7) antagonist with very low in vivo clearance and high oral bioavailability in all species examined. Some evidence to support the role of P2X(7) in the etiology of pain is also presented.

Structure-activity relationships and in vivo activity of (1H-pyrazol-4-yl)acetamide antagonists of the P2X7 receptor

Structure-activity relationships (SAR) of analogues of lead compound 1 were investigated and compound 16 was selected for further study in animal models of pain. Compound 16 was shown to be a potent antihyperalgesic agent in both the rat acute complete Freunds adjuvant (CFA) model of inflammatory pain [Iadarola, M. J.; Douglass, J.; Civelli, O.; Naranjo, J. R. rain Res.1988, 455, 205] and the knee joint model of chronic inflammatory pain [Wilson, A. W.; Medhurst, S. J.; Dixon, C. I.; Bontoft, N. C.; Winyard, L. A.; Brackenborough, K. T.; De Alba, J.; Clarke, C. J.; Gunthorpe, M. J.; Hicks, G. A.; Bountra, C.; McQueen, D. S.; Chessell, I. P. Eur. J. Pain2006, 10, 537].

Discovery of sodium 6-[(5-chloro-2-{[(4-chloro-2-fluorophenyl)methyl]oxy}phenyl)methyl]-2-pyridinecarboxylate (GSK269984A) an EP1 receptor antagonist for the treatment of inflammatory pain

We describe the medicinal chemistry programme that led to the identification of the EP(1) receptor antagonist GSK269984A (8h). GSK269984A was designed to overcome development issues encountered with previous EP(1) antagonists such as GW848687X and was found to display excellent activity in preclinical models of inflammatory pain. However, upon cross species pharmacokinetic profiling, GSK269984A was predicted to have suboptimal human pharmacokinetic and was thus progressed to a human microdose study.