Andrew Billinton

LABORAS™: Initial pharmacological validation of a system allowing continuous monitoring of laboratory rodent behaviour

A newly developed apparatus for automated behavioural analysis, Laboratory Animal Behaviour Observation, Registration and Analysis System (LABORAS), has been further validated with respect to the ability of the system to detect the pharmacodynamic effects of standard pharmacological tools. Data were obtained from rats administered with mCPP (reversal with SB242084), 8-OH-DPAT (reversal with WAY100635), amphetamine (reversal with haloperidol) and angiotensin, with the focus on locomotor activity, feeding and drinking behaviours. The data captured and analysed by LABORAS, suggests that the automated system is able to detect pharmacologically induced changes in behaviour, reliably and efficiently, with a significant reduction in the number of animals required, and reduced operator input.

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.

Further validation of LABORAS™ using various dopaminergic manipulations in mice including MPTP-induced nigro-striatal degeneration

The automated behavioural apparatus, LABORAS (Laboratory Animal Behaviour Observation, Registration and Analysis System), has been further validated with respect to the ability of the system to detect behavioural impairments in mice, following various dopaminergic manipulations. Initially data were obtained from mice administered with amphetamine, haloperidol, SCH23390, apomorphine and L-DOPA, with the focus on locomotor and grooming activities. The data recorded by LABORAS on administration of these pharmacological tool compounds, is comparable with published findings using standard LMA systems and conventional observer methods. In addition the home cage behaviour of mice administered with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) using an acute dosing regimen was also investigated. In LABORAS, mice subjected to MPTP lesioning showed deficits in spontaneous motor activity at day 6-7 post-MPTP administration, over a 24 h test period, as compared to saline treated controls. The data captured and analysed using LABORAS, suggests that the automated system is able to detect both pharmacologically and lesion-induced changes in behaviour of mice, reliably and efficiently.

High-performance liquid chromatography/tandem mass spectrometry assay for the determination of 1-methyl-4-phenyl pyridinium (MPP+) in brain tissue homogenates

A high-throughput liquid chromatography/tandem mass spectrometry method has been developed for the quantitative assessment of 1-methyl-4-phenylpyridinium (MPP+) in brain tissue samples. This separation is based on reversed phase chromatography using formic acid and acetonitrile as the mobile phase. Using gradient separation conditions, MPP+ was resolved within 5 min and detected using tandem mass spectrometry in the positive ion electrospray mode. The limit of detection for MPP+ was found to be 1 fmol on column with a signal to noise ratio of 3:1. The assay has been used routinely in our laboratory for the measurement of MPP+ levels in brain tissue from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, and can be used to distinguish neuroprotective efficacy and monoamine oxidase inhibition.

A novel behavioural registration system LABORAS and the social interaction paradigm detect long-term functional deficits following middle cerebral artery occlusion in the rat.

Following stroke, patients suffer a wide range of disabilities including motor impairment, anxiety and depression. However, to date, characterisation of rodent stroke models has concentrated mainly on the investigation of motor deficits. The aim of the present studies was therefore to investigate home cage behaviour (as assessed by a recently developed automatic behavioural classification system, LABORAS) and social behaviour (as a measure of anxiety) in rats following transient middle cerebral artery occlusion (tMCAO). Rats subjected to tMCAO (90 min) showed deficits in general home cage behaviours including locomotion, rearing, grooming and drinking for up to 7 weeks post occlusion, as compared with sham operated controls. In addition, a significant decrease in the total duration of social interaction was also observed in occluded rats compared with shams. The data shows that in addition to motor deficits, animals display changes in home cage behaviour and decreased social behaviour which, in contrast to motor function, are prolonged over time. Transient MCAO in rats may therefore provide a pre-clinical model to investigate agents offering symptomatic relief for ischaemia-induced motor deficits and anxiety over time following injury.

Lead Discovery for Microsomal Prostaglandin E Synthase Using a Combination of High-Throughput Fluorescent-Based Assays and RapidFire Mass Spectrometry

Microsomal prostaglandin E synthase-1 (mPGES-1) represents an attractive target for the treatment of rheumatoid arthritis and pain, being upregulated in response to inflammatory stimuli. Biochemical assays for prostaglandin E synthase activity are complicated by the instability of the substrate (PGH2) and the challenge of detection of the product (PGE2). A coupled fluorescent assay is described for mPGES-1where PGH2 is generated in situ using the action of cyclooxygenase 2 (Cox-2) on arachidonic acid. PGE2 is detected by coupling through 15-prostaglandin dehydrogenase (15-PGDH) and diaphorase. The overall coupled reaction was miniaturized to 1536-well plates and validated for high-throughput screening. For compound progression, a novel high-throughput mass spectrometry assay was developed using the RapidFire platform. The assay employs the same in situ substrate generation step as the fluorescent assay, after which both PGE2 and a reduced form of the unreacted substrate were detected by mass spectrometry. Pharmacology and assay quality were comparable between both assays, but the mass spectrometry assay was shown to be less susceptible to interference and false positives. Exploiting the throughput of the fluorescent assay and the label-free, direct detection of the RapidFire has proved to be a powerful lead discovery strategy for this challenging target.

Pyrazolopyridazine alpha-2-delta-1 ligands for the treatment of neuropathic pain

Optimization of the novel alpha-2-delta-1 ligand 4 provided compounds 37 and 38 which have improved DMPK profiles, good in vivo analgesic activity and in vitro selectivity over alpha-2-delta-2. An in-house P-gp prediction programme and the MetaSite software package were used to help solve the specific problems of high P-gp efflux and high in vivo clearance.

GABAB(1) splice variant mRNAs are differentially affected by electroshock induced seizure in rats

Receptor autoradiography with the high affinity antagonist radioligand [3H]CGP62349 and in situ hybridization with radiolabelled oligonucleotides were used to investigate GABAB receptor protein expression, and GABAB(1) mRNA splice variant (GABAB(1a) and GABAB(1b)) levels, in brain sections from rats 4 h following a single electroshock-induced generalized seizure. Densitometric analysis indicated that GABAB(1a) mRNA levels were not significantly altered by an acute electroshock seizure, but that GABAB(1b) mRNA levels were significantly increased throughout the brain. GABAB receptor expression at this time point was unaffected by the seizure. The observed up-regulation of GABAB(1b) mRNA levels may imply increased importance of this splice variant in the regulation of further seizure activity.

Central Inhibition of Granulocyte-Macrophage Colony-Stimulating Factor is Analgesic in Experimental Neuropathic Pain

Abstract With less than 50% of patients responding to the current standard of care and poor efficacy and selectivity of current treatments, neuropathic pain continues to be an area of considerable unmet medical need. Biological therapeutics such as monoclonal antibodies (mAbs) provide better intrinsic selectivity; however, delivery to the central nervous system (CNS) remains a challenge. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is well described in inflammation-induced pain, and early-phase clinical trials evaluating its antagonism have exemplified its importance as a peripheral pain target. Here, we investigate the role of this cytokine in a murine model of traumatic nerve injury and show that deletion of the GM-CSF receptor or treatment with an antagonizing mAb alleviates pain. We also demonstrate enhanced analgesic efficacy using an engineered construct that has greater capacity to penetrate the CNS. Despite observing GM-CSF receptor expression in microglia and astrocytes, the gliosis response in the dorsal horn was not altered in nerve injured knockout mice compared with wild-type littermate controls as evaluated by ionized calcium binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein, respectively. Functional analysis of glial cells revealed that pretreatment with GM-CSF potentiated lipopolysaccharide-induced release of proinflammatory cytokines. In summary, our data indicate that GM-CSF is a proinflammatory cytokine that contributes to nociceptive signalling through driving spinal glial cell secretion of proinflammatory mediators. In addition, we report a successful approach to accessing CNS pain targets, providing promise for central compartment delivery of analgesics.