register interest

Professor Chas Bountra

Research Area: Protein Science and Structural Biology
Technology Exchange: Computational biology, Crystallography, Drug discovery, Immunohistochemistry and In vivo imaging
Scientific Themes: Protein Science & Structural Biology and Physiology, Cellular & Molecular Biology
Keywords: target validation, drug discovery, translational science, structural biology, chemical probes and epigenetics
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SGC Oxford Chief Scientist

Chas Bountra, PhD

Chas is Professor of Translational Medicine in the Nuffield Department of Clinical Medicine and Associate Member of the Department of Pharmacology at the University of Oxford. He is also a Visiting Professor in Neuroscience and Mental Health at Imperial College, London. Chas is an invited expert on several government and charitable research funding bodies, and an advisor for many academic, biotech and pharma drug discovery programmes.

Prior to coming back to Oxford, Chas was Vice President and Head of Biology at GlaxoSmithKline. He was involved in the identification of more than 40 clinical candidates for many gastro-intestinal, inflammatory and neuro-psychiatric diseases. More than 20 of these molecules progressed into patient studies and more than five of these delivered successful “Proof of Concept” data and hence progressed into late stage development. He was involved in the launch and development of the first treatment for Irritable Bowel Syndrome (Alosetron) and was the first to show that neurokinin NK1 antagonists are anti-emetic in preclinical and clinical studies.

His current interests are i) using X ray structures of novel human proteins to generate small molecule inhibitors, screening in human cells to identify novel targets for drug discovery, and then developing clinical candidates for evaluation in patients, pre-competitively ii) focussing on epigenetic and genetically identified proteins, because these are likely to represent better targets for drug discovery, for many cancer, inflammatory, metabolic and neuro-psychiatric diseases iii) working with colleagues in Oxford to build major programmes in rare diseases and  in Alzheimers Disease, and creating a “BioEscalator” for the rapid translation of SGC science and iv) building stronger links with local hospitals, patient groups, regulatory agencies, private investors, CROs, biotechs and large pharma companies, to create a new, more efficient ecosystem for pioneer drug discovery.

Chas believes the SGC has become a leader in human protein structural biology and epigenetics chemical biology, and is arguably one of the most successful open innovation, public – private partnerships  in the world. Furthermore, with the many recent local developments (Target Discovery Institute, Kennedy Institute, Dementia Institute), he believes Oxford is emerging as one of the major academic drug discovery centres in Europe.

He has given over 300 invited lectures.  In 2012 he was voted one of the “top innovators in the industry”.

Name Department Institution Country
Professor Christopher Schofield Chemistry Oxford University United Kingdom
Rob Klose University of Oxford United Kingdom
Chris Austin National Chemical Genomic Centre, NIH, Washington United States
Professor Tim Willson SGC Center for Chemical Biology UNC Eshelman School of Pharmacy United States
David LLoyd Trinity College, Dublin Ireland
Luke O'Neill Trinity College, Dublin Ireland
David Gavaghan University of Oxford United Kingdom
Professor Liz Carpenter Structural Genomics Consortium University of Oxford United Kingdom
Dr Brian D Marsden Structural Genomics Consortium University of Oxford United Kingdom
Professor Stefan Knapp Structural Genomics Consortium University of Oxford United Kingdom
Dr Alex Bullock Structural Genomics Consortium University of Oxford United Kingdom
Dr Frank von Delft Structural Genomics Consortium University of Oxford United Kingdom
Dr Nicola A Burgess-Brown Structural Genomics Consortium University of Oxford United Kingdom
Dr Opher Gileadi Structural Genomics Consortium University of Oxford United Kingdom
Professor Panagis Filippakopoulos Structural Genomics Consortium University of Oxford United Kingdom
Professor Paul Brennan Target Discovery Institute University of Oxford United Kingdom
Dr Susanne Muller-Knapp Structural Genomics Consortium University of Oxford United Kingdom
Professor Wyatt W Yue Structural Genomics Consortium University of Oxford United Kingdom
Arshad Z, Smith J, Roberts M, Lee WH, Davies B, Bure K, Hollander GA, Dopson S, Bountra C, Brindley D. 2016. Open Access Could Transform Drug Discovery: A Case Study of JQ1. Expert Opin Drug Discov, 11 (3), pp. 321-332. | Show Abstract | Read more

INTRODUCTION: The cost to develop a new drug from target discovery to market is a staggering $1.8 billion, largely due to the very high attrition rate of drug candidates and the lengthy transition times during development. Open access is an emerging model of open innovation that places no restriction on the use of information and has the potential to accelerate the development of new drugs. AREAS COVERED: To date, no quantitative assessment has yet taken place to determine the effects and viability of open access on the process of drug translation. This need is addressed within this study. The literature and intellectual property landscapes of the drug candidate JQ1, which was made available on an open access basis when discovered, and conventionally developed equivalents that were not are compared using the Web of Science and Thomson Innovation software, respectively. EXPERT OPINION: Results demonstrate that openly sharing the JQ1 molecule led to a greater uptake by a wider and more multi-disciplinary research community. A comparative analysis of the patent landscapes for each candidate also found that the broader scientific diaspora of the publically released JQ1 data enhanced innovation, evidenced by a greater number of downstream patents filed in relation to JQ1. The authors' findings counter the notion that open access drug discovery would leak commercial intellectual property. On the contrary, JQ1 serves as a test case to evidence that open access drug discovery can be an economic model that potentially improves efficiency and cost of drug discovery and its subsequent commercialization.

Bavetsias V, Lanigan RM, Ruda GF, Atrash B, McLaughlin MG, Tumber A, Mok NY, Le Bihan YV et al. 2016. 8-Substituted Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatives As Potent, Cell Permeable, KDM4 (JMJD2) and KDM5 (JARID1) Histone Lysine Demethylase Inhibitors. J Med Chem, 59 (4), pp. 1388-1409. | Show Abstract | Read more

We report the discovery of N-substituted 4-(pyridin-2-yl)thiazole-2-amine derivatives and their subsequent optimization, guided by structure-based design, to give 8-(1H-pyrazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-ones, a series of potent JmjC histone N-methyl lysine demethylase (KDM) inhibitors which bind to Fe(II) in the active site. Substitution from C4 of the pyrazole moiety allows access to the histone peptide substrate binding site; incorporation of a conformationally constrained 4-phenylpiperidine linker gives derivatives such as 54j and 54k which demonstrate equipotent activity versus the KDM4 (JMJD2) and KDM5 (JARID1) subfamily demethylases, selectivity over representative exemplars of the KDM2, KDM3, and KDM6 subfamilies, cellular permeability in the Caco-2 assay, and, for 54k, inhibition of H3K9Me3 and H3K4Me3 demethylation in a cell-based assay.

Rekhi R, Smith JA, Arshad Z, Roberts M, Bountra C, Bingham I, Bure K, Brindley DA. 2015. Decision-support tools for monoclonal antibody and cell therapy bioprocessing: Current landscape and development opportunities BioProcess International, 13 (11),

Fedorov O, Castex J, Tallant C, Owen DR, Martin S, Aldeghi M, Monteiro O, Filippakopoulos P et al. 2015. Selective targeting of the BRG/PB1 bromodomains impairs embryonic and trophoblast stem cell maintenance. Sci Adv, 1 (10), pp. e1500723. | Show Abstract | Read more

Mammalian SWI/SNF [also called Brg/Brahma-associated factors (BAFs)] are evolutionarily conserved chromatin-remodeling complexes regulating gene transcription programs during development and stem cell differentiation. BAF complexes contain an ATP (adenosine 5'-triphosphate)-driven remodeling enzyme (either BRG1 or BRM) and multiple protein interaction domains including bromodomains, an evolutionary conserved acetyl lysine-dependent protein interaction motif that recruits transcriptional regulators to acetylated chromatin. We report a potent and cell active protein interaction inhibitor, PFI-3, that selectively binds to essential BAF bromodomains. The high specificity of PFI-3 was achieved on the basis of a novel binding mode of a salicylic acid head group that led to the replacement of water molecules typically maintained in other bromodomain inhibitor complexes. We show that exposure of embryonic stem cells to PFI-3 led to deprivation of stemness and deregulated lineage specification. Furthermore, differentiation of trophoblast stem cells in the presence of PFI-3 was markedly enhanced. The data present a key function of BAF bromodomains in stem cell maintenance and differentiation, introducing a novel versatile chemical probe for studies on acetylation-dependent cellular processes controlled by BAF remodeling complexes.

Arrowsmith CH, Audia JE, Austin C, Baell J, Bennett J, Blagg J, Bountra C, Brennan PE et al. 2015. Corrigendum: The promise and peril of chemical probes. Nat Chem Biol, 11 (11), pp. 887. | Read more

Arrowsmith CH, Audia JE, Austin C, Baell J, Bennett J, Blagg J, Bountra C, Brennan PE et al. 2015. The promise and peril of chemical probes. Nat Chem Biol, 11 (8), pp. 536-541. | Show Abstract | Read more

© 2015 Nature America, Inc. All rights reserved.Chemical probes are powerful reagents with increasing impacts on biomedical research. However, probes of poor quality or that are used incorrectly generate misleading results. To help address these shortcomings, we will create a community-driven wiki resource to improve quality and convey current best practice.

Anand U, Yiangou Y, Sinisi M, Fox M, MacQuillan A, Quick T, Korchev YE, Bountra C, McCarthy T, Anand P. 2015. Food for thought. Nat Chem Biol, 11 (1), pp. 1. | Show Abstract | Read more

© 2015 Anand et al.Background: The clinical efficacy of the Angiotensin II (AngII) receptor AT<inf>2</inf>R antagonist EMA401, a novel peripherally-restricted analgesic, was reported recently in post-herpetic neuralgia. While previous studies have shown that AT<inf>2</inf>R is expressed by nociceptors in human DRG (hDRG), and that EMA401 inhibits capsaicin responses in cultured hDRG neurons, the expression and levels of its endogenous ligands AngII and AngIII in clinical neuropathic pain tissues, and their signalling pathways, require investigation. We have immunostained AngII, AT<inf>2</inf>R and the capsaicin receptor TRPV1 in control post-mortem and avulsion injured hDRG, control and injured human nerves, and in cultured hDRG neurons. AngII, AngIII, and Ang-(1-7) levels were quantified by ELISA. The in vitro effects of AngII, AT<inf>2</inf>R agonist C21, and Nerve growth factor (NGF) were measured on neurite lengths; AngII, NGF and EMA401 effects on expression of p38 and p42/44 MAPK were measured using quantitative immunofluorescence, and on capsaicin responses using calcium imaging. Results: AngII immunostaining was observed in approximately 75% of small/medium diameter neurons in control (n=5) and avulsion injured (n=8) hDRG, but not large neurons i.e. similar to TRPV1. AngII was co-localised with AT<inf>2</inf>R and TRPV1 in hDRG and in vitro. AngII staining by image analysis showed no significant difference between control (n=12) and injured (n=13) human nerves. AngII levels by ELISA were also similar in control human nerves (4.09±0.36pmol/g, n=31), injured nerves (3.99±0.79pmol/g, n=7), and painful neuromas (3.43±0.73pmol/g, n=12); AngIII and Ang-(1-7) levels were undetectable (<0.03 and 0.05pmol/g respectively). Neurite lengths were significantly increased in the presence of NGF, AngII and C21 in cultured DRG neurons. AngII and, as expected, NGF significantly increased signal intensity of p38 and p42/44 MAPK, which was reversed by EMA401. AngII mediated sensitization of capsaicin responses was not observed in the presence of MAP kinase inhibitor PD98059, and the kinase inhibitor staurosporine. Conclusion: The major AT<inf>2</inf>R ligand in human peripheral nerves is AngII, and its levels are maintained in injured nerves. EMA401 may act on paracrine/autocrine mechanisms at peripheral nerve terminals, or intracrine mechanisms, to reduce neuropathic pain signalling in AngII/NGF/TRPV1-convergent pathways.

Chen P, Chaikuad A, Bamborough P, Bantscheff M, Bountra C, Chung CW, Fedorov O, Grandi P et al. 2016. Discovery and Characterization of GSK2801, a Selective Chemical Probe for the Bromodomains BAZ2A and BAZ2B. J Med Chem, 59 (4), pp. 1410-1424. | Show Abstract | Read more

Bromodomains are acetyl-lysine specific protein interaction domains that have recently emerged as a new target class for the development of inhibitors that modulate gene transcription. The two closely related bromodomain containing proteins BAZ2A and BAZ2B constitute the central scaffolding protein of the nucleolar remodeling complex (NoRC) that regulates the expression of noncoding RNAs. However, BAZ2 bromodomains have low predicted druggability and so far no selective inhibitors have been published. Here we report the development of GSK2801, a potent, selective and cell active acetyl-lysine competitive inhibitor of BAZ2A and BAZ2B bromodomains as well as the inactive control compound GSK8573. GSK2801 binds to BAZ2 bromodomains with dissociation constants (KD) of 136 and 257 nM for BAZ2B and BAZ2A, respectively. Crystal structures demonstrated a canonical acetyl-lysine competitive binding mode. Cellular activity was demonstrated using fluorescent recovery after photobleaching (FRAP) monitoring displacement of GFP-BAZ2A from acetylated chromatin. A pharmacokinetic study in mice showed that GSK2801 had reasonable in vivo exposure after oral dosing, with modest clearance and reasonable plasma stability. Thus, GSK2801 represents a versatile tool compound for cellular and in vivo studies to understand the role of BAZ2 bromodomains in chromatin biology.

Edwards AM, Arrowsmith CH, Bountra C, Bunnage ME, Feldmann M, Knight JC, Patel DD, Prinos P, Taylor MD, Sundström M, SGC Open Source Target-Discovery Partnership. 2015. Preclinical target validation using patient-derived cells. Nat Rev Drug Discov, 14 (3), pp. 149-150. | Show Abstract | Read more

The Structural Genomics Consortium (SGC) and its clinical, industry and disease-foundation partners are launching open-source preclinical translational medicine studies.

Perry D, Sperling R, Katz R, Berry D, Dilts D, Hanna D, Salloway S, Trojanowski JQ et al. 2015. Building a roadmap for developing combination therapies for Alzheimer's disease. Expert Rev Neurother, 15 (3), pp. 327-333. | Show Abstract | Read more

Combination therapy has proven to be an effective strategy for treating many of the world's most intractable diseases. A growing number of investigators in academia, industry, regulatory agencies, foundations and advocacy organizations are interested in pursuing a combination approach to treating Alzheimer's disease. A meeting co-hosted by the Accelerate Cure/Treatments for Alzheimer's Disease Coalition, the Critical Path Institute and the Alzheimer's Association addressed challenges in designing clinical trials to test multiple treatments in combination and outlined a roadmap for making such trials a reality.

Brindley DA, Arshad Z, Luo D, Dopson S, Hollander G, Frost S, Bountra C, Smith JA. 2015. 21(st) Century Cures Act: An Act of Cure or Diagnosis? Rejuvenation Res, 18 (4), pp. 295-298. | Read more

Anand U, Yiangou Y, Sinisi M, Fox M, MacQuillan A, Quick T, Korchev YE, Bountra C, McCarthy T, Anand P. 2015. Mechanisms underlying clinical efficacy of Angiotensin II type 2 receptor (AT2R) antagonist EMA401 in neuropathic pain: clinical tissue and in vitro studies. Mol Pain, 11 (1), pp. 38. | Show Abstract | Read more

BACKGROUND: The clinical efficacy of the Angiotensin II (AngII) receptor AT2R antagonist EMA401, a novel peripherally-restricted analgesic, was reported recently in post-herpetic neuralgia. While previous studies have shown that AT2R is expressed by nociceptors in human DRG (hDRG), and that EMA401 inhibits capsaicin responses in cultured hDRG neurons, the expression and levels of its endogenous ligands AngII and AngIII in clinical neuropathic pain tissues, and their signalling pathways, require investigation. We have immunostained AngII, AT2R and the capsaicin receptor TRPV1 in control post-mortem and avulsion injured hDRG, control and injured human nerves, and in cultured hDRG neurons. AngII, AngIII, and Ang-(1-7) levels were quantified by ELISA. The in vitro effects of AngII, AT2R agonist C21, and Nerve growth factor (NGF) were measured on neurite lengths; AngII, NGF and EMA401 effects on expression of p38 and p42/44 MAPK were measured using quantitative immunofluorescence, and on capsaicin responses using calcium imaging. RESULTS: AngII immunostaining was observed in approximately 75% of small/medium diameter neurons in control (n = 5) and avulsion injured (n = 8) hDRG, but not large neurons i.e. similar to TRPV1. AngII was co-localised with AT2R and TRPV1 in hDRG and in vitro. AngII staining by image analysis showed no significant difference between control (n = 12) and injured (n = 13) human nerves. AngII levels by ELISA were also similar in control human nerves (4.09 ± 0.36 pmol/g, n = 31), injured nerves (3.99 ± 0.79 pmol/g, n = 7), and painful neuromas (3.43 ± 0.73 pmol/g, n = 12); AngIII and Ang-(1-7) levels were undetectable (<0.03 and 0.05 pmol/g respectively). Neurite lengths were significantly increased in the presence of NGF, AngII and C21 in cultured DRG neurons. AngII and, as expected, NGF significantly increased signal intensity of p38 and p42/44 MAPK, which was reversed by EMA401. AngII mediated sensitization of capsaicin responses was not observed in the presence of MAP kinase inhibitor PD98059, and the kinase inhibitor staurosporine. CONCLUSION: The major AT2R ligand in human peripheral nerves is AngII, and its levels are maintained in injured nerves. EMA401 may act on paracrine/autocrine mechanisms at peripheral nerve terminals, or intracrine mechanisms, to reduce neuropathic pain signalling in AngII/NGF/TRPV1-convergent pathways.

Kruidenier L, Chung CW, Cheng Z, Liddle J, Che K, Joberty G, Bantscheff M, Bountra C et al. 2014. Kruidenier et al. reply. Nature, 514 (7520), pp. E2. | Show Abstract | Read more

©2014 Macmillan Publishers Limited. All rights reserved.Replying to B. Heinemann. Nature 514, http://dx.doi.org/10.1038/nature13688 (2014) We welcome the accompanying Comment by Heinemann et al., in which the authors use an extensive panel of sensitive KDM assays to independently confirm our results that GSK-J1 is a potent KDM6 inhibitor. Additionally, Heinemann et al. demonstrate that GSK-J1 has some, albeit weaker, activity towards KDM5B and KDM5C, for which we only had preliminary data available at the time of our original publication. As our jumonji assay portfolio expands, we have continued to update the GSK-J1 activity profile on the SGC website (http://www.thesgc.org/chemical-probes/GSKJ1); this includes KDM5 inhibition activity by GSK-J1 similar to that reported by Heinemann. In conclusion, GSK-J1 remains the most selective KDM inhibitor yet disclosed and thus a valuable chemical tool.

Grundy R, James L, Bountra C, Harrison T. 2014. Reconfiguring drug discovery through innovative partnerships Drug Discovery World, 15 (4), pp. 70-74. | Show Abstract

Attrition rates in drug discovery and development remain stubbornly high, despite large investments by the pharmaceutical industry.The cost of developing a new drug has been estimated at between $1.3 billion and upwards of $4 billion, if one takes into account the cost of failed development candidates'.

Borsook D, Hargreaves R, Bountra C, Porreca F. 2014. Lost but making progress--Where will new analgesic drugs come from? Sci Transl Med, 6 (249), pp. 249sr3. | Show Abstract | Read more

There is a critical need for effective new pharmacotherapies for pain. The paucity of new drugs successfully reaching the clinic calls for a reassessment of current analgesic drug discovery approaches. Many points early in the discovery process present significant hurdles, making it critical to exploit advances in pain neurobiology to increase the probability of success. In this review, we highlight approaches that are being pursued vigorously by the pain community for drug discovery, including innovative preclinical pain models, insights from genetics, mechanistic phenotyping of pain patients, development of biomarkers, and emerging insights into chronic pain as a disorder of both the periphery and the brain. Collaborative efforts between pharmaceutical, academic, and public entities to advance research in these areas promise to de-risk potential targets, stimulate investment, and speed evaluation and development of better pain therapies.

Rice AS, Dworkin RH, McCarthy TD, Anand P, Bountra C, McCloud PI, Hill J, Cutter G et al. 2014. EMA401, an orally administered highly selective angiotensin II type 2 receptor antagonist, as a novel treatment for postherpetic neuralgia: a randomised, double-blind, placebo-controlled phase 2 clinical trial. Lancet, 383 (9929), pp. 1637-1647. | Show Abstract | Read more

BACKGROUND: Existing treatments for postherpetic neuralgia, and for neuropathic pain in general, are limited by modest efficacy and unfavourable side-effects. The angiotensin II type 2 receptor (AT2R) is a new target for neuropathic pain. EMA401, a highly selective AT2R antagonist, is under development as a novel neuropathic pain therapeutic agent. We assessed the therapeutic potential of EMA401 in patients with postherpetic neuralgia. METHODS: In this multicentre, placebo-controlled, double-blind, randomised, phase 2 clinical trial, we enrolled patients (aged 22-89 years) with postherpetic neuralgia of at least 6 months' duration from 29 centres across six countries. We randomly allocated 183 participants to receive either oral EMA401 (100 mg twice daily) or placebo for 28 days. Randomisation was done according to a centralised randomisation schedule, blocked by study site, which was generated by an independent, unmasked statistician. Patients and staff at each site were masked to treatment assignment. We assessed the efficacy, safety, and pharmacokinetics of EMA401. The primary efficacy endpoint was change in mean pain intensity between baseline and the last week of dosing (days 22-28), measured on an 11-point numerical rating scale. The primary efficacy analysis was intention to treat. This trial is registered with the Australian New Zealand Clinical Trials Registry, number ACTRN12611000822987. FINDINGS: 92 patients were assigned to EMA401 and 91 were assigned to placebo. The patients given EMA401 reported significantly less pain compared with baseline values in the final week of treatment than did those given placebo (mean reductions in pain scores -2.29 [SD 1.75] vs -1.60 [1.66]; difference of adjusted least square means -0.69 [SE 0.25]; 95% CI -1.19 to -0.20; p=0.0066). No serious adverse events related to EMA401 occurred. Overall, 32 patients reported 56 treatment-emergent adverse events in the EMA401 group compared with 45 such events reported by 29 patients given placebo. INTERPRETATION: EMA401 (100 mg twice daily) provides superior relief of postherpetic neuralgia compared with placebo at the end of 28 days of treatment. EMA401 was well tolerated by patients. FUNDING: Spinifex Pharmaceuticals.

Cited:

38

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Rice ASC, Dworkin RH, McCarthy TD, Anand P, Bountra C, McCloud PI, Hill J, Cutter G, Kitson G, Desem N, Raff M. 2014. EMA401, an orally administered highly selective angiotensin II type 2 receptor antagonist, as a novel treatment for postherpetic neuralgia: A randomised, double-blind, placebo-controlled phase 2 clinical trial The Lancet, 383 (9929), pp. 1637-1647. | Show Abstract | Read more

Background: Existing treatments for postherpetic neuralgia, and for neuropathic pain in general, are limited by modest efficacy and unfavourable side-effects. The angiotensin II type 2 receptor (AT2R) is a new target for neuropathic pain. EMA401, a highly selective AT2R antagonist, is under development as a novel neuropathic pain therapeutic agent. We assessed the therapeutic potential of EMA401 in patients with postherpetic neuralgia. Methods: In this multicentre, placebo-controlled, double-blind, randomised, phase 2 clinical trial, we enrolled patients (aged 22-89 years) with postherpetic neuralgia of at least 6 months' duration from 29 centres across six countries. We randomly allocated 183 participants to receive either oral EMA401 (100 mg twice daily) or placebo for 28 days. Randomisation was done according to a centralised randomisation schedule, blocked by study site, which was generated by an independent, unmasked statistician. Patients and staff at each site were masked to treatment assignment. We assessed the efficacy, safety, and pharmacokinetics of EMA401. The primary efficacy endpoint was change in mean pain intensity between baseline and the last week of dosing (days 22-28), measured on an 11-point numerical rating scale. The primary efficacy analysis was intention to treat. This trial is registered with the Australian New Zealand Clinical Trials Registry, number ACTRN12611000822987. Findings: 92 patients were assigned to EMA401 and 91 were assigned to placebo. The patients given EMA401 reported significantly less pain compared with baseline values in the final week of treatment than did those given placebo (mean reductions in pain scores -2·29 [SD 1·75] vs -1·60 [1·66]; difference of adjusted least square means -0·69 [SE 0·25]; 95% CI -1·19 to -0·20; p=0·0066). No serious adverse events related to EMA401 occurred. Overall, 32 patients reported 56 treatment-emergent adverse events in the EMA401 group compared with 45 such events reported by 29 patients given placebo. Interpretation: EMA401 (100 mg twice daily) provides superior relief of postherpetic neuralgia compared with placebo at the end of 28 days of treatment. EMA401 was well tolerated by patients.

Picaud S, Wells C, Felletar I, Brotherton D, Martin S, Savitsky P, Diez-Dacal B, Philpott M et al. 2013. RVX-208, an inhibitor of BET transcriptional regulators with selectivity for the second bromodomain. Proc Natl Acad Sci U S A, 110 (49), pp. 19754-19759. | Show Abstract | Read more

Bromodomains have emerged as attractive candidates for the development of inhibitors targeting gene transcription. Inhibitors of the bromo and extraterminal (BET) family recently showed promising activity in diverse disease models. However, the pleiotropic nature of BET proteins regulating tissue-specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. Here, we report that RVX-208, a compound currently in phase II clinical trials, is a BET bromodomain inhibitor specific for second bromodomains (BD2s). Cocrystal structures revealed binding modes of RVX-208 and its synthetic precursor, and fluorescent recovery after photobleaching demonstrated that RVX-208 displaces BET proteins from chromatin. However, gene-expression data showed that BD2 inhibition only modestly affects BET-dependent gene transcription. Our data demonstrate the feasibility of specific targeting within the BET family resulting in different transcriptional outcomes and highlight the importance of BD1 in transcriptional regulation.

Newey PJ, Gorvin CM, Cleland SJ, Willberg CB, Bridge M, Azharuddin M, Drummond RS, van der Merwe PA, Klenerman P, Bountra C, Thakker RV. 2013. Mutant prolactin receptor and familial hyperprolactinemia. N Engl J Med, 369 (21), pp. 2012-2020. | Show Abstract | Read more

Hyperprolactinemia that is not associated with gestation or the puerperium is usually due to tumors in the anterior pituitary gland and occurs occasionally in hereditary multiple endocrine neoplasia syndromes. Here, we report data from three sisters with hyperprolactinemia, two of whom presented with oligomenorrhea and one with infertility. These symptoms were not associated with pituitary tumors or multiple endocrine neoplasia but were due to a heterozygous mutation in the prolactin receptor gene, PRLR, resulting in an amino acid change from histidine to arginine at codon 188 (His188Arg). This substitution disrupted the high-affinity ligand-binding interface of the prolactin receptor, resulting in a loss of downstream signaling by Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5). Thus, the familial hyperprolactinemia appears to be due to a germline, loss-of-function mutation in PRLR, resulting in prolactin insensitivity.

Bountra C. 2013. Working together to identify new epigenetic targets for cancer MOLECULAR CANCER THERAPEUTICS, 12 (11), pp. PL03-01-PL03-01. | Read more

Shintre CA, Pike AC, Li Q, Kim JI, Barr AJ, Goubin S, Shrestha L, Yang J et al. 2013. Structures of ABCB10, a human ATP-binding cassette transporter in apo- and nucleotide-bound states. Proc Natl Acad Sci U S A, 110 (24), pp. 9710-9715. | Show Abstract | Read more

ABCB10 is one of the three ATP-binding cassette (ABC) transporters found in the inner membrane of mitochondria. In mammals ABCB10 is essential for erythropoiesis, and for protection of mitochondria against oxidative stress. ABCB10 is therefore a potential therapeutic target for diseases in which increased mitochondrial reactive oxygen species production and oxidative stress play a major role. The crystal structure of apo-ABCB10 shows a classic exporter fold ABC transporter structure, in an open-inwards conformation, ready to bind the substrate or nucleotide from the inner mitochondrial matrix or membrane. Unexpectedly, however, ABCB10 adopts an open-inwards conformation when complexed with nonhydrolysable ATP analogs, in contrast to other transporter structures which adopt an open-outwards conformation in complex with ATP. The three complexes of ABCB10/ATP analogs reported here showed varying degrees of opening of the transport substrate binding site, indicating that in this conformation there is some flexibility between the two halves of the protein. These structures suggest that the observed plasticity, together with a portal between two helices in the transmembrane region of ABCB10, assist transport substrate entry into the substrate binding cavity. These structures indicate that ABC transporters may exist in an open-inwards conformation when nucleotide is bound. We discuss ways in which this observation can be aligned with the current views on mechanisms of ABC transporters.

Quigley A, Dong YY, Pike AC, Dong L, Shrestha L, Berridge G, Stansfeld PJ, Sansom MS et al. 2013. The structural basis of ZMPSTE24-dependent laminopathies. Science, 339 (6127), pp. 1604-1607. | Show Abstract | Read more

Mutations in the nuclear membrane zinc metalloprotease ZMPSTE24 lead to diseases of lamin processing (laminopathies), such as the premature aging disease progeria and metabolic disorders. ZMPSTE24 processes prelamin A, a component of the nuclear lamina intermediate filaments, by cleaving it at two sites. Failure of this processing results in accumulation of farnesylated, membrane-associated prelamin A. The 3.4 angstrom crystal structure of human ZMPSTE24 has a seven transmembrane α-helical barrel structure, surrounding a large, water-filled, intramembrane chamber, capped by a zinc metalloprotease domain with the catalytic site facing into the chamber. The 3.8 angstrom structure of a complex with a CSIM tetrapeptide showed that the mode of binding of the substrate resembles that of an insect metalloprotease inhibitor in thermolysin. Laminopathy-associated mutations predicted to reduce ZMPSTE24 activity map to the zinc metalloprotease peptide-binding site and to the bottom of the chamber.

Cited:

25

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Quigley A, Dong YY, Pike ACW, Dong L, Shrestha L, Berridge G, Stansfeld PJ, Sansom MSP et al. 2013. The structural basis of ZMPSTE24-dependent laminopathies Science, 340 (6127), pp. 1604-1607. | Show Abstract | Read more

Mutations in the nuclear membrane zinc metalloprotease ZMPSTE24 lead to diseases of lamin processing (laminopathies), such as the premature aging disease progeria and metabolic disorders. ZMPSTE24 processes prelamin A, a component of the nuclear lamina intermediate filaments, by cleaving it at two sites. Failure of this processing results in accumulation of farnesylated, membrane-associated prelamin A. The 3.4 angstrom crystal structure of human ZMPSTE24 has a seven transmembrane α-helical barrel structure, surrounding a large, water-filled, intramembrane chamber, capped by a zinc metalloprotease domain with the catalytic site facing into the chamber. The 3.8 angstrom structure of a complex with a CSIM tetrapeptide showed that the mode of binding of the substrate resembles that of an insect metalloprotease inhibitor in thermolysin. Laminopathy-associated mutations predicted to reduce ZMPSTE24 activity map to the zinc metalloprotease peptide - binding site and to the bottom of the chamber.

Anand U, Facer P, Yiangou Y, Sinisi M, Fox M, McCarthy T, Bountra C, Korchev YE, Anand P. 2013. Angiotensin II type 2 receptor (AT2 R) localization and antagonist-mediated inhibition of capsaicin responses and neurite outgrowth in human and rat sensory neurons. Eur J Pain, 17 (7), pp. 1012-1026. | Show Abstract | Read more

BACKGROUND: The angiotensin II (AngII) receptor subtype 2 (AT2 R) is expressed in sensory neurons and may play a role in nociception and neuronal regeneration. METHODS: We used immunostaining with characterized antibodies to study the localization of AT2 R in cultured human and rat dorsal root ganglion (DRG) neurons and a range of human tissues. The effects of AngII and AT2 R antagonist EMA401 on capsaicin responses in cultured human and rat (DRG) neurons were measured with calcium imaging, on neurite length and density with Gap43 immunostaining, and on cyclic adenosine monophosphate (cAMP) expression using immunofluorescence. RESULTS: AT2 R expression was localized in small-/medium-sized cultured neurons of human and rat DRG. Treatment with the AT2 R antagonist EMA401 resulted in dose-related functional inhibition of capsaicin responses (IC50  = 10 nmol/L), which was reversed by 8-bromo-cAMP, and reduced neurite length and density; AngII treatment significantly enhanced capsaicin responses, cAMP levels and neurite outgrowth. The AT1 R antagonist losartan had no effect on capsaicin responses. AT2 R was localized in sensory neurons of human DRG, and nerve fibres in peripheral nerves, skin, urinary bladder and bowel. A majority sub-population (60%) of small-/medium-diameter neuronal cells were immunopositive in both control post-mortem and avulsion-injured human DRG; some very small neurons appeared to be intensely immunoreactive, with TRPV1 co-localization. While AT2 R levels were reduced in human limb peripheral nerve segments proximal to injury, they were preserved in painful neuromas. CONCLUSIONS: AT2 R antagonists could be particularly useful in the treatment of chronic pain and hypersensitivity associated with abnormal nerve sprouting.

Arrowsmith CH, Bountra C, Fish PV, Lee K, Schapira M. 2012. Epigenetic protein families: a new frontier for drug discovery. Nat Rev Drug Discov, 11 (5), pp. 384-400. | Show Abstract | Read more

Epigenetic regulation of gene expression is a dynamic and reversible process that establishes normal cellular phenotypes but also contributes to human diseases. At the molecular level, epigenetic regulation involves hierarchical covalent modification of DNA and the proteins that package DNA, such as histones. Here, we review the key protein families that mediate epigenetic signalling through the acetylation and methylation of histones, including histone deacetylases, protein methyltransferases, lysine demethylases, bromodomain-containing proteins and proteins that bind to methylated histones. These protein families are emerging as druggable classes of enzymes and druggable classes of protein-protein interaction domains. In this article, we discuss the known links with disease, basic molecular mechanisms of action and recent progress in the pharmacological modulation of each class of proteins.

Kruidenier L, Chung CW, Cheng Z, Liddle J, Che K, Joberty G, Bantscheff M, Bountra C et al. 2012. A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. Nature, 488 (7411), pp. 404-408. | Show Abstract | Read more

The jumonji (JMJ) family of histone demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance, as well as in development, physiology and disease. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.

Cited:

419

Scopus

Arrowsmith CH, Bountra C, Fish PV, Lee K, Schapira M. 2012. Epigenetic protein families: A new frontier for drug discovery Nature Reviews Drug Discovery, 11 (5), pp. 384-400. | Show Abstract | Read more

Epigenetic regulation of gene expression is a dynamic and reversible process that establishes normal cellular phenotypes but also contributes to human diseases. At the molecular level, epigenetic regulation involves hierarchical covalent modification of DNA and the proteins that package DNA, such as histones. Here, we review the key protein families that mediate epigenetic signalling through the acetylation and methylation of histones, including histone deacetylases, protein methyltransferases, lysine demethylases, bromodomain-containing proteins and proteins that bind to methylated histones. These protein families are emerging as druggable classes of enzymes and druggable classes of proteing-protein interaction domains. In this article, we discuss the known links with disease, basic molecular mechanisms of action and recent progress in the pharmacological modulation of each class of proteins. © 2012 Macmillan Publishers Limited. All rights reserved.

Norman TC, Bountra C, Edwards AM, Yamamoto KR, Friend SH. 2011. Leveraging crowdsourcing to facilitate the discovery of new medicines. Sci Transl Med, 3 (88), pp. 88mr1. | Show Abstract | Read more

Gloomy predictions about the future of pharma have forced the industry to investigate alternative models of drug discovery. Public-private partnerships (PPPs) have the potential to revitalize the discovery and development of first-in-class therapeutics. The new PPP Arch2POCM hopes to foster biomedical innovation through precompetitive validation of pioneer therapeutic targets for human diseases. In this meeting report, we capture insights garnered from the April 2011 Arch2POCM conference.

Norman T, Edwards A, Bountra C, Friend S. 2011. The precompetitive space: time to move the yardsticks. Sci Transl Med, 3 (76), pp. 76cm10. | Show Abstract | Read more

Industry, government, patient advocacy groups, public funders, and academic thought leaders met in Toronto, Canada, to set into motion an initiative that addresses some of the scientific and organizational challenges of modern therapeutics discovery. What emerged from the meeting was a public-private partnership that seeks to establish proof of clinical mechanism (POCM) for selected "pioneer" disease targets using lead compounds-all accomplished in the precompetitive space. The group will reconvene in April 2011 to create a business plan that specifies the generation of two positive POCM results per year.

Bountra C, Oppermann U, Heightman TD. 2011. Animal models of epigenetic regulation in neuropsychiatric disorders. Curr Top Behav Neurosci, 7 (1), pp. 281-322. | Show Abstract | Read more

Epigenetics describes the phenomenon of heritable changes in gene regulation that are governed by non-Mendelian processes, primarily through biochemical modifications to chromatin structure that occur during cell development and differentiation. Numerous lines of evidence link abnormal levels of chromatin modifications (either to DNA, histones, or both) in patients with a wide variety of diseases including cancer, psychiatry, neurodegeneration, metabolic and inflammatory disorders. Drugs that target the proteins controlling chromatin modifications can modulate the expression of clusters of genes, potentially offering higher therapeutic efficacy than classical agents with single target pharmacologies that are susceptible to biochemical pathway degeneracy. Here, we summarize recent research linking epigenetic dysregulation with diseases in neurosciences, the application of relevant animal models, and the potential for small molecule modulator development to facilitate target discovery, validation and translation into clinical treatments.

Sanger GJ, Chang L, Bountra C, Houghton LA. 2010. Challenges and prospects for pharmacotherapy in functional gastrointestinal disorders. Therap Adv Gastroenterol, 3 (5), pp. 291-305. | Show Abstract | Read more

Functional gastrointestinal disorders, such as irritable bowel syndrome and functional dyspepsia, are complex conditions with multiple factors contributing to their pathophysiology. As a consequence they are difficult to treat and have posed significant challenges to the pharmaceutical industry when trying to develop new and effective treatments. This review provides an overview of these difficulties and how the industry is reshaping its drug developmental strategies. It describes some of the more significant and encouraging advances that have occurred, and discusses how future research might embrace the opportunities provided by advances in genetic and in particular, epigenetic research.

Toronto International Data Release Workshop Authors, Birney E, Hudson TJ, Green ED, Gunter C, Eddy S, Rogers J, Harris JR et al. 2009. Prepublication data sharing. Nature, 461 (7261), pp. 168-170. | Show Abstract | Read more

Rapid release of prepublication data has served the field of genomics well. Attendees at a workshop in Toronto recommend extending the practice to other biological data sets.

Swarbrick ME, Beswick PJ, Gleave RJ, Green RH, Bingham S, Bountra C, Carter MC, Chambers LJ et al. 2009. Identification of [4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)-2-pyrimidinyl] amines and ethers as potent and selective cyclooxygenase-2 inhibitors. Bioorg Med Chem Lett, 19 (15), pp. 4504-4508. | Show Abstract | Read more

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.

Beswick PJ, Blackaby AP, Bountra C, Brown T, Browning K, Campbell IB, Corfield J, Gleave RJ et al. 2009. Identification and optimisation of a novel series of pyrimidine based cyclooxygenase-2 (COX-2) inhibitors. Utilisation of a biotransformation approach. Bioorg Med Chem Lett, 19 (15), pp. 4509-4514. | Show Abstract | Read more

Many years of work have been invested in the identification of potent and selective COX-2 inhibitors for the treatment of chronic inflammatory pain. One issue faced by workers is the balance between the lipophilicity required for potent enzyme inhibition and the physical properties necessary for drug absorption and distribution in vivo. Frequently approaches to reduce lipophilicity through introduction of polar functionality is hampered by highly challenging chemistry to prepare key molecules. We have complemented traditional synthetic chemistry with a biotransformations approach which efficiently provided access to an array of key target molecules.

Edwards AM, Bountra C, Kerr DJ, Willson TM. 2009. Open access chemical and clinical probes to support drug discovery. Nat Chem Biol, 5 (7), pp. 436-440. | Show Abstract | Read more

Drug discovery resources in academia and industry are not used efficiently, to the detriment of industry and society. Duplication could be reduced, and productivity could be increased, by performing basic biology and clinical proofs of concept within open access industry-academia partnerships. Chemical biologists could play a central role in this effort.

Anand U, Otto WR, Sanchez-Herrera D, Facer P, Yiangou Y, Korchev Y, Birch R, Benham C, Bountra C, Chessell IP, Anand P. 2008. Cannabinoid receptor CB2 localisation and agonist-mediated inhibition of capsaicin responses in human sensory neurons. Pain, 138 (3), pp. 667-680. | Show Abstract | Read more

Cannabinoid receptor 2 (CB2) agonists provide the potential for treating chronic pain states without CNS effects associated with CB1 receptor activation. Animal models suggest that they act mainly via non-neuronal cells, possibly inhibition of inflammatory cells in the periphery or CNS, or via release of beta-endorphin; however, the clinical relevance and mechanism of analgesic action is uncertain. Here, we demonstrate colocalisation of CB2 with CB1 and the capsaicin receptor TRPV1 in human dorsal root ganglion (DRG) sensory neurons and increased levels of CB2 receptors in human peripheral nerves after injury, particularly painful neuromas. In primary cultures of human DRG neurons, selective CB2 agonists blocked activation of inward cation currents and elevation of cytoplasmic Ca2+ in response to capsaicin. These inhibitory effects were reversed by GW818646X a CB2 antagonist, and 8-bromo cAMP, but not by SR141716 a CB1 antagonist, or naloxone. Thus CB2 receptor agonists functionally inhibited nociceptive signalling in human primary sensory neurons via a mechanism shared with opioids, of adenylyl cyclase inhibition, but not via mu-opioid receptors. We conclude that CB2 agonists deserve imminent clinical trials for nociceptive, inflammatory and neuropathic chronic pain, in which capsaicin or heat-activated responses via TRPV1 may provide a clinical marker.

Langley CK, Aziz Q, Bountra C, Gordon N, Hawkins P, Jones A, Langley G, Nurmikko T, Tracey I. 2008. Volunteer studies in pain research--opportunities and challenges to replace animal experiments: the report and recommendations of a Focus on Alternatives workshop. Neuroimage, 42 (2), pp. 467-473. | Show Abstract | Read more

Despite considerable research, effective and safe treatments for human pain disorders remain elusive. Understanding the biology of different human pain conditions and researching effective treatments continue to be dominated by animal models, some of which are of limited value. British and European legislation demands that non-animal approaches should be considered before embarking on research using experimental animals. Recent scientific and technical developments, particularly in human neuroimaging, offer the potential to replace some animal procedures in the study of human pain. A group of pain research experts from academia and industry met with the aim of exploring creatively the tools, strategies and challenges of replacing some animal experiments in pain research with ethically conducted studies of human patients and healthy volunteers, in combination with in vitro methods. This report considers how a range of neuroimaging techniques including functional magnetic resonance imaging, magnetoencephalography and positron emission tomography, singly and combined, can address human pain conditions. In addition, microdialysis in human subjects; genome-wide association research, twin studies and other epidemiological approaches; and in vitro cell and tissue research, are examined for their replacement potential in combination with neuroimaging. Recommendations highlight further opportunities to advance the replacement of animal studies with robust methods of relevance to understanding and treating human pain.

Anand U, Otto WR, Bountra C, Chessell I, Sinisi M, Birch R, Anand P. 2008. Cytosine arabinoside affects the heat and capsaicin receptor TRPV1 localisation and sensitivity in human sensory neurons. J Neurooncol, 89 (1), pp. 1-7. | Show Abstract | Read more

BACKGROUND: Cytosine arabinoside (Ara C) is a useful chemotherapy agent, used for treating acute myeloid leukaemia, although it may be associated with side effects including painful neuropathy. It is also used for in vitro neuronal studies to limit the proliferation of non-neuronal cells and thereby select nondividing neuronal cells. We studied the effects of Ara C on human dorsal root ganglion (DRG) neurons, especially the expression and sensitivity of the ion channel TRPV1, which responds to noxious heat and capsaicin and is a key mediator of neuropathic pain. METHODS: Human DRG neurons were cultured with or without Ara C for 2 weeks, after which Ara C was discontinued. Double immunostaining for the regenerative neuronal marker Gap43 and the capsaicin receptor TRPV1 showed that the normal membrane-bound localisation of TRPV1 was absent in neurons with Ara C treatment, and as expected there was massive diminution of dividing non-neuronal cells. Calcium imaging studies showed that during exposure to Ara C the neurons lost responsiveness to capsaicin, although ionomycin responses were intact, indicating general cell viability and responsiveness. Between 2 days and up to 3 weeks after removal of Ara C, the neuronal responses to capsaicin were regained and were observed to be four times (P = 0.0008, Student's t-test) that of controls, but there was only a gradual recovery of non-neuronal cells. Three to six weeks after Ara C removal, capsaicin responses were comparable to controls. CONCLUSIONS: It is postulated that Ara C treatment blocked insertion of TRPV1 in the cell membrane, resulting in accumulation of the receptors in the cytoplasm, loss of capsaicin sensitivity, and membrane-bound immunostaining, which was restored with a rebound on withdrawal of Ara C. The observed pattern of loss of capsaicin sensitivity, followed by hypersensitivity and recovery, appears to reflect some of the features observed in chemotherapy-induced neuropathy, and may provide a model for developing new treatments and prophylaxis.

Worsley MA, Clayton NM, Bountra C, Boissonade FM. 2008. The effects of ibuprofen and the neurokinin-1 receptor antagonist GR205171A on Fos expression in the ferret trigeminal nucleus following tooth pulp stimulation. Eur J Pain, 12 (3), pp. 385-394. | Show Abstract | Read more

We have developed a model to study central changes following inflammation of the tooth pulp in the ferret and have examined Fos expression in the trigeminal nucleus following stimulation of non-inflamed and inflamed tooth pulps. The aim of this study was to establish the ability of this model to predict analgesic efficacy in clinical studies of inflammatory pain. We addressed this by assessing the effects of the neurokinin-1 receptor antagonist GR205171A and ibuprofen on Fos expression following stimulation of the inflamed pulp and comparing this with known analgesic efficacy. Adult ferrets were prepared under anaesthesia to allow tooth pulp stimulation, recording from the digastric muscle and intravenous injections at a subsequent experiment. In some animals pulpal inflammation was induced, by introducing human caries into a deep buccal cavity. After 5 days, animals were reanaesthetised, treated with vehicle, GR205171A or ibuprofen and the teeth were stimulated at ten times the threshold of the jaw-opening reflex. Stimulation of all tooth pulps induced ipsilateral Fos in trigeminal subnuclei caudalis and oralis. GR205171A had no significant effect on Fos expression in the trigeminal nucleus of animals with either non-inflamed or inflamed tooth pulps. Ibuprofen reduced Fos expression in the trigeminal nucleus and this effect was most marked in animals with pulpal inflammation. These results differ from those previously described using a range of other animal models, but agree with known clinical efficacy of neurokinin-1 receptor antagonists and ibuprofen. Therefore this model is likely to be of use in accurately predicting the analgesic efficacy of novel compounds.

Davis JB, Bountra C, Richardson J. 2008. Perspectives of Alzheimer's disease treatments. Handb Clin Neurol, 89 pp. 273-290. | Read more

Yiangou Y, Facer P, Chessell IP, Bountra C, Chan C, Fertleman C, Smith V, Anand P. 2007. Voltage-gated ion channel Nav1.7 innervation in patients with idiopathic rectal hypersensitivity and paroxysmal extreme pain disorder (familial rectal pain). Neurosci Lett, 427 (2), pp. 77-82. | Show Abstract | Read more

Faecal urgency and incontinence with rectal hypersensitivity is a chronic, unexplained condition that is difficult to treat. The aim of this study was to determine if there was an altered level of the voltage gated tetrodotoxin-sensitive (TTX-s) sodium channel Na(v)1.7 in rectal sensory fibres, since this channel has been implicated in clinical nociceptive disorders. Full thickness rectal biopsies from patients with physiologically characterised rectal hypersensitivity (n=7) were compared with control tissues (n=10). Formalin fixed specimens were studied by immunohistochemistry using affinity purified antibodies to Na(v)1.7 and the pan-neuronal structural marker PGP9.5, and the immunoreactive nerve fibres quantified by computerised image analysis. In rectal hypersensitivity, Na(v)1.7 immunoreactive nerve fibres were significantly increased in mucosal (P=0.0004), sub-mucosal (P=0.019), and muscle layers (P=0.0076), while PGP9.5 immunoreactive nerve fibres were increased significantly only in the mucosa (P=0.04); ratios of Na(v)1.7:PGP9.5 showed a significant increase in all layers, suggesting increased expression of Na(v)1.7, and nerve sprouting in the mucosa. The cause of this increase remains uncertain, but may be due to increase of nerve growth factor (NGF), which regulates the expression of both Na(v)1.7 and TRPV1, which we have previously reported to be increased in this condition. In paroxysmal extreme pain disorder (familial rectal pain), where the gene that encodes Na(v)1.7 is mutated, Na(v)1.7 protein was undetectable in the rectum (n=2), which suggests reduced Na(v)1.7 immunoreactivity or expression. Drugs that target Na(v)1.7-expressing nerve terminals may be useful for treating rectal hypersensitivity, and combining these with TRPV1 antagonists may enhance efficacy.

Yilmaz Z, Renton T, Yiangou Y, Zakrzewska J, Chessell IP, Bountra C, Anand P. 2007. Burning mouth syndrome as a trigeminal small fibre neuropathy: Increased heat and capsaicin receptor TRPV1 in nerve fibres correlates with pain score. J Clin Neurosci, 14 (9), pp. 864-871. | Show Abstract | Read more

Burning mouth syndrome (BMS) is often an idiopathic chronic and intractable pain condition, affecting 1.5-5.5% of middle-aged and elderly women. We have studied the heat and capsaicin receptor TRPV1, and its regulator nerve growth factor (NGF), in BMS. Patients with BMS (n=10) and controls (n=10) were assessed for baseline and post-topical capsaicin pain scores, and their tongue biopsies immunostained for TRPV1, NGF, and structural nerve markers neurofilament and peripherin. Nerve fibres penetrating the epithelium were less abundant in BMS (p<0.0001), indicating a small fibre neuropathy. TRPV1-positive fibres were overall significantly increased in BMS (p=0.0011), as were NGF fibres (p<0.0001) and basal epithelial cell NGF staining (p<0.0147). There was a significant correlation between the baseline pain score and TRPV1 (p=0.0143) and NGF fibres (p=0.0252). A significant correlation was observed between baseline and post-capsaicin pain (p=0.0006). Selective TRPV1 and NGF blockers may provide a new therapy for BMS.

Staton PC, Wilson AW, Bountra C, Chessell IP, Day NC. 2007. Changes in dorsal root ganglion CGRP expression in a chronic inflammatory model of the rat knee joint: differential modulation by rofecoxib and paracetamol. Eur J Pain, 11 (3), pp. 283-289. | Show Abstract | Read more

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 Freund's 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.

Connor SC, Gray RA, Hodson MP, Clayton NM, Haselden JN, Chessell IP, Bountra C. 2007. An NMR-based metabolic profiling study of inflammatory pain using the rat FCA model METABOLOMICS, 3 (1), pp. 29-39. | Show Abstract | Read more

The objective measurement of chronic pain in pre-clinical animal models and in the clinical arena is problematic. The multifactorial nature of pain can adversely affect the accuracy with which it can be quantitatively assessed. This is due to diverse aetiology, environmental factors and psychological status which can significantly affect the measurement of pain in animal models and presentation in the clinic. In pre-clinical studies, the objectivity of pain measurement is affected by variability in behavioural phenotype and the lack of verbal description, while clinical pain is subject to variance by a considerable placebo effect, thought to be related to merely being in a clinical setting. Therefore, it would be advantageous to identify a biological marker which is sensitive to pain pathology and modulated by an efficacious analgesic/anti-inflammatory agent. The present study highlights several metabolites present in urine that are modulated from basal levels by pain and inflammation induced following hindpaw injection of Freund's Complete Adjuvant. Nuclear magnetic resonance (NMR) analysis of urine and multivariate statistical data analysis (MVDA) were used to examine in detail the modulation of small molecule candidate biomarkers or surrogates. Several molecules were shown by NMR to be modulated by FCA injection including carboxylates, acetylated metabolites, choline metabolites, tricarboxylic acid cycle intermediates, creatine and creatinine, taurine, N-methylnicotinamide and its metabolites and several unassigned peaks. © Springer Science+Business Media, LLC 2006.

Worsley MA, Davies SL, Clayton NM, Bountra C, Loescher AR, Robinson PP, Boissonade FM. 2007. The effect of inflammation on Fos expression in the ferret trigeminal nucleus. Eur J Oral Sci, 115 (1), pp. 40-47. | Show Abstract | Read more

We have previously carried out detailed characterization and identification of Fos expression within the trigeminal nucleus after tooth pulp stimulation in ferrets. The aim of this study was to determine the effect of pulpal inflammation on the excitability of central trigeminal neurons following tooth pulp stimulation. Adult ferrets were prepared under anesthesia to allow tooth pulp stimulation, recording from the digastric muscle, and intravenous injections at a subsequent experiment. In some animals, pulpal inflammation was induced by introducing human caries into a deep buccal cavity. After 5 d, animals were re-anaethetized, and the teeth were stimulated at 10 times the threshold of the jaw-opening reflex. Stimulation of all tooth pulps induced ipsilateral Fos in the trigeminal subnuclei caudalis and oralis. All non-stimulated animals showed negligible Fos labeling, with no differences recorded between inflamed and non-inflamed groups. Following tooth pulp stimulation, Fos expression was greater in animals with inflamed teeth than in animals with non-inflamed teeth, with the greatest effect seen in the subnucleus caudalis. These results suggest that inflammation increases the number of trigeminal brainstem neurons activated by tooth pulp stimulation; this may be mediated by peripheral or central mechanisms.

Sánchez D, Anand U, Gorelik J, Benham CD, Bountra C, Lab M, Klenerman D, Birch R, Anand P, Korchev Y. 2007. Localized and non-contact mechanical stimulation of dorsal root ganglion sensory neurons using scanning ion conductance microscopy. J Neurosci Methods, 159 (1), pp. 26-34. | Show Abstract | Read more

Mechanosensitive ion channels convert external mechanical force into electrical and chemical signals in cells, but their physiological function in different tissues is not clearly understood. One reason for this is that there is as yet no satisfactory physiological method to stimulate these channels in living cells. Using the nanopipette-probe of the Scanning Ion Conductance Microscope (SICM), we have developed a new technique to apply local mechanical stimulus to living cells to an area of about 0.385 microm2, determined by the pipette diameter. Our method prevents any physical contact and damage to the cell membrane by use of a pressure jet applied via the nanopipette. The study used whole-cell patch-clamp recordings and measurements of intracellular Ca2+ concentration to validate the application of the mechanical stimulation protocols in human and rat dorsal root ganglia (DRG) sensory neurons. We were able, for the first time, to produce a non-contact, controlled mechanical stimulation on living neurites of human DRG neurons. Our methods will enable the identification and characterisation of compounds being developed for the treatment of clinical mechanical hypersensitivity states.

Facer P, Casula MA, Smith GD, Benham CD, Chessell IP, Bountra C, Sinisi M, Birch R, Anand P. 2007. Differential expression of the capsaicin receptor TRPV1 and related novel receptors TRPV3, TRPV4 and TRPM8 in normal human tissues and changes in traumatic and diabetic neuropathy. BMC Neurol, 7 (1), pp. 11. | Show Abstract | Read more

BACKGROUND: Transient receptor potential (TRP) receptors expressed by primary sensory neurons mediate thermosensitivity, and may play a role in sensory pathophysiology. We previously reported that human dorsal root ganglion (DRG) sensory neurons co-expressed TRPV1 and TRPV3, and that these were increased in injured human DRG. Related receptors TRPV4, activated by warmth and eicosanoids, and TRPM8, activated by cool and menthol, have been characterised in pre-clinical models. However, the role of TRPs in common clinical sensory neuropathies needs to be established. METHODS: We have studied TRPV1, TRPV3, TRPV4, and TRPM8 in nerves (n = 14) and skin from patients with nerve injury, avulsed dorsal root ganglia (DRG) (n = 11), injured spinal nerve roots (n = 9), diabetic neuropathy skin (n = 8), non-diabetic neuropathic nerve biopsies (n = 6), their respective control tissues, and human post mortem spinal cord, using immunohistological methods. RESULTS: TRPV1 and TRPV3 were significantly increased in injured brachial plexus nerves, and TRPV1 in hypersensitive skin after nerve repair, whilst TRPV4 was unchanged. TRPM8 was detected in a few medium diameter DRG neurons, and was unchanged in DRG after avulsion injury, but was reduced in axons and myelin in injured nerves. In diabetic neuropathy skin, TRPV1 expressing sub- and intra-epidermal fibres were decreased, as was expression in surviving fibres. TRPV1 was also decreased in non-diabetic neuropathic nerves. Immunoreactivity for TRPV3 was detected in basal keratinocytes, with a significant decrease of TRPV3 in diabetic skin. TRPV1-immunoreactive nerves were present in injured dorsal spinal roots and dorsal horn of control spinal cord, but not in ventral roots, while TRPV3 and TRPV4 were detected in spinal cord motor neurons. CONCLUSION: The accumulation of TRPV1 and TRPV3 in peripheral nerves after injury, in spared axons, matches our previously reported changes in avulsed DRG. Reduction of TRPV1 levels in nerve fibres in diabetic neuropathy skin may result from the known decrease of nerve growth factor (NGF) levels. The role of TRPs in keratinocytes is unknown, but a relationship to changes in NGF levels, which is produced by keratinocytes, deserves investigation. TRPV1 represents a more selective therapeutic target than other TRPs for pain and hypersensitivity, particularly in post-traumatic neuropathy.

Atherton DD, Facer P, Roberts KM, Misra VP, Chizh BA, Bountra C, Anand P. 2007. Use of the novel Contact Heat Evoked Potential Stimulator (CHEPS) for the assessment of small fibre neuropathy: correlations with skin flare responses and intra-epidermal nerve fibre counts. BMC Neurol, 7 (1), pp. 21. | Show Abstract | Read more

BACKGROUND: The Contact Heat Evoked Potential Stimulator (CHEPS) rapidly stimulates cutaneous small nerve fibres, and resulting evoked potentials can be recorded from the scalp. We have studied patients with symptoms of sensory neuropathy and controls using CHEPS, and validated the findings using other objective measures of small nerve fibres i.e. the histamine-induced skin flare response and intra-epidermal fibres (IEF), and also quantitative sensory testing (QST), a subjective measure. METHODS: In patients with symptoms of sensory neuropathy (n = 41) and healthy controls (n = 9) we performed clinical examination, QST (monofilament, vibration and thermal perception thresholds), nerve conduction studies, histamine-induced skin flares and CHEPS. Skin punch biopsies were immunostained using standard ABC immunoperoxidase for the nerve marker PGP 9.5 or the heat and capsaicin receptor TRPV1. Immunoreactive IEF were counted per length of tissue section and epidermal thickness recorded. RESULTS: Amplitudes of Adelta evoked potentials (muV) following face, arm or leg stimulation were reduced in patients (e.g. for the leg: mean +/- SEM - controls 11.7 +/- 1.95, patients 3.63 +/- 0.85, p = 0.0032). Patients showed reduced leg skin flare responses, which correlated with Adelta amplitudes (rs = 0.40, p = 0.010). In patient leg skin biopsies, PGP 9.5- and TRPV1-immunoreactive IEF were reduced and correlated with Adelta amplitudes (PGP 9.5, rs = 0.51, p = 0.0006; TRPV1, rs = 0.48, p = 0.0012). CONCLUSION: CHEPS appears a sensitive measure, with abnormalities observed in some symptomatic patients who did not have significant IEF loss and/or QST abnormalities. Some of the latter patients may have early small fibre dysfunction or ion channelopathy. CHEPS provides a clinically practical, non-invasive and objective measure, and can be a useful additional tool for the assessment of sensory small fibre neuropathy. Although further evaluation is required, the technique shows potential clinical utility to differentiate neuropathy from other chronic pain states, and provide a biomarker for analgesic development.

Davies SL, Loescher AR, Clayton NM, Bountra C, Robinson PP, Boissonade FM. 2006. Changes in sodium channel expression following trigeminal nerve injury. Exp Neurol, 202 (1), pp. 207-216. | Show Abstract | Read more

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.

Schweinhardt P, Bountra C, Tracey I. 2006. Pharmacological FMRI in the development of new analgesic compounds. NMR Biomed, 19 (6), pp. 702-711. | Show Abstract | Read more

Chronic pain is a major problem for the individual and for society. Despite a range of drugs being available to treat chronic pain, only inadequate pain relief can be achieved for many patients. There is therefore a need for the development of new analgesic compounds. The assessment of pain depends to date entirely on the subjective report of the patient, in contrast to many other clinical conditions where biomarkers that help determine the severity and stage of the disease enable the physician to monitor the course of the disease and treatment effects longitudinally. In this article, we illustrate that magnetic resonance-based imaging techniques have the potential to provide sensitive and specific biomarkers of the pain experience, as well as clarifying disease mechanisms. Functional magnetic resonance imaging (FMRI) is particularly suited to investigating the effects of pharmacological agents on pain processing within the human central nervous system. Combination of FMRI and drug administration is termed pharmacological FMRI (phFMRI). In addition to outlining several methodological considerations that have to be taken into account when performing phFMRI, we discuss phFMRI studies that have already used this technique to study the effects of analgesic compounds. These studies provide promising data for the use of phFMRI as sensitive tool in assessing a potential drug effect. Such pharmacodynamic readouts obtained early in the process of drug development would not only save the pharmaceutical industry substantial amounts of money, but would also avoid the unnecessary exposure of patients to molecules with limited or no therapeutic value. We are therefore optimistic that phFMRI will be used as a tool with high sensitivity and specificity for evaluating analgesic agents in early drug development and clinical studies.

Wilson AW, Medhurst SJ, Dixon CI, Bontoft NC, Winyard LA, Brackenborough KT, De Alba J, Clarke CJ et al. 2006. An animal model of chronic inflammatory pain: pharmacological and temporal differentiation from acute models. Eur J Pain, 10 (6), pp. 537-549. | Show Abstract | Read more

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 (3mg/kg, t.i.d.), dexamethasone (1mg/kg, b.i.d.), ibuprofen (30mg/kg, t.i.d.), etoricoxib (5mg/kg, b.i.d.) and rofecoxib (0.3-10mg/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.

Schweinhardt P, Glynn C, Brooks J, McQuay H, Jack T, Chessell I, Bountra C, Tracey I. 2006. An fMRI study of cerebral processing of brush-evoked allodynia in neuropathic pain patients. Neuroimage, 32 (1), pp. 256-265. | Show Abstract | Read more

Previous human imaging studies have revealed a network of brain regions involved in the processing of allodynic pain; this includes prefrontal areas, insula, cingulate cortex, primary and secondary somatosensory cortices and parietal association areas. In this study, the neural correlates of the perceived intensity of allodynic pain in neuropathic pain patients were investigated. In eight patients, dynamic mechanical allodynia was provoked and brain responses recorded using functional magnetic resonance imaging (fMRI). Voxels in which the magnitude of fMRI signal correlated linearly with the ratings of allodynic pain across the group were determined in a whole brain analysis using a general linear model. To ensure that activation reflected only allodynic pain ratings, a nuisance variable containing ratings of ongoing pain was included in the analysis. We found that the magnitude of activation in the caudal anterior insula (cAI) correlates with the perceived intensity of allodynic pain across subjects, independent of the level of ongoing pain. However, the peak of activation in the allodynic condition was located in the rostral portion (rAI). This matches the representation of other clinical pain syndromes, confirmed by a literature review. In contrast, experimental pain in healthy volunteers resides predominantly in the cAI, as shown by the same literature review. Taken together, our data and the literature review suggest a functional segregation of anterior insular cortex.

Anand U, Otto WR, Casula MA, Day NC, Davis JB, Bountra C, Birch R, Anand P. 2006. The effect of neurotrophic factors on morphology, TRPV1 expression and capsaicin responses of cultured human DRG sensory neurons. Neurosci Lett, 399 (1-2), pp. 51-56. | Show Abstract | Read more

We have studied the effect of key neurotrophic factors (NTFs) on morphology, levels of the vanilloid receptor-1 (TRPV1) and responses to capsaicin in adult human sensory neurons in vitro. Avulsed dorsal root ganglia (DRG, n = 5) were cultured with or without a combination of nerve growth factor (NGF), glial cell (line)-derived growth factor (GDNF) and neurotrophin3 (NT3) for 5 days. In the absence of NTFs, the diameter of neurons ranged from 20 to 100 microm (mean 42 +/- 4 microm). Adding NTFs caused a significant increase in neuronal sizes, up to 120 microm (mean diameter 62 +/- 5 microm, P < 0.01, t-test), an overall 35% increase of TRPV1-positive neurons (P < 0.003), and notably of large TRPV1-positive neurons > 80 microm (P < 0.05). Responses to capsaicin were significantly enhanced with calcium ratiometry (P < 0.0001). Short duration (1h) exposure of dissociated sensory neurons to NTFs increased numbers of TRPV1-positive neurons, but not of TRPV3, Nav 1.8 and IK1 and the morphological size-distribution remained similar to intact post-mortem DRG neurons. NTFs thus increase size, elevate TRPV1 levels and enhance capsaicin responses in cultured human DRG neurons; these changes may relate to pathophysiology in disease states, and provide an in vitro model to study novel analgesics.

Yiangou Y, Facer P, Durrenberger P, Chessell IP, Naylor A, Bountra C, Banati RR, Anand P. 2006. COX-2, CB2 and P2X7-immunoreactivities are increased in activated microglial cells/macrophages of multiple sclerosis and amyotrophic lateral sclerosis spinal cord. BMC Neurol, 6 (1), pp. 12. | Show Abstract | Read more

BACKGROUND: While multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS) are primarily inflammatory and degenerative disorders respectively, there is increasing evidence for shared cellular mechanisms that may affect disease progression, particularly glial responses. Cyclooxygenase 2 (COX-2) inhibition prolongs survival and cannabinoids ameliorate progression of clinical disease in animal models of ALS and MS respectively, but the mechanism is uncertain. Therefore, three key molecules known to be expressed in activated microglial cells/macrophages, COX-2, CB2 and P2X7, which plays a role in inflammatory cascades, were studied in MS and ALS post-mortem human spinal cord. METHODS: Frozen human post mortem spinal cord specimens, controls (n = 12), ALS (n = 9) and MS (n = 19), were available for study by immunocytochemistry and Western blotting, using specific antibodies to COX-2, CB2 and P2X7, and markers of microglial cells/macrophages (CD 68, ferritin). In addition, autoradiography for peripheral benzodiazepine binding sites was performed on some spinal cord sections using [3H] (R)-PK11195, a marker of activated microglial cells/macrophages. Results of immunostaining and Western blotting were quantified by computerized image and optical density analysis respectively. RESULTS: In control spinal cord, few small microglial cells/macrophages-like COX-2-immunoreactive cells, mostly bipolar with short processes, were scattered throughout the tissue, whilst MS and ALS specimens had significantly greater density of such cells with longer processes in affected regions, by image analysis. Inflammatory cell marker CD68-immunoreactivity, [3H] (R)-PK11195 autoradiography, and double-staining against ferritin confirmed increased production of COX-2 by activated microglial cells/macrophages. An expected 70-kDa band was seen by Western blotting which was significantly increased in MS spinal cord. There was good correlation between the COX-2 immunostaining and optical density of the COX-2 70-kDa band in the MS group (r = 0.89, P = 0.0011, n = 10). MS and ALS specimens also had significantly greater density of P2X7 and CB2-immunoreactive microglial cells/macrophages in affected regions. CONCLUSION: It is hypothesized that the known increase of lesion-associated extracellular ATP contributes via P2X7 activation to release IL-1 beta which in turn induces COX-2 and downstream pathogenic mediators. Selective CNS-penetrant COX-2 and P2X7 inhibitors and CB2 specific agonists deserve evaluation in the progression of MS and ALS.

Mukerji G, Yiangou Y, Corcoran SL, Selmer IS, Smith GD, Benham CD, Bountra C, Agarwal SK, Anand P. 2006. Cool and menthol receptor TRPM8 in human urinary bladder disorders and clinical correlations. BMC Urol, 6 (1), pp. 6. | Show Abstract | Read more

BACKGROUND: The recent identification of the cold-menthol sensory receptor (TRPM8; CMR1), provides us with an opportunity to advance our understanding of its role in the pathophysiology of bladder dysfunction, and its potential mediation of the bladder cooling reflex. In this study, we report the distribution of the cool and menthol receptor TRPM8 in the urinary bladder in patients with overactive and painful bladder syndromes, and its relationship with clinical symptoms. METHODS: Bladder specimens obtained from patients with painful bladder syndrome (PBS, n = 16), idiopathic detrusor overactivity (IDO, n = 14), and asymptomatic microscopic hematuria (controls, n = 17), were immunostained using specific antibodies to TRPM8; nerve fibre and urothelial immunostaining were analysed using fibre counts and computerized image analysis respectively. The results of immunohistochemistry were compared between the groups and correlated with the Pain, Frequency and Urgency scores. RESULTS: TRPM8-immunoreactive staining was observed in the urothelium and nerve fibres scattered in the suburothelium. The nerve fibre staining was seen in fine-calibre axons and thick (myelinated) fibres. There was marked increase of TRPM8-immunoreactive nerve fibres in IDO (P = 0.0249) and PBS (P < 0.0001) specimens, compared with controls. A significantly higher number of TRPM8-immunoreactive axons were also seen in the IDO (P = 0.0246) and PBS (P < 0.0001) groups. Urothelial TRPM8 and TRPM8-immunoreactive thick myelinated fibres appeared unchanged in IDO and PBS. The relative density of TRPM8-immunoreactive nerve fibres significantly correlated with the Frequency (r = 0.5487, P = 0.0004) and Pain (r = 0.6582, P < 0.0001) scores, but not Urgency score. CONCLUSION: This study demonstrates increased TRPM8 in nerve fibres of overactive and painful bladders, and its relationship with clinical symptoms. TRPM8 may play a role in the symptomatology and pathophysiology of these disorders, and may provide an additional target for future overactive and painful bladder pharmacotherapy.

Mukerji G, Waters J, Chessell IP, Bountra C, Agarwal SK, Anand P. 2006. Pain during ice water test distinguishes clinical bladder hypersensitivity from overactivity disorders. BMC Urol, 6 pp. 31. | Show Abstract | Read more

BACKGROUND: The Bladder cooling reflex (BCR) i.e. uninhibited detrusor contractions evoked by intravesical instillation of cold saline, is a segmental reflex believed to be triggered by menthol sensitive cold receptors in the bladder wall, with the afferent signals transmitted by C fibres. The BCR is a neonatal reflex that becomes suppressed by descending signals from higher centres at approximately the time when the child gains full voluntary control of voiding. It re-emerges in adults with neurogenic detrusor overactivity as a consequence of loss of central descending inhibition, resulting from conditions such as spinal cord injury or multiple sclerosis. We have recently shown an increase of nerve fibres expressing the cool and menthol receptor TRPM8 in both overactive (IDO) and painful bladder syndrome (PBS), but its functional significance is unknown. We have therefore studied the bladder cooling reflex and associated sensory symptoms in patients with PBS and overactivity disorders. METHODS: The BCR, elicited by ice water test (IWT) was performed in patients with painful bladder syndrome (PBS, n = 17), idiopathic detrusor overactivity (IDO, n = 22), neurogenic detrusor overactivity (NDO, n = 4) and stress urinary incontinence (as controls, n = 21). The IWT was performed by intravesical instillation of cold saline (0 - 4 degrees C). A positive IWT was defined as presence of uninhibited detrusor contraction evoked by cold saline, associated with urgency or with fluid expulsion. Patients were asked to report and rate any pain and cold sensation during the test. RESULTS: A positive IWT was observed in IDO (6/22, 27.3%) and NDO (4/4, 100%) patients, but was negative in all control and PBS patients. Thirteen (76.5%) PBS patients reported pain during the IWT, with significantly higher pain scores during ice water instillation compared to the baseline (P = 0.0002), or equivalent amount of bladder filling (100 mls) with saline at room temperature (P = 0.015). None of the control or overactive (NDO/IDO) patients reported any pain during the IWT. CONCLUSION: The BCR in DO may reflect loss of central inhibition, which appears necessary to elicit this reflex; the pain elicited in PBS suggests afferent sensitisation, hence sensory symptoms are evoked but not reflex detrusor contractions. The ice water test may be a useful and simple marker for clinical trials in PBS, particularly for novel selective TRPM8 antagonists.

Renton T, Yiangou Y, Plumpton C, Tate S, Bountra C, Anand P. 2005. Sodium channel Nav1.8 immunoreactivity in painful human dental pulp. BMC Oral Health, 5 (1), pp. 5. | Show Abstract | Read more

BACKGROUND: The tetrodotoxin-resistant voltage-gated sodium channel Nav1.8 (SNS1/PN3) is expressed by nociceptors and may play a role in pain states. METHODS: Using specific antibodies for immunohistochemistry, we studied Nav1.8 immunoreactivity in human dental pulp in relation to the neuronal marker neurofilament. Human tooth pulp was extracted from teeth harvested from a total of twenty-two patients (fourteen without dental pain, eight patients with dental pain). RESULTS: Fibres immunoreactive for Nav1.8, were significantly increased on image analysis in the painful group: median (range) Nav1.8 to Neurofilament % area ratio, non-painful 0.059 (0.006-0.24), painful 0.265 (0.13-0.5), P = 0.0019. CONCLUSION: Nav1.8 sodium channels may thus represent a therapeutic target in trigeminal nerve pain states.

Chessell IP, Hatcher JP, Bountra C, Michel AD, Hughes JP, Green P, Egerton J, Murfin M et al. 2005. Disruption of the P2X7 purinoceptor gene abolishes chronic inflammatory and neuropathic pain. Pain, 114 (3), pp. 386-396. | Show Abstract | Read more

The P2X(7) purinoceptor is a ligand-gated cation channel, expressed predominantly by cells of immune origin, with a unique phenotype which includes release of biologically active inflammatory cytokine, interleukin (IL)-1beta following activation, and unique ion channel biophysics observed only in this receptor family. Here we demonstrate that in mice lacking this receptor, inflammatory (in an adjuvant-induced model) and neuropathic (in a partial nerve ligation model) hypersensitivity is completely absent to both mechanical and thermal stimuli, whilst normal nociceptive processing is preserved. The knockout animals were unimpaired in their ability to produce mRNA for pro-IL-1beta, and cytometric analysis of paw and systemic cytokines from knockout and wild-type animals following adjuvant insult suggests a selective effect of the gene deletion on release of IL-1beta and IL-10, with systemic reductions in adjuvant-induced increases in IL-6 and MCP-1. In addition, we show that this receptor is upregulated in human dorsal root ganglia and injured nerves obtained from chronic neuropathic pain patients. We hypothesise that the P2X(7) receptor, via regulation of mature IL-1beta production, plays a common upstream transductional role in the development of pain of neuropathic and inflammatory origin. Drugs which block this target may have the potential to deliver broad-spectrum analgesia.

Gopinath P, Wan E, Holdcroft A, Facer P, Davis JB, Smith GD, Bountra C, Anand P. 2005. Increased capsaicin receptor TRPV1 in skin nerve fibres and related vanilloid receptors TRPV3 and TRPV4 in keratinocytes in human breast pain. BMC Womens Health, 5 (1), pp. 2. | Show Abstract | Read more

BACKGROUND: Breast pain and tenderness affects 70% of women at some time. These symptoms have been attributed to stretching of the nerves with increase in breast size, but tissue mechanisms are poorly understood. METHODS: Eighteen patients (n = 12 breast reduction and n = 6 breast reconstruction) were recruited and assessed for breast pain by clinical questionnaire. Breast skin biopsies from each patient were examined using immunohistological methods with specific antibodies to the capsaicin receptor TRPV1, related vanilloid thermoreceptors TRPV3 and TRPV4, and nerve growth factor (NGF). RESULTS: TRPV1-positive intra-epidermal nerve fibres were significantly increased in patients with breast pain and tenderness (TRPV1 fibres / mm epidermis, median [range] - no pain group, n = 8, 0.69 [0-1.27]; pain group, n = 10, 2.15 [0.77-4.38]; p = 0.0009). Nerve Growth Factor, which up-regulates TRPV1 and induces nerve sprouting, was present basal keratinocytes: some breast pain specimens also showed NGF staining in supra-basal keratinocytes. TRPV4-immunoreactive fibres were present in sub-epidermis but not significantly changed in painful breast tissue. Both TRPV3 and TRPV4 were significantly increased in keratinocytes in breast pain tissues; TRPV3, median [range] - no pain group, n = 6, 0.75 [0-2]; pain group, n = 11, 2 123, p = 0.008; TRPV4, median [range] - no pain group, n = 6, [0-1]; pain group, n = 11, 1 [0.5-2], p = 0.014). CONCLUSION: Increased TRPV1 intra-epidermal nerve fibres could represent collateral sprouts, or re-innervation following nerve stretch and damage by polymodal nociceptors. Selective TRPV1-blockers may provide new therapy in breast pain. The role of TRPV3 and TRPV4 changes in keratinocytes deserve further study.

Bingham S, Beswick PJ, Bountra C, Brown T, Campbell IB, Chessell IP, Clayton N, Collins SD et al. 2005. The cyclooxygenase-2 inhibitor GW406381X [2-(4-ethoxyphenyl)-3-[4-(methylsulfonyl)phenyl]-pyrazolo[1,5-b]pyridazine] is effective in animal models of neuropathic pain and central sensitization. J Pharmacol Exp Ther, 312 (3), pp. 1161-1169. | Show Abstract | Read more

The pathogenic form of the cyclooxygenase (COX) enzyme, COX-2, is also constitutively present in the spinal cord and has been implicated in chronic pain states in rat and man. A number of COX-2 inhibitors, including celecoxib and rofecoxib, are already used in man for the treatment of inflammatory pain. Preclinically, the dual-acting COX-2 inhibitor, GW406381X [2-(4-ethoxyphenyl)-3-[4-(methylsulfonyl)phenyl]-pyrazolo[1,5-b]pyridazine, where X denotes the free base], is as effective as rofecoxib and celecoxib in the rat established Freund's Complete Adjuvant model with an ED(50) of 1.5 mg/kg p.o. compared with 1.0 mg/kg p.o. for rofecoxib and 6.6 mg/kg p.o. for celecoxib. However, in contrast to celecoxib (5 mg/kg p.o. b.i.d.) and rofecoxib (5 mg/kg p.o. b.i.d.), which were without significant effect, GW406381X (5 mg/kg p.o. b.i.d.) fully reversed mechanical allodynia in the chronic constriction injury model and reversed thermal hyperalgesia in the mouse partial ligation model, both models of neuropathic pain. GW406381X, was also effective in a rat model of capsaicin-induced central sensitization, when given intrathecally (ED(50) = 0.07 mug) and after chronic but not acute oral dosing. Celecoxib and rofecoxib had no effect in this model. Several hypotheses have been proposed to try to explain these differences in efficacy, including central nervous system penetration, enzyme kinetics, and potency. The novel finding of effectiveness of GW406381X in these models of neuropathic pain/central sensitization, in addition to activity in inflammatory pain models and together with its central efficacy, suggests dual activity of GW406381X compared with celecoxib and rofecoxib, which may translate into greater efficacy in a broader spectrum of pain states in the clinic.

Davies SL, Loescher AR, Clayton NM, Bountra C, Robinson PP, Boissonade FM. 2004. nNOS expression following inferior alveolar nerve injury in the ferret. Brain Res, 1027 (1-2), pp. 11-17. | Show Abstract | Read more

Damage to the inferior alveolar nerve (IAN) may result in permanent painful dysaesthesia, and there is compelling evidence to suggest that ectopic activity from the injury site plays a crucial role in the initiation of this disorder. The aim of this study was to determine whether neuronal nitric oxide synthase (nNOS), a regulator of neuronal excitability, could be involved in the development of the abnormal activity. In seven ferrets, the left IAN was exposed and a retrograde tracer, fluorogold, was applied to the nerve for the identification of cell bodies in the trigeminal ganglion with axons in the IAN. In four animals, the nerve was sectioned distal to the injection site, and three served as controls. After 3 days, the animals were perfused with fixative, and the left and right IANs and trigeminal ganglia were processed using indirect immunofluorescence for nNOS. Image analysis was used to quantify the percentage area of staining (PAS) at the injury site. In the ganglia, counts were made of positively labelled cells in the fluorogold population. At the injury site, PAS was significantly greater in injured nerves than in either contralateral or control nerves, and contralateral PAS was elevated compared to control. In the ganglia, the proportion of nNOS-labelled cells was significantly reduced following injury. These results indicate a possible translocation of the nNOS protein from the cell body to the site of nerve injury, where it accumulates. Thus, nNOS could play a role in the development of ectopic activity at a site of trigeminal nerve injury.

Beswick P, Bingham S, Bountra C, Brown T, Browning K, Campbell I, Chessell I, Clayton N et al. 2004. Identification of 2,3-diaryl-pyrazolo[1,5-b]pyridazines as potent and selective cyclooxygenase-2 inhibitors. Bioorg Med Chem Lett, 14 (21), pp. 5445-5448. | Show Abstract | Read more

GW406381 (8), currently undergoing clinical evaluation for the treatment of inflammatory pain is a member of a novel series of 2,3-diaryl-pyrazolo[1,5-b]pyridazine based cyclooxygenase-2 (COX-2) inhibitors, which have been shown to be highly potent and selective. Several examples of the series, in addition to possessing favourable pharmacokinetic profiles and analgesic activity in vivo, have also demonstrated relatively high brain penetration in the rat compared with the clinically available compounds, which may ultimately prove beneficial in the treatment of pain.

Dalziel RG, Bingham S, Sutton D, Grant D, Champion JM, Dennis SA, Quinn JP, Bountra C, Mark MA. 2004. Allodynia in rats infected with varicella zoster virus--a small animal model for post-herpetic neuralgia. Brain Res Brain Res Rev, 46 (2), pp. 234-242. | Show Abstract | Read more

The most common complication of herpes zoster is post-herpetic neuralgia (PHN), which has been defined as severe pain occurring 1 month after rash onset or persisting for greater than 3 months. PHN is classed as a neuropathic pain that is associated with mechanical allodynia where normally innocuous tactile stimuli are perceived as painful. The development of therapies to treat PHN has been hampered by the lack of animal models, which mimic the clinical situation. We have previously reported that varicella zoster virus (VZV) infection in the rat results in mechanical allodynia and thermal hyperalgesia. Here, we report that following VZV infection of the left footpad rats develop a chronic mechanical allodynia, which is present for longer than 60 days post-infection and which resolves by 100 days PI. The model is robust and reproducible with animals consistently developing allodynia by 3 days PI and continuing to present with symptoms for at least 30 days. The reproducible nature of the induction and course of the allodynia allows the use of this model to determine the effect of various compounds on, and to investigate the pathogenic mechanisms underlying the development of VZV-induced allodynia. Comparative studies using HSV-1 show that the induction of the chronic allodynia is VZV-specific and is not a result is of virus replication-induced tissue damage or accompanying inflammation. Therefore, we propose that the rat VZV infection model could prove useful in studying the mechanisms underlying post-herpetic neuralgia.

Casula MA, Facer P, Powell AJ, Kinghorn IJ, Plumpton C, Tate SN, Bountra C, Birch R, Anand P. 2004. Expression of the sodium channel beta3 subunit in injured human sensory neurons. Neuroreport, 15 (10), pp. 1629-1632. | Show Abstract | Read more

Voltage-gated sodium channel alpha-subunits play a key role in pain pathophysiology, and are modulated by beta-subunits. We previously reported that beta1- and beta2-subunits were decreased in human sensory neurons after spinal root avulsion injury. We have now detected, by immunohistochemistry, beta3-subunits in 82% of small/medium and 67% of large diameter sensory neurons in intact human dorsal root ganglia: 54% of beta3 small/medium neurons were NGF receptor trkA negative. Unlike beta1- and beta2, beta3-immunoreactivity did not decrease after avulsion injury, and the beta3:neurofilament ratio was significantly increased in proximal injured human nerves. beta3-subunit expression may thus be regulated differently from beta1, beta2 and Nav1.8. Targeting beta3 interactions with key alpha-subunits, particularly Nav1.3 and Nav1.8, may provide novel selective analgesics.

Durrenberger PF, Facer P, Gray RA, Chessell IP, Naylor A, Bountra C, Banati RB, Birch R, Anand P. 2004. Cyclooxygenase-2 (Cox-2) in injured human nerve and a rat model of nerve injury. J Peripher Nerv Syst, 9 (1), pp. 15-25. | Show Abstract | Read more

Inflammation associated with nerve injury produces a number of pathogenic chemical mediators of which prostanoids are a potent component. Cyclooxygenases (Cox-1 and Cox-2) are the enzymes responsible for prostanoid production. We have investigated Cox-2 immunoreactivity (Cox-2-IR) and glial activation in human injured (n = 16) and uninjured (n = 8) nerves and in the chronic constriction injury (CCI) model of nerve injury in the rat, using immunohistological and autoradiographic methods. Tissues were immunostained with antibodies to Cox-2, CD-68 (human macrophage marker), OX42 (rat microglial marker), or incubated with tritiated PK11195 (marker of glial activation), prior to image analysis. In human nerves, Cox-2-IR was detected in cells with morphology and distribution similar to macrophages/microglia - these were increased significantly in human nerve proximal to injury (p < 0.002), reaching a peak at 4-6 weeks after injury. In the rat CCI model, at 40 days after injury, microglia-like cells with Cox-2-IR were increased significantly in the injured nerve (p < 0.004) and ipsilateral dorsal spinal cord (p < 0.008). PK11195-binding results were similar for Cox-2-IR in chronic injured human nerve and rat tissues. These findings suggest that Cox-2-immunoreactive cells could play a role in processes associated with Wallerian degeneration, nerve regeneration, and the development of persistent pain. Selection of patients 4-6 weeks after nerve injury would be more likely to show any efficacy of Cox-2 inhibitors.

Yates JM, Loescher AR, Boissonade FM, Clayton N, Bountra C, Robinson PP. 2003. Sodium channel blacker 4030W92 reduces spontaneous discharge from damaged axons in the lingual nerve of ferrets. JOURNAL OF DENTAL RESEARCH, 82 pp. B160-B160.

Davies SL, Worsley MA, Loescher AR, Clayton N, Bountra C, Robinson PP, Boissonade FM. 2003. The effect of the sodium channel blocker 4030W92 on fos expression the jaw-opening reflex following stimulation of normal and inflamed ferret tooth pulps. JOURNAL OF DENTAL RESEARCH, 82 pp. B160-B160.

Worsley MA, Davies SL, Clayton N, Bountra C, Boissonade FM. 2003. The effects of inflammation and the neurokinin-1 receptor antagonist GR205171A on fos expression in the ferret trigeminal nucleus following tooth pulp stimulation. JOURNAL OF DENTAL RESEARCH, 82 pp. B161-B161.

Arnold SJ, Facer P, Yiangou Y, Chen MX, Plumpton C, Tate SN, Bountra C, Chan CL, Williams NS, Anand P. 2003. Decreased potassium channel IK1 and its regulator neurotrophin-3 (NT-3) in inflamed human bowel. Neuroreport, 14 (2), pp. 191-195. | Show Abstract | Read more

Calcium-activated potassium currents of intermediate conductance (IK1) have been described in the rodent enteric nervous system, where they may regulate afterhyperpolarisation of intrinsic primary afferent neurons. Using specific antibodies for immuno-cytochemistry, we now report IK1-like immunoreactivity for the first time in enteric neurons of human colon, and a significant decrease of IK1-positive cells in myenteric plexus in inflamed colon from patients with Crohn's disease and ulcerative colitis (p = 0.031). Neurotrophin-3 (NT-3), which regulates IK1 expression, was also observed in fewer neurons of the myenteric ganglia in Crohn's bowel (p = 0.048), and in inflamed colonic extracts by Western blotting (p = 0.004); the numbers of neurons expressing the NT-3 high affinity receptor trk C were unchanged. Our findings may explain the diarrhoea and colicky abdominal pain produced by inflammatory bowel disease, and by IK1-blocking pyridine drugs prescribed for neuromuscular disorders.

Chan CL, Facer P, Davis JB, Smith GD, Egerton J, Bountra C, Williams NS, Anand P. 2003. Sensory fibres expressing capsaicin receptor TRPV1 in patients with rectal hypersensitivity and faecal urgency. Lancet, 361 (9355), pp. 385-391. | Show Abstract | Read more

BACKGROUND: Faecal urgency and incontinence with rectal hypersensitivity is a distressing, unexplained disorder that is inadequately treated. We aimed to determine whether expression of the heat and capsaicin receptor vanilloid receptor 1 (TRPV1 or VR1) was changed in rectal sensory fibres, and to correlate nerve fibre density with sensory abnormalities. METHODS: We compared full-thickness rectal biopsy samples from nine patients with physiologically characterised rectal hypersensitivity with tissue samples from 12 controls. Sensory thresholds to rectal balloon distension and heating the rectal mucosa were measured before biopsy. We assessed specimens with immunohistochemistry and image analysis using specific antibodies to TRPV1; nerve growth factor (NGF) receptor tyrosine kinase A; glial cell line-derived neurotrophic factor (GDNF); neuropeptides calcitonin gene-related peptide (CGRP) and substance P; the related vanilloid receptor-like protein (VRL) 2; glial markers S-100 and glial fibrillary acid protein (GFAP); and the nerve structural marker peripherin. FINDINGS: In rectal hypersensitivity, nerve fibres immunoreactive to TRPV1 were increased in muscle, submucosal, and mucosal layers: in the mucosal layer, the median% area positive was 0.44 (range 0.30-0.59) in patients who were hypersensitive and 0.11 (0.00-0.21) in controls (p=0.0005). The numbers of peripherin-positive fibres also increased in the mucosal layer (hypersensitive 3.00 [1.80-6.50], controls 1.20 [0.39-2.10]: (p=0.0002). The increase in TRVP1 correlated significantly with the decrease in rectal heat (p=0.03) and the distension (p=0.02) sensory thresholds. The thresholds for heat and distension were also significantly correlated (p=0.0028). Expression of nerve fibres positive for GDNF (p=0.001) and tyrosine kinase A (p=0.002) was also increased, as were cell bodies of the submucosal ganglia immunoreactive to CGRP (p=0.0009). INTERPRETATION: Faecal urgency and rectal hypersensitivity could result from increased numbers of polymodal sensory nerve fibres expressing TRPV1. The triggering factor or factors remain uncertain, but drugs that target nerve terminals that express this receptor, such as topical resiniferatoxin, deserve consideration.

Clayton N, Marshall FH, Bountra C, O'Shaughnessy CT. 2002. CB1 and CB2 cannabinoid receptors are implicated in inflammatory pain. Pain, 96 (3), pp. 253-260. | Show Abstract | Read more

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 g/kg-1 i.p. (in the carrageenan model). The selective CB1 antagonist, AM281 (0.5 microg/kg-1 i.p.) reversed effects of HU210 (10 and 30 microg/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 microg/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.

Boettger MK, Till S, Chen MX, Anand U, Otto WR, Plumpton C, Trezise DJ, Tate SN et al. 2002. Calcium-activated potassium channel SK1- and IK1-like immunoreactivity in injured human sensory neurones and its regulation by neurotrophic factors. Brain, 125 (Pt 2), pp. 252-263. | Show Abstract | Read more

Calcium-activated potassium ion channels SK and IK (small and intermediate conductance, respectively) may be important in the pathophysiology of pain following nerve injury, as SK channels are known to impose a period of reduced excitability after each action potential by afterhyperpolarization. We studied the presence and changes of human SK1 (hSK1)- and hIK1-like immunoreactivity in control and injured human dorsal root ganglia (DRG) and peripheral nerves and their regulation by key neurotrophic factors in cultured rat sensory neurones. Using specific antibodies, hSK-1 and hIK-1-like immunoreactivity was detected in a majority of large and small/medium-sized cell bodies of human DRG. hSK1 immunoreactivity was decreased significantly in cell bodies of avulsed human DRG (n = 8, surgery delay 8 h to 12 months). There was a decrease in hIK1-like immunoreactivity predominantly in large cells acutely (<3 weeks after injury), but also in small/medium cells of chronic cases. Twenty-three injured peripheral nerves were studied (surgery delay 8 h to 12 months); in five of these, hIK1-like immunoreactivity was detected proximally but not distally to injury, whereas neurofilament staining confirmed the presence of nerve fibres in both regions. These five nerves, unlike the others, had all undergone Wallerian degeneration previously and the loss of hIK1-like immunoreactivity may therefore reflect reduced axonal transport of this ion channel across the injury site in regenerated fibres, as well as decreased expression in the cell body. In vitro studies of neonatal rat DRG neurones showed that nerve growth factor (NGF) significantly increased the percentage of hSK1-positive cells, whereas neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor (GDNF) failed to show a significant effect. NT-3 stimulated hIK1 expression, while NGF and GDNF were ineffective. As expected, NGF increased expression of the voltage-gated sodium channel SNS1/PN3 in this system. Decreased retrograde transport of these neurotrophic factors in injured sensory neurones may thus reduce expression of these ion channels and increase excitability. Blockade of IK1-like and other potassium channels by aminopyridines (4-AP and 3,4-DAP) may also explain the paraesthesiae induced by these medications. Selective potassium channel openers are likely to represent novel therapies for pain following nerve injury.

Bucknill AT, Coward K, Plumpton C, Tate S, Bountra C, Birch R, Sandison A, Hughes SP, Anand P. 2002. Nerve fibers in lumbar spine structures and injured spinal roots express the sensory neuron-specific sodium channels SNS/PN3 and NaN/SNS2. Spine (Phila Pa 1976), 27 (2), pp. 135-140. | Show Abstract | Read more

STUDY DESIGN: This prospective study examined the innervation of lumbar spine in tissues from patients with lower back pain and spine nerve roots from patients with traumatic brachial plexus injuries. OBJECTIVES: To demonstrate the presence of nerve fibers in lumbar spine structures and spine nerve roots, and to determine whether they express the sensory neuron-specific sodium channels SNS/PN3 and NaN/SNS2. SUMMARY OF BACKGROUND DATA: The anatomic and molecular basis of low back pain and sciatica is poorly understood. Previous studies have demonstrated sensory nerves in the facet joint capsule and prolapsed intervertebral disc, but not in the ligamentum flavum. The voltage-gated sodium channels SNS/PN3 and NaN/SNS2 are expressed by sensory neurone that mediate pain, but their presence in the lumbar spine is unknown. METHODS: Tissue samples of ligamentum flavum (n = 32), facet joint capsule (n = 20), intervertebral disc (n = 15), and spine roots (n = 8) were immunostained with specific antibodies to protein gene product 9.5 (a panneuronal marker), SNS/PN3, and NaN/SNS2. RESULTS: Protein gene product 9.5 immunoreactive nerve fibers were detected in 72% of the ligamentum flavum specimens and 70% of the facet joint capsule specimens, but in only 20% of the intervertebral disc specimens. The study detected SNS/PN3- and NaN/SNS2-positive fibers, respectively, in 28% and 3% of the ligamentum flavum specimens and 25% and 15% of the facet joint capsule specimens. Numerous SNS/PN3- and NaN/SNS2-positive fibers were found in the acutely injured spine roots, and some were still present in the dorsal roots in the chronic state. CONCLUSIONS: As the findings showed, SNS/PN3- and NaN/SNS2-immunoreactivity is present in a subset of nerve fibers in lumbar spine structures, including ligamentum flavum, and in injured spine roots. Selective SNS/PN3- and NaN/SNS2-blocking agents may provide new therapy for back pain and sciatica.

Clayton NM, O'Shaughnessy CT, Marshall F, Bountra C. 2001. CB1 and CB2 cannabinoid receptors are implicated in inflammatory hypersensitivity to pain BRITISH JOURNAL OF PHARMACOLOGY, 134

Brown T, Clayton N, Brazdil R, Wiseman J, Naylor A, Bountra C. 2001. The effects of a selective Cox-2 inhibitor GR253035X in rat models of inflammatory hyperalgesia assessed using behavioural readouts and by quantitation of spinal immunochemistry BRITISH JOURNAL OF PHARMACOLOGY, 134

Brazdil R, Kozlowski CM, Clayton NM, Stratton SC, Bountra C. 2001. Inhibition of colorectal distension induced c-Fos expression in the spinal cord following MK801 and GV 196771 in the anaesthetised rat. BRITISH JOURNAL OF PHARMACOLOGY, 134

Brown T, Clayton N, Stratton S, Bountra C, Sheehan M. 2001. The anti-hyperalgesic effects of the high and low intrinsic efficacy Adenosine(1) receptor agonists GR79236X and GR190178X in a mouse model of inflammatory hyperalgesia BRITISH JOURNAL OF PHARMACOLOGY, 134

Collins SD, Clayton NM, Sheehan MJ, Bountra C. 2001. The effect of GR190178, a selective low-efficacy adenosine A1 receptor agonist, on the treatment of neuropathic hyperalgesia in the rat. BRITISH JOURNAL OF PHARMACOLOGY, 133

Clayton NM, Brown TA, Sargent RS, Brazdil R, Collins SD, Sheehan MJ, Bountra C. 2001. The effect of the selective low-efficacy adenosine A1 agonist GR190178, in models of nociceptive, acute and established pain hypersensitivity in the rat BRITISH JOURNAL OF PHARMACOLOGY, 133

Clayton N, Kozlowski CM, Brazdil R, Bountra C. 2001. Colorectal distension-evoked iNOS, nitrotyrosine and C-FOS expression in the anaesthetised rat is inhibited by the selective iNOS inhibitor GW274150 BRITISH JOURNAL OF PHARMACOLOGY, 133 pp. U79-U79.

O'Sullivan MA, Clayton N, Hurle M, Bountra C, Buckley M, O'Morain CA. 2001. Activation of nuclear factor-kappa B (NF-kappa B)in diarrhoea predominant irritable bowel syndrome GASTROENTEROLOGY, 120 (5), pp. A641-A642. | Read more

Davies SL, Loescher AR, Clayton N, Bountra C, Robinson PP, Boissonade FM. 2001. Sodium channel and nNOS expression following inferior alveolar nerve JOURNAL OF DENTAL RESEARCH, 80 (4), pp. 1167-1167.

Coward K, Jowett A, Plumpton C, Powell A, Birch R, Tate S, Bountra C, Anand P. 2001. Sodium channel beta1 and beta2 subunits parallel SNS/PN3 alpha-subunit changes in injured human sensory neurons. Neuroreport, 12 (3), pp. 483-488. | Show Abstract | Read more

Voltage-gated sodium channels consist of a pore-containing alpha-subunit and one or more auxiliary beta-subunits, which may modulate channel function. We previously demonstrated that sodium channel SNS/PN3 alpha-subunits were decreased in human sensory cell bodies after spinal root avulsion injury, and accumulated at injured nerve terminals in pain states. Using specific antibodies for immunohistochemistry, we have now detected sodium channel beta1 and beta2 subunits in sensory cell bodies within control human postmortem sensory ganglia (78% of small/medium (< or = 50 microm) and 68% of large (> or = 50 microm) cells); their changes in cervical sensory ganglia after avulsion injury paralleled those described for SNS/PN3 alpha-subunits. Our results suggest that alpha- and beta-subunits share common regulatory mechanisms, but present distinct targets for novel analgesics.

Coward K, Aitken A, Powell A, Plumpton C, Birch R, Tate S, Bountra C, Anand P. 2001. Plasticity of TTX-sensitive sodium channels PN1 and brain III in injured human nerves. Neuroreport, 12 (3), pp. 495-500. | Show Abstract | Read more

Sensory neurones co-express voltage-gated sodium channels that mediate TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) currents, which may contribute to chronic pain after nerve injury. We previously demonstrated that TTX-R channels were decreased acutely in human sensory cell bodies after central axotomy, but accumulated in nerve terminals after peripheral axotomy. We have now studied the TTX-S channels PN1 and Brain III, using specific antibodies for immunohistochemistry, in dorsal root ganglia (DRG) from 10 patients with traumatic central axotomy, nerves from 16 patients with peripheral axotomy, and controls. PN1 showed temporal changes similar to the TTX-R channels in sensory cell bodies of injured DRG. In contrast, Brain III was found only in injured nerves (not control nerves, or control/central axotomy DRG). PNI and Brain III are distinct targets for novel analgesics.

Coward K, Mosahebi A, Plumpton C, Facer P, Birch R, Tate S, Bountra C, Terenghi G, Anand P. 2001. Immunolocalisation of sodium channel NaG in the intact and injured human peripheral nervous system. J Anat, 198 (Pt 2), pp. 175-180. | Show Abstract | Read more

The voltage-gated 'glial' sodium channel NaG belongs to a distinct molecular class within the multi-gene family of mammalian sodium channels. Originally found in central and peripheral glia, NaG has since been detected in neurons in rat dorsal root ganglia (DRG) and may play a role in Schwann cell-axon interactions. We have studied the presence of NaG-like immunoreactivity in the intact and injured human peripheral nervous system using a specific affinity-purified antibody. Nerve fibres in normal and injured peripheral nerves and normal skin exhibited intense NaG-immunoreactivity. Numerous NaG-immunoreactive nerve fibres surrounded neuronal cell bodies within postmortem control DRG, and in DRG avulsed from the spinal cord (i.e. after traumatic central axotomy). There were no significant differences in the pattern of NaG immunostaining between control and avulsed DRG, or with delay after injury. Generally, the neuronal cell bodies were only very weakly immunoreactive to NaG, indicating that the NaG immunoreactivity was predominantly in Schwann cells/myelin. In accord, we demonstrated NaG immunostaining in cultured human and rat Schwann cells, and in distal nerve after wallerian degeneration. NaG thus appears to be a useful new marker for Schwann cells in the human PNS, and a role in neuropathy deserves investigation.

Shembalkar PK, Till S, Boettger MK, Terenghi G, Tate S, Bountra C, Anand P. 2001. Increased sodium channel SNS/PN3 immunoreactivity in a causalgic finger. Eur J Pain, 5 (3), pp. 319-323. | Show Abstract | Read more

The sodium channels SNS/PN3 and NaN/SNS2 are regulated by the neurotrophic factors-nerve growth factor (NGF) and glial-derived neurotrophic factor (GDNF), and may play an important role in the development of pain after nerve injury or inflammation. These key molecules have been studied in an amputated causalgic finger and control tissues by immunohistochemistry. There was a marked increase in the number and intensity of SNS/PN3-immunoreactive nerve terminals in the affected finger, while GDNF-immunoreactivity was not observed, in contrast to controls. No differences were observed for NGF, trk A, NT-3 or NaN/SNS2-immunoreactivity. While further studies are required, these findings suggest that accumulation of SNS/PN3 and/or loss of GDNF may contribute to pain in causalgia, and that selective blockers of SNS/PN3 and/or rhGDNF may provide effective novel treatments.

O'Sullivan M, Clayton N, Breslin NP, Harman I, Bountra C, McLaren A, O'Morain CA. 2000. Increased mast cells in the irritable bowel syndrome. Neurogastroenterol Motil, 12 (5), pp. 449-457. | Show Abstract | Read more

Mast cells (MC) release potent mediators which alter enteric nerve and smooth muscle function and may play a role in the pathogenesis of the irritable bowel syndrome (IBS). The aim of this study was to determine if MC were increased in the colon of IBS patients compared to controls. Biopsy specimens were obtained from the caecum, ascending colon, descending colon and rectum of 28 patients: 14 IBS (Rome criteria); seven normal; and seven inflammatory controls. Tissue was stained immunohistochemically using a monoclonal mouse antibody for human mast cell tryptase (AA1). Tissue area occupied by tryptase-positive MC (volume density of mast cells) was quantified by image analysis. The number of plasma cells, lymphocytes, eosinophils, neutrophils and macrophages were each graded semiquantitatively (0-4) in haematoxylin and eosin stained sections. Mast cell volume density was significantly (P < 0.05) higher in IBS (0.91 +/- 0.18; CI 0.79; 1.0) than normal controls (0.55 +/- 0.14; CI 0.40; 0.69) in the caecum but not at other sites. Apart from MC, there was no evidence of increased cellular infiltrate in the IBS group. MC were significantly increased in the caecum of IBS patients compared to controls. The multiple effects of the intestinal mast cell alone, or as a participant of a persistent inflammatory response, may be fundamental to the pathogenesis of IBS.

Asghar AU, Wheeldon A, Coleman RA, Bountra C, McQueen DS. 2000. Hoe 140 and pseudo-irreversible antagonism in the rat vas deferens in vitro. Eur J Pharmacol, 398 (1), pp. 131-138. | Show Abstract | Read more

The effects of bradykinin and the bradykinin B(2) receptor antagonists D-Arg-[Hyp(3),Thi(5,8),D-Phe(7)]-bradykinin (NPC 349) and D-Arg-[Hyp(3),Thi(5),D-Tic(7),Oic(8)]-bradykinin (Hoe 140) were examined in the electrically-stimulated rat vas deferens. Cumulative additions of bradykinin (1-3000 nM) produced two distinct responses: an enhancement in the magnitude of the basal electrically-induced twitch response (neurogenic response) and an increase in the baseline tension (musculotropic response). NPC 349 (10-100 microM) produced concentration-dependent surmountable rightward shifts of both the bradykinin neurogenic and musculotropic response curves. In contrast, while Hoe 140 (10-100 nM) caused an apparently surmountable antagonism of the bradykinin neurogenic response, it caused an apparent insurmountable antagonism of the bradykinin musculotropic response. Interestingly, co-incubation of Hoe 140 (30 nM) with NPC 349 (30 and 100 microM) resulted in a concentration-related upwards displacement of the Hoe 140-suppressed bradykinin musculotropic response curve. Thus, Hoe 140 can be described as a pseudo-irreversible antagonist against the bradykinin musculotropic response. No time-dependent changes were observed in the maximum bradykinin musculotropic response attainable when NPC 349 (100 microM) additions were made for the final 2 or 18 min of the Hoe 140 incubation (20 min). These findings indicate that slow reversibility of Hoe 140 from the bradykinin B(2) receptor is unlikely to be the mechanism responsible for the pseudo-irreversible antagonism of the bradykinin-induced musculotropic response. Instead, we propose an alternative explanation involving a third, unstable and inactive form of the bradykinin B(2) receptor.

Kozlowski CM, Bountra C, Grundy D. 2000. The effect of fentanyl, DNQX and MK-801 on dorsal horn neurones responsive to colorectal distension in the anaesthetized rat. Neurogastroenterol Motil, 12 (3), pp. 239-247. | Show Abstract | Read more

Certain dorsal horn neurones respond in a graded manner to noxious colorectal distension (CRD). Morphine inhibits these responses in the spinalized rat, but the role of excitatory amino acids in baseline visceral nociceptive transmission is less clear. This study examines the effect of the mu-opiate receptor agonist fentanyl, and the non-NMDA and NMDA antagonists DNQX and MK-801, respectively, on such responses to CRD in the sodium pentobarbitone-anaesthetized rat. Male rats were prepared for extracellular recording from the lumbosacral spinal cord. 90 neurones responsive to CRD, located throughout the dorsal horn, were classified according to their response duration and latency to 60 mmHg distension, as SL-A (short latency-abrupt; 59%), SL-S (short latency-sustained; 23%), L-L (long-latency; 10%) and Inhib (inhibited; 8%). Convergent cutaneous receptive fields were mapped for 79/90 neurones and classified as LT (low threshold), WDR (wide dynamic range) or HT (high threshold). CRD (20-100 mm Hg) elicited graded responses in most neurones. In 6/6 SL-S neurones, fentanyl (1-8 microg kg-1) dose-dependently inhibited the response to 60 mm Hg CRD, in a naloxone-sensitive manner, with an ID50 value (+/-95% confidence limits) of 2.48 (1.7-3. 7) microg kg-1. In 6/6 SL-A neurones, fentanyl had no significant effect on the response to CRD. DNQX (0.03-3 mg kg-1) produced a dose-dependent inhibition of the response to CRD in 5/5 SL-A neurones, with an ID50 value of 0.32 (0.01-41.1) mg kg-1. MK-801 (0. 03-0.3 mg kg-1) had no significant effect on responses to CRD in 6/6 SL-A neurones. The differential inhibitory effects of fentanyl on two neuronal subtypes may indicate functional differences. In SL-A neurones AMPA/kainate, but not NMDA receptors are involved in mediating baseline nociceptive neurotransmission.

Kozlowski CM, Green A, Grundy D, Boissonade FM, Bountra C. 2000. The 5-HT(3) receptor antagonist alosetron inhibits the colorectal distention induced depressor response and spinal c-fos expression in the anaesthetised rat. Gut, 46 (4), pp. 474-480. | Show Abstract | Read more

BACKGROUND: Noxious intestinal distention elicits a reflex depressor response in the sodium pentobarbitone anaesthetised rat, which can be used as an index of visceral nociception. 5-HT(3) receptor antagonists inhibit this reflex. Repeated colorectal distention (CRD) induces Fos like immunoreactivity (Fos-LI) in the rat spinal cord. AIMS: To examine the effect of the 5-HT(3) receptor antagonist alosetron on the depressor response to CRD, and on Fos expression in the lumbosacral spinal cord. METHODS: Male rats were anaesthetised with sodium pentobarbitone, and mean arterial blood pressure monitored during repeated colorectal balloon inflation before and after treatment with alosetron or saline. Rats anaesthetised with urethane and treated with alosetron or saline underwent a repeated CRD paradigm, after which the lumbosacral spinal cord was removed and processed for visualisation of Fos-LI. RESULTS: CRD elicited reproducible, volume dependent falls in arterial blood pressure, and repeated distention-effect curves were constructed. Alosetron (1-100 microg/kg intravenously) inhibited the depressor response to CRD in a dose related manner, with an ID(50) value of 3.0 microg/kg. Following repeated CRD, numbers of Fos-LI neurones were significantly increased to 1246 (total in 12 sections at 120 microm intervals from L6 to S1) compared with 49 in sham distended animals. Pretreatment with alosetron (100 microg/kg) significantly reduced numbers of Fos-LI neurones to 479.8. CONCLUSION: The 5-HT(3) receptor antagonist alosetron inhibits the depressor response to CRD in a potent and dose dependent manner. It also inhibits CRD induced Fos-LI in the spinal cord. These results suggest that 5-HT(3) receptors are involved in visceral nociceptive transmission, perhaps located on primary afferent or spinal neurones.

O'Sullivan MA, Clayton N, Wong T, Bountra C, Buckley MM, O'Morain CA. 2000. Increased iNOS and nitrotyrosine expression in irritable bowel syndrome (IBS). GASTROENTEROLOGY, 118 (4), pp. A702-A702.

Coward K, Plumpton C, Facer P, Birch R, Carlstedt T, Tate S, Bountra C, Anand P. 2000. Immunolocalization of SNS/PN3 and NaN/SNS2 sodium channels in human pain states. Pain, 85 (1-2), pp. 41-50. | Show Abstract | Read more

The tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel SNS/PN3 and the newly discovered NaN/SNS2 are expressed in sensory neurones, particularly in nociceptors. Using specific antibodies, we have studied, for the first time in humans, the presence of SNS/PN3 and NaN/SNS2 in peripheral nerves, including tissues from patients with chronic neurogenic pain. In brachial plexus injury patients, there was an acute decrease of SNS/PN3- and NaN/SNS2-like immunoreactivity in sensory cell bodies of cervical dorsal root ganglia (DRG) whose central axons had been avulsed from spinal cord, with gradual return of the immunoreactivity to control levels over months. In contrast, there was increased intensity of immunoreactivity to both channels in some peripheral nerve fibers just proximal to the site of injury in brachial plexus trunks, and in neuromas. These findings suggest that the expression of these sodium channels in neuronal cell bodies is reduced after spinal cord root avulsion injury in man, but that pre-synthesized channel proteins may undergo translocation with accumulation at sites of nerve injury, as in animal models of peripheral axotomy. The latter may contribute to positive symptoms, as our patients all showed a positive Tinel's sign. Nerve terminals in distal limb neuromas and skin from patients with chronic local hyperalgesia and allodynia all showed marked increases of SNS/PN3-immunoreactive fibers, but little or no NaN/SNS2-immunoreactivity, suggesting that the former may be related to the persistent hypersensitive state. Axonal immunoreactivity to both channels was similar to control nerves in sural nerve biopsies in a selection of neuropathies, irrespective of nerve inflammation, demyelination or spontaneous pain, including a patient with congenital insensitivity to pain. Our studies suggest that the best target for SNS/PN3 blocking agents is likely to be chronic local hypersensitivity.

Clayton NM, Brown T, Brazdil R, Collins SD, Pass M, Sheehan M, Bountra C. 2000. The effect of the highly selective adenosine A1 agonist GR79236 in models of nociceptive, acute and chronic inflammatory pain BRITISH JOURNAL OF PHARMACOLOGY, 129 pp. U45-U45.

Collins SD, Clayton NM, Sheehan MJ, Pass M, Bountra C. 2000. The effect of GR79236, a highly selective adenosine A1 receptor agonist, on the treatment of neuropathic pain in the rat BRITISH JOURNAL OF PHARMACOLOGY, 129

Clayton NM, Alderton W, Angel A, Craig C, Dawson J, Brown T, Collins SD, Russell L, Knowles RG, Bountra C. 2000. The effect of the potent and selective iNOS (NOS-2) inhibitor GW274150 in models of acute and establised inflammatory and neuropathic pain in the rat EUROPEAN JOURNAL OF NEUROSCIENCE, 12 pp. 123-123.

Collins SD, Clayton NM, Brown T, Brazdil R, Sheehan MJ, Pass M, Bountra C. 2000. The effect of GR79236, a highly selective adenosine al receptor agonist, in models of nociceptive, acute and chronic inflammatory and neuropathic pain. EUROPEAN JOURNAL OF NEUROSCIENCE, 12 pp. 125-125.

Duffy C, Collins SD, Nobbs M, Bountra C. 2000. Neuronal apoptosis in rat dorsal root ganglia may contribute to the development of chronic hypersensitivity after chronic constriction injury EUROPEAN JOURNAL OF NEUROSCIENCE, 12 pp. 230-230.

Clayton NM, Sargent R, Butler A, Gale J, Maxwell MP, Hunt AA, Barrett VJ, Cambridge D, Bountra C, Humphrey PP. 1999. The pharmacological properties of the novel selective 5-HT3 receptor antagonist, alosetron, and its effects on normal and perturbed small intestinal transit in the fasted rat. Neurogastroenterol Motil, 11 (3), pp. 207-217. | Show Abstract | Read more

The purpose of this study was to investigate the pharmacological properties of the novel, selective 5-HT3 receptor antagonist, alosetron, and its effects on transit time in both the normal and perturbed small intestine of the rat. Alosetron concentration-dependently inhibited radioligand binding in membranes containing rat and human 5-HT3 receptors with estimated pKi values of 9.8 (n = 3) and 9.4 (n = 6), respectively. In selectivity studies alosetron had little or no significant affinity for any of the many other receptors and ion channels studied. Alosetron potently antagonized the depolarization produced by 5-HT in the rat vagus nerve (estimated pKB value of 9.8, n = 25). In anaesthetized rats, i. v. administration of alosetron inhibited 2-methyl-5-HT induced bradycardia (Bezold Jarisch index) at 1 and 3 microg kg-1, with an agonist dose ratio of approximately 3.0 at 1.0 microg kg-1, = 3-5). Alosetron administered via the duodenum also inhibited this reflex, with duration of action that was significantly longer than that seen with ondansetron (120-60 min, respectively, n = 6). Alosetron had no significant effect on normal small intestinal propulsion in the rat, but fully reversed the increase in intestinal propulsion (96%, n = 3) produced by egg albumin challenge. Alosetron is a highly selective 5-HT3 antagonist which normalizes perturbed small intestinal propulsion. Previous clinical data in IBS patients together with the transit data provide a good rationale for further studies with alosetron in IBS patients.

Humphrey PP, Bountra C, Clayton N, Kozlowski K. 1999. Review article: the therapeutic potential of 5-HT3 receptor antagonists in the treatment of irritable bowel syndrome. Aliment Pharmacol Ther, 13 Suppl 2 (s1), pp. 31-38. | Show Abstract | Read more

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. Alosetron is a highly selective, potent 5-HT3 receptor antagonist which is well absorbed with a long pharmacodynamic half-life. Its ability to provide long-lasting blockade of 5-HT3 receptors throughout the body make it an ideal candidate within its class to evaluate the clinical hypothesis that sustained and ubiquitous 5-HT3 receptor blockade is of value in the treatment of IBS.

O'Sullivan M, Breslin NP, Harman I, Clayton N, Bountra C, McLaren A, O'Morain CA. 1999. Increased mucosal mast cells - Evidence of an inflammatory response in IBS? GUT, 44 pp. A132-A132.

Munglani R, Hudspith MJ, Fleming B, Harrisson S, Smith G, Bountra C, Elliot PJ, Birch PJ, Hunt SP. 1999. Effect of pre-emptive NMDA antagonist treatment on long-term Fos expression and hyperalgesia in a model of chronic neuropathic pain. Brain Res, 822 (1-2), pp. 210-219. | Show Abstract | Read more

The unilateral sciatic nerve chronic constriction injury (CCI) model of Bennett and Xie [G.J. Bennett, Y.-K. Xie, A peripheral neuropathy in rat that produces disorders of pain sensation like those seen in man, Pain, 33 (1988) 87-108] shows features of a neuropathic pain state. We examined mechanical hyperalgesia and Fos protein staining in the lumbar spinal cord 1, 7, 14 and 28 days after unilateral CCI to the sciatic nerve or sham operation. In addition, we examined the effect of the NMDA antagonist MK-801 (0.3 mg/kg s.c. administered 30 min prior to and 6 h following operation) on Fos expression and hyperalgesia at 28 days. CCI animals were hyperalgesic compared to the sham operated animals at 14 and 28 days post injury. MK-801 reduced hyperalgesia by 68% in CCI animals on day 28 (p=0.0001). In the spinal cord, Fos positive cells were present bilaterally in deeper laminae in both sham and CCI animals at all time points examined. Relatively few Fos positive cells were present in laminae 1-2 at any time point examined. At days 1 and 7, there were increased numbers of Fos positive cells ipsilaterally in the deeper laminae of the spinal cord in CCI animals compared to sham animals, but by 14 and 28 days Fos counts were similar in sham and CCI despite the obvious behavioural differences between the two groups. Fos counts ipsilateral to the injury in laminae 3-10 correlated with hyperalgesia scores in the CCI but not sham animals. Analysis at the 28-day time point showed that MK-801 differentially affected Fos expression: MK-801 significantly reduced the Fos count bilaterally in laminae 3-10 in the CCI but not in the sham group animals. These results indicate that Fos expression is initiated by different peripheral and central mechanisms following nerve injury or sham operation.

Hudspith MJ, Harrisson S, Smith G, Bountra C, Elliot PJ, Birch PJ, Hunt SP, Munglani R. 1999. Effect of post-injury NMDA antagonist treatment on long-term Fos expression and hyperalgesia in a model of chronic neuropathic pain. Brain Res, 822 (1-2), pp. 220-227. | Show Abstract | Read more

Chronic constriction injury (CCI) of the sciatic nerve results in persistent mechanical hyperalgesia together with Fos protein expression in the lumbar spinal cord. We have examined the relationship between mechanical hyperalgesia and Fos expression within the lumbar spinal cord on days 14, 35 and 55 after either CCI or sham operation. To determine the role of NMDA receptor mechanisms in the maintenance of hyperalgesia and Fos expression, the NMDA antagonist MK-801 (0.3 mg kg-1 s.c.) was administered daily on days 28 to 34 after operation. CCI animals developed unilateral hind limb hyperalgesia that persisted unchanged from days 14 to 55 of the study. MK-801 treatment reduced hyperalgesia by 57% (p=0.02) on day 35 in CCI animals but did influence hyperalgesia at day 55. In the spinal cord, Fos positive cells were present bilaterally throughout laminae 3-10 at all time points examined in both CCI and sham group animals. Fos counts ipsilateral to the side of injury in laminae 3-10 correlated significantly with hyperalgesia scores in the CCI but not sham animals. MK-801 treatment resulted in a suppression of Fos expression in ipsilateral laminae 3-4 (p=0.0017) and laminae 5-10 (p=0.0026) of CCI animals on day 35. Fos expression in sham group animals was not inhibited by MK-801 treatment at day 35. These results indicate that Fos expression is maintained by differing mechanisms following nerve injury or sham operation. The functional consequences of Fos expression following nerve injury and sham operation are discussed.

Cougnon-Aptel N, Munglani R, Clayton NM, Ward P, Bountra C. 1999. Changes in spinal cord neuropeptides in the adjuvant model of chronic inflammatory hyperalgesia in the presence and absence of the NK1 receptor antagonist GR205171 BRITISH JOURNAL OF PHARMACOLOGY, 126 pp. U148-U148.

Tate S, Benn S, Hick C, Trezise D, John V, Mannion RJ, Costigan M, Plumpton C et al. 1998. Two sodium channels contribute to the TTX-R sodium current in primary sensory neurons. Nat Neurosci, 1 (8), pp. 653-655. | Read more

O'Sullivan M, Breslin NP, Harman I, Clayton N, Bountra C, McLaren A, O'Morain CA. 1998. Increased mucosal mast cells in irritable bowel syndrome (IBS). GASTROENTEROLOGY, 114 (4), pp. A816-A816. | Read more

Collins SD, Clayton NM, Nobbs M, Bountra C. 1998. The effect of 4030W92, a novel sodium channel blocker, on the treatment of neuropathic pain in the rat BRITISH JOURNAL OF PHARMACOLOGY, 123

Clayton NM, Collins SD, Sargent R, Brown T, Nobbs M, Bountra C, Trezise DJ. 1998. The effect of the novel sodium channel blocker 4030W92 in models of acute and chronic inflammatory pain in the rat BRITISH JOURNAL OF PHARMACOLOGY, 123

Evans KS, Kozlowski CM, Bountra C. 1998. The novel sodium channel blocker 4030W92 inhibits carrageenan-induced cutaneous hypersensitivity in the anaesthetised rat BRITISH JOURNAL OF PHARMACOLOGY, 123 pp. U126-U126.

Kozlowski CM, Smith EJ, Grundy D, Bountra C. 1998. The sodium channel blocker 4030W92 inhibits spinal c-Fos expression in response to somatic and visceral noxious stimulation in the rat BRITISH JOURNAL OF PHARMACOLOGY, 123 pp. U126-U126.

Kozlowski CM, Smith EJ, Grundy D, Bountra C. 1998. The sodium channel blocker 4030W92 inhibits deoxycholic acid-induced sensitisation of dorsal horn neurone responses to visceral and cutaneous mechanical stimulation in the anaesthetised rat BRITISH JOURNAL OF PHARMACOLOGY, 123 pp. U127-U127.

Young MR, Fleetwood-Walker SM, Dickinson T, Blackburn-Munro G, Sparrow H, Birch PJ, Bountra C. 1997. Behavioural and electrophysiological evidence supporting a role for group I metabotropic glutamate receptors in the mediation of nociceptive inputs to the rat spinal cord. Brain Res, 777 (1-2), pp. 161-169. | Show Abstract | Read more

A combined study of behavioural and electrophysiological tests was carried out in order to assess the role of metabotropic glutamate receptors (mGluRs) in mediating sensory inputs to the spinal cord of the rat. In the behavioural study the responses of conscious animals, with or without carrageenan-induced inflammation, to noxious mechanical and thermal stimuli were observed both before and after the intrathecal administration of mGluR antagonists L(+)-2-amino-3-phosphonopropionic acid (L-AP3) and (S)-4-carboxy-3-hydroxyphenylglycine (CHPG). It was found that the mGluR antagonist (S)-CHPG was capable of increasing both mechanical threshold and thermal latency in both groups of animals, and L-AP3 did so in those with inflammation induced in their hindpaw. Following this study, the responses of single lamina III-V dorsal horn neurons to an innocuous A beta fibre brush stimulus and a noxious C fibre (mustard oil) stimulus were extracellularly recorded and the effect of ionophoretically applied drugs was examined. Cyclothiazide (CTZ), a selective antagonist at mGluR1, markedly reduced the activity evoked by mustard oil, but not that elicited by brushing of the receptive field. Activity induced in dorsal horn neurons by ionophoresing various mGluR subgroup agonists was examined. CTZ successfully inhibited the activity evoked by group I mGluR agonist 3,5-dihydroxyphenylglycine (DHPG). In comparison to the neurons which responded to the ionophoresis of DHPG, less were activated by the selective mGluR5 agonist trans-azetidine dicarboxylic acid (t-ADA). Together these results indicate that group I mGlu receptors, in particular mGluR1, play a crucial role in mediating nociception, particularly following a sustained noxious input.

Evans KS, Scott CM, Bountra C. 1997. The effect of ibuprofen, morphine and amitriptyline on carrageenan-induced cutaneous hypersensitivity in the anaesthetised rat BRITISH JOURNAL OF PHARMACOLOGY, 122 pp. P26-P26.

Clayton NM, Ward P, Munglani R, Keeling S, Bountra C. 1997. Effect of the neurokinin 1 (NK1) receptor antagonist GR205171 on adjuvant-induced inflammatory pain in the rat BRITISH JOURNAL OF PHARMACOLOGY, 122 pp. P27-P27.

Kozlowski CM, Grundy D, Bountra C. 1997. The effect of deoxycholic acid on dorsal horn responses to colorectal distension and cutaneous mechanical stimulation, and the effect of MK-801, in the anaesthetised rat BRITISH JOURNAL OF PHARMACOLOGY, 122 pp. P30-P30.

Smith EJ, Scott CM, Bountra C. 1997. The neurokinin 1 (NK) receptor antagonist GR205171 reduces carrageenan-evoked Fos-like immunoreactivity in the rat lumbar spinal cord BRITISH JOURNAL OF PHARMACOLOGY, 122 pp. P75-P75.

Scott CM, Grundy D, Boissonade F, Bountra C. 1997. Alosetron inhibits the colorectal distension-evoked depressor response and spinal c-Fos expression in the anaesthetised rat. GASTROENTEROLOGY, 112 (4), pp. A822-A822.

Evans KS, Scott CM, Bountra C. 1997. Sensitisation of cutaneous afferent neurotransmission to innocuous and noxious mechanical stimuli using topical prostaglandin E(2) (PGE(2)) BRITISH JOURNAL OF PHARMACOLOGY, 120 pp. P218-P218.

Clayton NM, Oakley I, Thompson S, Wheeldon A, Sargent B, Bountra C. 1997. Validation of the dual channel weight averager as an instrument of the measurement of clinically relevant pain BRITISH JOURNAL OF PHARMACOLOGY, 120 pp. P219-P219.

Thompson SL, Clayton NM, Oakley IG, Bountra C. 1997. The use of locomotor activity equipment to assess analgesic and anti-inflammatory activity BRITISH JOURNAL OF PHARMACOLOGY, 120 pp. P220-P220.

Scott CM, Grundy D, Bountra C. 1997. The differential effects of fentanyl on dorsal horn neurones responsive to colorectal distension within the lumbosacral cord of the anaesthetised rat BRITISH JOURNAL OF PHARMACOLOGY, 120 pp. P221-P221.

Munglani R, Harrison SM, Smith GD, Bountra C, Birch PJ, Elliot PJ, Hunt SP. 1996. Neuropeptide changes persist in spinal cord despite resolving hyperalgesia in a rat model of mononeuropathy. Brain Res, 743 (1-2), pp. 102-108. | Show Abstract | Read more

We have previously described the changes in spinal cord neuropeptides in the unilateral sciatic chronic constriction injury (CCI) model of Bennett and Xie [Pain, 33 (1988) 87-108] at 28 days, a time of maximum mechanical hyperalgesia. In this study we examine the same model 100-120 days post injury by which time resolution of the hyperalgesia and peripheral nerve injury has occurred according to previous studies. Rats underwent either CCI of the sciatic nerve (n = 12) or else sham operation (n = 8) which involved exposure but no ligation of the nerve. Mechanical hyperalgesia was assessed with a Ugo-Basile analgesymeter and immunohistochemistry performed on the spinal cord sections of the animals and quantified using a confocal microscope. At this late time point CCI rats were no longer significantly mechanically hyperalgesic compared to the sham animals (P > or = 0.09). However, examination of the lumbar spinal cord revealed the following changes. (i) The neuropeptides substance P (SP) (P < 0.0001) and galanin (P < 0.003) both showed decreases of about 30% ipsilaterally in immunoreactivity in laminae 1 and 2 of the dorsal horn compared to the sham operated animals. (ii) Calcitonin gene-related peptide (CGRP) and neuropeptide Y (NPY) in laminae 1 and 2 showed no significant changes compared to sham animals. (iii) NPY levels in laminae 3 and 4 of the spinal cord showed a 15% increase in immunoreactivity compared to sham animals (P = 0.008). These results indicate that changes in neuronal markers in the spinal cord can persist after apparent resolution of a peripheral nerve injury. We suggest that these changes may form a substrate for subsequent development of abnormal pain states.

Hudspith MJ, Harrison S, Smith G, Elliot PJ, Birch PJ, Bountra C, Jones JG, Hunt SP, Munglani R. 1996. Effects of post-injury MK-801 on behaviour and neuropeptides in the spinal cord dorsal horn of the mononeuropathic rat BRITISH JOURNAL OF ANAESTHESIA, 77 (5), pp. P691-P692.

Bountra C, Gale JD, Gardner CJ, Jordan CC, Kilpatrick GJ, Twissell DJ, Ward P. 1996. Towards understanding the aetiology and pathophysiology of the emetic reflex: novel approaches to antiemetic drugs. Oncology, 53 Suppl 1 (1), pp. 102-109. | Show Abstract | Read more

The introduction of 5-HT3 antagonists, such as ondansetron, as antiemetic agents has transformed the management of patients receiving chemotherapy or radiation therapy. Studies in animal models with NK1 antagonists suggest that these represent a new class of antiemetic agents having a broader spectrum of activity than 5-HT3 antagonists. Compounds of this class may prove to be more effective in man against delayed emesis induced by cisplatin, post-operative nausea and vomiting and motion sickness. Thus, they have the potential to complement 5-HT3 antagonists and so provide a further advance in the management of nausea and vomiting.

Ward P, Armour DR, Bays DE, Evans B, Giblin GM, Heron N, Hubbard T, Liang K, Middlemiss D, Mordaunt J. 1995. Discovery of an orally bioavailable NK1 receptor antagonist, (2S,3S)-(2-methoxy-5-tetrazol-1-ylbenzyl)(2-phenylpiperidin-3-yl)amine (GR203040), with potent antiemetic activity. J Med Chem, 38 (26), pp. 4985-4992. | Show Abstract | Read more

The antiemetic, pharmacokinetic, and metabolic profile of CP-99,994, a potent NK1 receptor antagonist, has been carefully evaluated. As a result we began a medicinal chemistry program which initially identified a 3-furanyl analogue (6) with improved antiemetic potency and a methyl sulfone (5) with enhanced metabolic stability and oral bioavailability. The improved pharmacokinetic profile of methyl sulfone (5) was associated with its low lipophilicity, and a therefore a number of heterocyclic analogues with reduced log D were synthesized. Out of this program emerged 19 (GR203040), a tetrazolyl-substituted analogue. Tetrazole 19 inhibits radiation-induced emesis in the ferret with high potency when administered both subcutaneously and orally, has a long duration of action, and has high oral bioavailability in the dog. Tetrazole 19 is currently undergoing evaluation as a novel approach for the control of emesis associated with, for example, cancer chemotherapy.

Gardner CJ, Twissell DJ, Dale TJ, Gale JD, Jordan CC, Kilpatrick GJ, Bountra C, Ward P. 1995. The broad-spectrum anti-emetic activity of the novel non-peptide tachykinin NK1 receptor antagonist GR203040. Br J Pharmacol, 116 (8), pp. 3158-3163. | Show Abstract | Read more

1. Following our earlier observations that the tachykinin NK1 receptor antagonist CP-99,994 is an effective anti-emetic in ferrets, we have examined the anti-emetic effects of a more potent and novel NK1 receptor antagonist, GR203040, against various emetic stimuli in the ferret, dog and house musk shrew (Suncus murinus). 2. In ferrets, GR203040 (0.1 mg kg-1 s.c. or i.v.) is effective against emesis induced by radiation, cisplatin, cyclophosphamide, copper sulphate, ipecacuanha or morphine. 3. In animals in which emesis had been established with cisplatin, GR203040 (1 mg kg-1 s.c.) was fully effective as an interventional treatment. No further emesis was seen in animals treated with GR203040 whilst saline-treated animals continued to vomit. 4. GR203040 (0.1 mg kg-1 s.c.) retains anti-emetic efficacy in the ferret, even when given as a 6 h pretreatment, indicating that this compound has a long duration of action. The compound is also effective orally at the same dose, when given as a 90 min pretreatment. 5. GR203040 (0.1 mg kg-1 i.v.) is fully effective against ipecacuanha-induced emesis in the dog. 6. GR203040 is effective against motion- and cisplatin-induced emesis in Suncus murinus. These effects were seen at doses an order of magnitude greater than those shown to be effective against cisplatin in the ferret. 7. In conclusion, GR203040 is a novel anti-emetic agent, and the broad spectrum of anti-emetic activity, together with activity observed in three species, suggests that this compound is worthy of clinical investigation.

YOUNG M, FLEETWOODWALKER S, BIRCH P, BOUNTRA C, BOWERS J. 1995. THE ROLE OF NEUROKININ AND METABOTROPIC GLUTAMATE RECEPTORS IN THERMAL AND MECHANICAL PAW WITHDRAWAL RESPONSES IN NORMAL AND CARRAGEENAN-TREATED RATS BRITISH JOURNAL OF PHARMACOLOGY, 114 pp. P316-P316.

MUNGLANI R, HARRISSON S, SMITH G, BOUNTRA C, BIRCH P, JONES J, HUNT S. 1995. EFFECT OF DIFFERENT PREEMPTIVE TREATMENTS ON LONG-TERM NEUROPEPTIDE-EXPRESSION IN THE DORSAL-ROOT GANGLIA IN A MODEL OF NEUROPATHIC PAIN BRITISH JOURNAL OF ANAESTHESIA, 74 (4), pp. P482-P482.

GARDNER C, BOUNTRA C, BUNCE K, DALE T, JORDAN C, TWISSELL D, WARD P. 1994. ANTIEMETIC ACTIVITY OF NEUROKININ NK1 RECEPTOR ANTAGONISTS IS MEDIATED CENTRALLY IN THE FERRET BRITISH JOURNAL OF PHARMACOLOGY, 112 pp. U46-U46.

GROSSMAN C, TREVETHICK M, WISEMAN J, BOUNTRA C, BIRCH P. 1994. BACTERIAL LIPOPOLYSACCHARIDE INDUCES DE-NOVO SYNTHESIS OF CYCLOOXYGENASE ACTIVITY IN FRESHLY ISOLATED HUMAN MONONUCLEAR-CELLS BRITISH JOURNAL OF PHARMACOLOGY, 112 pp. U219-U219.

Freeman AJ, Bountra C, Dale TJ, Gardner CJ, Twissell DJ. 1993. The vomiting reflex and the role of 5-HT3 receptors. Anticancer Drugs, 4 Suppl 2 (Supplement 2), pp. 9-15. | Read more

Bountra C, Bunce K, Dale T, Gardner C, Jordan C, Twissell D, Ward P. 1993. Anti-emetic profile of a non-peptide neurokinin NK1 receptor antagonist, CP-99,994, in ferrets. Eur J Pharmacol, 249 (1), pp. R3-R4. | Show Abstract | Read more

In the ferret, 5-HT3 receptor antagonists are effective in controlling emesis produced by cytotoxic agents or radiation. To investigate the possibility that substance P has a role, as well as 5-HT, in the emetic reflex pathway, we have examined the anti-emetic effects of a NK1 receptor antagonist (racemic CP-99,994) in the ferret. Racemic CP-99,994 was effective against a range of emetogens, comprising cytotoxic drugs, radiation, morphine, ipecacuanha and copper sulphate.

DENYER J, THORN P, BOUNTRA C, JORDAN C. 1993. ACETYLCHOLINE AND CHOLECYSTOKININ INDUCED ACID EXTRUSION IN MOUSE ISOLATED PANCREATIC ACINAR-CELLS AS MEASURED BY THE MICROPHYSIOMETER JOURNAL OF PHYSIOLOGY-LONDON, 459 pp. P390-P390.

Pasternack M, Bountra C, Voipio J, Kaila K. 1992. Influence of extracellular and intracellular pH on GABA-gated chloride conductance in crayfish muscle fibres. Neuroscience, 47 (4), pp. 921-929. | Show Abstract | Read more

The effect of intracellular and extracellular pH on GABA-gated Cl- conductance was studied using H(+)-selective microelectrodes and a three-microelectrode voltage clamp in crayfish leg opener muscle fibres in bicarbonate-free solutions. Experimental variation of intracellular pH in the range 6.4-8.0 did not affect the GABA-gated conductance. In contrast to this, the GABA-gated conductance was sensitive to changes in external pH. Raising the external pH from 7.4 to 8.4 decreased the GABA-gated peak conductance observed immediately following application of GABA by 30%, and a change from 7.4 to 6.4 produced an increase of 26%. The effect of extracellular pH on the GABA-gated peak conductance was approximately linear in the pH range 6.4-8.9. A slight decrease in the slope of the pH-conductance relationship was evident in the pH range 5.4-6.4. The desensitization of the GABA-gated conductance was also affected by external pH. At pH 6.9 the conductance produced by 1 mM GABA showed a desensitization of about 15%, and at pH 8.9 this value was 34%. Raising the external pH in the presence of GABA decreased the GABA-gated peak conductance and increased the fractional desensitization, while lowering the external pH produced opposite effects, and was capable of repriming the conductance from a desensitized state to the non-desensitized state. The above results show that the GABA-gated conductance is sensitive to changes in external pH in the physiological range, and suggest that pH-dependent changes in the postsynaptic efficacy of GABA-mediated inhibition may contribute to H+ modulation of neuronal excitability.

Bountra C, Powell T, Vaughan-Jones RD. 1990. Comparison of intracellular pH transients in single ventricular myocytes and isolated ventricular muscle of guinea-pig. J Physiol, 424 (1), pp. 343-365. | Show Abstract | Read more

1. Intracellular pH was recorded (double-barrelled pH-selective microelectrodes) in single ventricular myocytes and whole papillary muscles isolated from guinea-pig heart. Both preparations were acid-loaded by various manoeuvres (addition and removal of external NH4Cl or CO2) in order that a comparison could be made of the size and speed of intracellular pH changes and hence of the apparent intracellular buffering power (beta). 2. For the same acid-loading procedure, the size of intracellular pH (pHi) changes was about threefold larger in the isolated myocyte than in whole papillary muscle. The rate of initial acid loading as well as the subsequent rate of pHi recovery (caused by acid extrusion from the cell) were also threefold faster in the myocyte. Estimates of apparent intrinsic (non-CO2) buffering power, based upon the size of pHi changes during acid loading, were 15-20 mmol l-1 for the myocyte and about 70 mmol l-1 for whole muscle. This latter value is similar to previous estimates of beta in heart. 3. When acid extrusion was reduced by applying a high dose of amiloride (1 mmol l-1), then the size of the pHi change during acid loading increased greatly in papillary muscle but changed much less in the myocyte; beta now appeared to be about 30 mmol l-1 in whole muscle but remained essentially unchanged in the myocyte. 4. We conclude that previous values for beta in cardiac muscle have been greatly overestimated because of the presence of sarcolemmal acid extrusion. Paradoxically, this error in estimating beta is far less evident in the isolated myocyte. We suggest that this is because a much more rapid acid loading is achievable in the myocyte so that acid loading will be blunted less by acid extrusion than in whole muscle. We present a simple mathematical model that demonstrates this phenomenon. We conclude that beta in ventricular muscle is likely to resemble that measured in the isolated myocyte, i.e. 15-20 mmol l-1. 5. Slow acid loading in whole ventricular muscle will also affect the kinetics of pHi changes. The model indicates that the rate of pHi recovery from an acid load in papillary muscle does not reflect the pHi dependence of acid extrusion. Instead, it is heavily influenced by the slow rate of acid loading. This emphasises that great care should be taken when interpreting the kinetics of pHi changes in multicellular ventricular preparations.

Bountra C, Vaughan-Jones RD. 1989. Effect of intracellular and extracellular pH on contraction in isolated, mammalian cardiac muscle. J Physiol, 418 (1), pp. 163-187. | Show Abstract | Read more

1. Intracellular pH (pHi) and Na+ activity were recorded (ion-selective microelectrodes) in guinea-pig papillary muscle and the sheep cardiac Purkinje fibre while simultaneously recording twitch tension. The effects of intracellular acidosis and alkalosis upon contraction were investigated. 2. A fall of pHi produced by reducing pHo was associated with a fall of twitch tension. Similarly, a rise of pHi produced by raising pHo produced a rise of twitch tension. The time course of the changes in tension correlated with the time course of changes of pHi rather than pHo. These results are consistent with previous work showing that acidosis inhibits contraction and that the inhibition depends upon a fall of pHi. 3. Changes of pHi were produced while maintaining pHo constant at 7.4. Removal of NH4Cl or addition of sodium acetate (pHo 7.4) reduced pHi but this gave either an increase of tension (papillary muscle) or an initial fall followed by a subsequent recovery of tension (Purkinje fibre). The increase or recovery of tension occurred despite the fact that there was an intracellular acid load. Thus, reducing pHi at constant pHo can increase tension whereas reducing pHi at low pHo (6.4, see paragraph 2) inhibits tension. 4. The increase of recovery of tension during intracellular acidosis produced at a constant pHo (7.4) was associated with a rise of intracellular sodium activity (aiNa). Amiloride (1.5 mmol/l), an inhibitor of Na(+)-H+ exchange, prevented the rise of aiNa during intracellular acidosis and also prevented the recovery of tension. It is concluded that the increase or recovery of tension at low pHi is secondary to a rise of aiNa caused by stimulation of Na(+)-H+ exchange. A rise of aiNa will elevate Ca2+ via sarcolemmal Na(+)-Ca2+ exchange and thus will elevate tension. 5. An intracellular acidosis produced by reducing pHo (6.4) does not elevate aiNa in the Purkinje fibre. In papillary muscle, aiNa rises but this occurs slowly and the rise is 50% smaller than that seen when the same intracellular acidosis is induced at normal pHo (7.4). The net depression of tension under these conditions thus correlates with the lack of a large rise of aiNa. 6. Knowing the quantitative dependence of tension upon both aiNa and pHi in the two tissues it is possible to predict the recovery of twitch tension during intracellular acidosis at constant pHo (7.4), using the changes of pHi and aiNa measured under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Vaughan-Jones RD, Wu ML, Bountra C. 1989. Sodium-hydrogen exchange and its role in controlling contractility during acidosis in cardiac muscle. Mol Cell Biochem, 89 (2), pp. 157-162. | Show Abstract | Read more

Intracellular pH (pHi) and Na (aina) were recorded in isolated sheep cardiac Purkinje fibres using ion-selective microelectrodes while simultaneously recording twitch tension. A fall of pHi stimulated acid-extrusion via sarcolemmal Na-H exchange but the extrusion was inhibited by reducing extracellular pH (pHo), indicating an inhibitory effect of external H ions upon the exchanger. Intracellular acidosis can reduce contraction by directly reducing myofibrillar Ca2+ sensitivity. The activation of Na-H exchange at low pHi can offset this direct inhibitory effect of H+ ions since exchange-activation elevates aina which then indirectly elevates Ca2+i (via Na-Ca exchange) thus tending to restore tension. This protection of contraction during intracellular acidosis can be removed if extracellular pH is also allowed to fall since, under these conditions, Na-H exchange is inhibited.

Bountra C, Kaila K, Vaughan-Jones RD. 1988. Mechanism of rate-dependent pH changes in the sheep cardiac Purkinje fibre. J Physiol, 406 (1), pp. 483-501. | Show Abstract | Read more

1. The mechanism of the rate-dependent decrease in intracellular pH (pHi) and its recovery were studied in isolated sheep cardiac Purkinje fibres. Intracellular Na+ activity (aiNa) and pHi were measured using ion-selective microelectrodes. Twitches were elicited by field stimulation or by depolarizing pulses applied using a two-microelectrode voltage clamp. 2. A 3 Hz train of short (50 ms) depolarizing voltage-clamp pulses induced a reversible fall in pHi which was accompanied by a reversible increase in aiNa. A train of longer (200 ms) pulses also produced a fall in pHi which was now paralleled by a decrease in aiNa. These observations indicate that the rate-dependent acidosis is not dependent upon a rise in aiNa. 3. Neither the fall in pHi nor the increase in aiNa seen upon an increase in action potential frequency was inhibited by amiloride (1 mmol l-1) which indicates that Na+-H+ exchange is not involved in the generation of the acidosis. Furthermore, the rate-dependent acidosis was not abolished in Na+-free solution (Li+ or N-methyl glucamine substituted) indicating that other Na+-requiring processes (such as Na+-Ca2+ exchange) are not a necessary requirement. Rate-dependent pHi changes were also unaffected by the stilbene compound DIDS indicating no participation by Cl--HCO-3 exchange. 4. The rate-dependent acidosis was inhibited by the organic calcium antagonist D600 (20 mumol l-1) which also inhibited twitch tension. This suggests that the acidosis is related to the activation by Ca2+ of developed tension. D600 also inhibited the rate-dependent rise in aiNa (field stimulation). 5. The rate-dependent acidosis was not inhibited by cyanide (2 mmol l-1) but it was blocked by iodoacetate (0.5 mmol l-1) and by 2-deoxyglucose (DOG) (10 mmol l-1, applied in glucose-free solution). These results suggest that the acidosis is generated metabolically via stimulation of glycolysis, following an increase in contraction. Contributions from aerobic metabolism are likely to be small. 6. Twitch tension was inhibited by ryanodine (10 mumol l-1) but the drug had little inhibitory effect on the rate-dependent acidosis. A tonic component of tension was observed, however, in the presence of ryanodine. The lack of effect of ryanodine upon the rate-induced acidosis is discussed. 7. The half-time of pHi recovery from the frequency-dependent acidosis was consistently shorter than that from an intracellular acid load induced by adding and then removing external NH4Cl (10 mmol l-1).(ABSTRACT TRUNCATED AT 400 WORDS)

BOUNTRA C, HILGEMANN D, VAUGHANJONES R. 1988. EXTRACELLULAR PH TRANSIENTS MEASURED WITH PHENOL RED AND PH-SELECTIVE MICROELECTRODES IN ISOLATED SHEEP CARDIAC PURKINJE-FIBERS JOURNAL OF PHYSIOLOGY-LONDON, 406 pp. P125-P125.

BOUNTRA C, VAUGHANJONES R. 1988. EFFECT OF INTRACELLULAR ACIDOSIS ON CONTRACTION IN MAMMALIAN CARDIAC-MUSCLE INVITRO JOURNAL OF PHYSIOLOGY-LONDON, 407 pp. P118-P118.

Bountra C, Kaila K, Vaughan-Jones RD. 1988. Effect of repetitive activity upon intracellular pH, sodium and contraction in sheep cardiac Purkinje fibres. J Physiol, 398 (1), pp. 341-360. | Show Abstract | Read more

1. The influence of repetitive activity upon intracellular pH (pHi), intracellular Na+ activity (aNA(i)) and contraction was examined in isolated sheep cardiac Purkinje fibres. Ion-selective microelectrodes were used to measure intracellular Na+ and H+ ion activity. Twitch tension was elicited by field stimulation or by depolarizing pulses applied using a two-microelectrode voltage clamp. Experiments were performed in HEPES-buffered solution equilibrated either with air or 100% O2. 2. An increase in action potential frequency from a basal rate of 0.1 to 1-4 Hz induced a reversible fall in pHi and a reversible rise in aNa(i). These effects reached a steady state 3-10 min following an increase in stimulation frequency, and showed a linear dependence on frequency with a mean slope of 0.023 pH units Hz-1 and 0.57 mmol l-1 Hz-1, respectively. The rise in total intracellular acid and aNa(i) associated with a single action potential was estimated as 5.3 mu equiv l-1 of acid and 3.5 mu equiv l-1 of Na+. 3. At action potential frequencies greater than 1 Hz, the rate-dependent rise in aNa(i) was usually accompanied by a positive force staircase. 4. The fall in pHi following a rate increase also occurred when fibres were bathed in Tyrode solution equilibrated with 23 mM-HCO3- plus nominally 5% CO2/95% O2. In these cases, however, the fall in pHi was halved in magnitude. 5. In fibres exposed to strophanthidin (0.5 microM), the rate-dependent fall in pHi was doubled in magnitude and its time course was more variable than under drug-free conditions. The rate-dependent rise in aiNa was also usually larger in strophanthidin. 6. In order to examine the influence of the rate-dependent acidosis on developed tension, the acidosis was reversed experimentally by adding 2 mmol l-1 NH4Cl to the bathing solution. This produced a rise in pHi accompanied by a large increase in twitch tension. Such an effect of pHi upon tension was quantitatively similar to that observed in previous work on Purkinje fibres (Vaughan-Jones, Eisner & Lederer, 1987). 7. It is concluded that the rate dependence of pHi will influence both the magnitude and the time course of an inotropic response to a change in heart rate.

Bountra C, Martin RJ. 1987. Single-channel currents from zona-free mouse eggs. Q J Exp Physiol, 72 (4), pp. 483-492. | Show Abstract

Cell-attached patch recordings were made from zona-free mouse eggs to examine currents at the single-channel level; two types were identified. A channel with a mean conductance of 130 pS showed changes in reversal potential on dilution of the patch pipette solution consistent with a cation channel; the channel current persisted when the only cation in the pipette was K+: it was concluded that this channel was permeable to K+. The binomial distribution was used to describe the proportion of time 0, 1, 2, ..., n channels were open in multichannel recordings of the K+ channel. Bursts of inward currents of about 1 pA, which showed voltage activation and could be recorded in the presence of CsCl and tetraethylammonium (TEA), were identified as Ca2+ channels.

BOUNTRA C, POWELL T, VAUGHANJONES R. 1987. COMPARISON OF MICROELECTRODE MEASUREMENT OF INTRACELLULAR PH IN CARDIAC VENTRICULAR TISSUE AND ISOLATED VENTRICULAR CELLS OF GUINEA-PIG JOURNAL OF PHYSIOLOGY-LONDON, 390 pp. P58-P58.

Kaila K, Vaughan-Jones RD, Bountra C. 1987. Regulation of intracellular pH in sheep cardiac Purkinje fibre: interactions among Na+, H+, and Ca2+1. Can J Physiol Pharmacol, 65 (5), pp. 963-969. | Show Abstract | Read more

Experiments were performed on sheep cardiac Purkinje fibres using pH- and sodium-selective microelectrodes, while simultaneously measuring tension, to determine if the fall in intracellular pH (pHi) following a rise in intracellular Na+ activity (aiNa) is caused by inhibition or reversal of acid extrusion on Na+-H+ exchange. A rise in aiNa was induced either by using the cardioactive steroid strophanthidin to inhibit the sarcolemmal Na+-K+ pump or by increasing the frequency of stimulation (0-4 Hz). Both of these manoeuvres led to an increase in aiNa and a decrease in pHi. Following exposure to strophanthidin, amiloride (an inhibitor of sarcolemmal Na+-H+ exchange) produced a decrease in both pHi and aiNa. These effects of amiloride increased with decreasing pHi, indicating that acid extrusion on Na+-H+ exchange is stimulated by the fall in pHi. The changes in intracellular Na+ and H+ caused by amiloride were quantitatively consistent with an electroneutral stoichiometry. The fall in pHi during strophanthidin exposure is therefore not caused by inhibition or reversal of acid extrusion Na+-H+ exchange. It is likely that the fall in pHi during a rate increase is also independent of Na+-H+ exchange. This is because (i) it has been shown previously to occur in the presence of amiloride and (ii) the calcium antagonist D600 completely abolished the stimulation-dependent fall in pHi. It is concluded that the intracellular acidosis following inhibition of the sarcolemmal Na+-K+ pump or following an increase in the rate of stimulation is secondary to a rise in intracellular calcium.

Georgiou P, Bountra C, McNiven A, House CR. 1987. The effect of lanthanum, quercetin and dinitrophenol on calcium-evoked electrical responses in hamster eggs. Q J Exp Physiol, 72 (2), pp. 227-241. | Show Abstract

At room temperature micro-injections of calcium or strontium produced transient hyperpolarizations with an associated rise in input conductance. By contrast micro-injections of potassium, barium, magnesium, cobalt or lanthanum did not produce hyperpolarizations. The reversal potential for the hyperpolarizing response was about -80 mV. Some responses to calcium injections appeared to suffer from an additional transient leak conductance generated by the injected current. In these cases the recovery of the potential and the conductance to normal values was prolonged. The reversal potential of this additional leak pathway was about -10 mV. Experiments designed to investigate the role of active calcium extrusion from the cells showed that extracellular lanthanum or quercetin caused a pronounced slowing of the recovery phase of the potential and conductance response to calcium injection. The metabolic uncoupler dinitrophenol also prolonged the calcium-evoked responses. The replacement of extracellular sodium by lithium or choline produced no alteration in the time course of the calcium-evoked responses, thus suggesting that sodium-calcium exchange exerts no rate control on the recovery phase of those responses.

Georgiou P, Bountra C, House CR. 1987. Intracellular and whole-cell recordings from zona-free hamster eggs: significance of leak impalement artifact. Q J Exp Physiol, 72 (1), pp. 105-118. | Show Abstract

Measurements have been made of membrane potential and input resistance of zona-free hamster eggs from single micro-electrode recordings. At room temperature (20-23 degrees C) the mean (+/- S.D.) values for the potential and resistance were -30 +/- 8 mV and 280 +/- 130 M omega (n = 94 eggs). At 37 degrees C the mean (+/- S.D.) values for the potential and resistance were -39 +/- 13 mV and 230 +/- 60 M omega (n = 60 eggs). The most negative potential recorded at room temperature was -51 mV in a cell which had an input resistance of 620 M omega. At 37 degrees C six eggs out of sixty had potentials more negative than -50 mV and three of these gave all-or-none action potentials in response to depolarizing current pulses. In a separate series of experiments with high resistance micro-electrodes (ca. 100 M omega) six eggs out of twenty-one had potentials more negative than -50 mV and four of these were electrically excitable. Transient potential recordings during impalement indicated that the potential was more negative than -30 mV but that the insertion of a micro-electrode produced a leak pathway with a resistance of about 10 M omega, substantially smaller than the steady-state estimates of the input resistance (see above). Whole-cell recordings with patch pipettes gave potentials in the range -30 to -80 mV and input resistances in the range 180 to 350 M omega (n = 8); four eggs gave action potentials in response to depolarizing current pulses passed through the patch pipette. It is concluded that the leak impalement artifact is so significant in micro-electrode recordings from hamster eggs that it prevents routine reliable potential measurements.

BOUNTRA C, VAUGHANJONES R. 1987. THE INFLUENCE OF PHI ON TWITCH TENSION IN ISOLATED GUINEA-PIG PAPILLARY-MUSCLE JOURNAL OF PHYSIOLOGY-LONDON, 382 pp. P109-P109.

BOUNTRA C, KAILA K, VAUGHANJONES R. 1986. A CA-ANTAGONIST INHIBITS THE INTRACELLULAR ACIDIFICATION OBSERVED DURING STIMULATION OF ISOLATED SHEEP CARDIAC PURKINJE-FIBERS JOURNAL OF PHYSIOLOGY-LONDON, 377 pp. P114-P114.

Green K, Bountra C, Georgiou P, House CR. 1985. An electrophysiologic study of rabbit ciliary epithelium. Invest Ophthalmol Vis Sci, 26 (3), pp. 371-381. | Show Abstract

Microelectrode recordings from cells in rabbit ciliary epithelium have been made in vitro. Ionophoresis of Lucifer Yellow dye from microelectrodes during measurements of potential confirmed that the recordings were intracellular. Dye passed from the impaled cells into adjacent cells in both the nonpigmented and pigmented layers of the epithelium. Electrical coupling between epithelial cells also was observed. The mean (+/- SD) values of the potential measured across the basolateral membranes of the nonpigmented cells was -65 +/- -15 mV (n = 77); the mean value of the input resistance at this intracellular recording site was 37 +/- 28 M omega (n = 17). The membrane potential was reduced by raising the concentration of extracellular potassium but unaffected by changes in the concentrations of sodium, chloride, or bicarbonate ions. After a period of deprivation of extracellular potassium, the cells hyperpolarized without a measurable change in membrane resistance when potassium was restored to the bathing solution; this transient response to potassium was abolished by preincubation with ouabain or by bathing the epithelium in a solution lacking sodium. It was concluded that the ciliary epithelial cells are permeable to potassium but exhibit only a low permeability to sodium, chloride, or bicarbonate ions; that the cells possess an electrogenic Na/K pump; and finally, that all of the cells in the epithelium function as a syncytium.

Georgiou P, Bountra C, Bland KP, House CR. 1984. Calcium action potentials in unfertilized eggs of mice and hamsters. Q J Exp Physiol, 69 (2), pp. 365-380. | Show Abstract

Measurements of membrane potential and resistance have been made in zona-free eggs of mice and hamsters. The mean +/- S.D. values for membrane potential were -91 +/- 28 mV (mouse) and -97 +/- 29 mV (hamster) and for input resistance were 430 +/- 230 M omega (mouse) and 410 +/- 150 M omega (hamster) respectively. Large fluctuations (20 mV) of membrane potential occurred apparently at random and these were accompanied by changes of membrane resistance. Depolarizing current pulses passed through the recording micro-electrode evoked action potentials in eggs of both species. The threshold for excitation was about -50 mV, the maximum rate of rise of the action potential was about 3 V.s-1 and its peak value was about +13 mV. Action potentials could be evoked in eggs bathed in sodium-free solution or in normal solution containing tetrodotoxin (3 microM). The presence of cobalt (5-20 mM), lanthanum (1 mM) or verapamil (200-400 microM) in the bathing solution suppressed the action potential. Raising the extracellular calcium concentration from 4 to 40 mM increased the peak value of the action potential by 25 mV. It is concluded that the plasma membranes of mouse and hamster eggs have voltage-dependent calcium channels.

BLAND K, BOUNTRA C, GEORGIOU P, HOUSE C, MARTIN R. 1984. SINGLE-CHANNEL CURRENTS IN UNFERTILIZED MOUSE EGGS JOURNAL OF PHYSIOLOGY-LONDON, 353 (AUG), pp. P84-P84.

BOUNTRA C, GEORGIOU P, GREEN K, HOUSE C. 1984. MICRO-ELECTRODE RECORDINGS FROM RABBIT CILIARY EPITHELIUM INVITRO JOURNAL OF PHYSIOLOGY-LONDON, 354 (SEP), pp. P45-P45.

Georgiou P, Bountra C, Bland KP, House CR. 1983. Calcium-evoked opening of potassium channels in hamster eggs. Q J Exp Physiol, 68 (4), pp. 687-700. | Show Abstract

Measurements of membrane potential and resistance have been made in zona-free hamster eggs. The resting potential lay in the range -9 to -100 mV and the input resistance fell in the range 14 to 440 M omega; high resting potentials were associated with large input resistances. Calcium injected ionophoretically into an egg from an intracellular micro-electrode caused a reduction of the membrane resistance. The estimated reversal potential for the calcium-evoked response was about -80 mV and its amplitude depended on the extracellular concentration of potassium but not on the chloride concentration. We conclude that membrane potassium channels open in response to a rise in the cytosolic concentration of calcium ions. Evidence is presented to suggest that micro-electrode recordings of the membrane potential and resistance of eggs suffer from an impalement leak artifact. The presence of the artifact lowers the resting potential and resistance of the cell so that intracellular calcium injection causes a hyperpolarization. We conclude that a hyperpolarizing response to calcium would be unlikely in the absence of an impalement artifact.

BLAND K, BOUNTRA C, GEORGIOU P, HOUSE C. 1983. ACTION-POTENTIAL IN INFERTILIZED MOUSE EGG JOURNAL OF PHYSIOLOGY-LONDON, 343 (OCT), pp. P103-P103.

BLAND K, BOUNTRA C, GEORGIOU P, HOUSE C. 1983. CALCIUM INJECTION INTO HAMSTER EGGS CAUSES HYPERPOLARIZATION AND DECREASE IN MEMBRANE RESISTANCE JOURNAL OF PHYSIOLOGY-LONDON, 342 (SEP), pp. P78-P79.

Pettitt D, Smith J, Meadows N, Arshad Z, Schuh A, DiGiusto D, Bountra C, Holländer G, Barker R, Brindley D. 2016. Regulatory barriers to the advancement of precision medicine Expert Review of Precision Medicine and Drug Development, 1 (3), pp. 319-329. | Read more

Picaud S, Fedorov O, Thanasopoulou A, Leonards K, Jones K, Meier J, Olzscha H, Monteiro O et al. 2015. Generation of a Selective Small Molecule Inhibitor of the CBP/p300 Bromodomain for Leukemia Therapy. Cancer Res, 75 (23), pp. 5106-5119. | Show Abstract | Read more

The histone acetyltransferases CBP/p300 are involved in recurrent leukemia-associated chromosomal translocations and are key regulators of cell growth. Therefore, efforts to generate inhibitors of CBP/p300 are of clinical value. We developed a specific and potent acetyl-lysine competitive protein-protein interaction inhibitor, I-CBP112, that targets the CBP/p300 bromodomains. Exposure of human and mouse leukemic cell lines to I-CBP112 resulted in substantially impaired colony formation and induced cellular differentiation without significant cytotoxicity. I-CBP112 significantly reduced the leukemia-initiating potential of MLL-AF9(+) acute myeloid leukemia cells in a dose-dependent manner in vitro and in vivo. Interestingly, I-CBP112 increased the cytotoxic activity of BET bromodomain inhibitor JQ1 as well as doxorubicin. Collectively, we report the development and preclinical evaluation of a novel, potent inhibitor targeting CBP/p300 bromodomains that impairs aberrant self-renewal of leukemic cells. The synergistic effects of I-CBP112 and current standard therapy (doxorubicin) as well as emerging treatment strategies (BET inhibition) provide new opportunities for combinatorial treatment of leukemia and potentially other cancers.

Grundy R, James L, Bountra C, Harrison T. 2014. Reconfiguring drug discovery through innovative partnerships Drug Discovery World, 15 (4), pp. 70-74. | Show Abstract

Attrition rates in drug discovery and development remain stubbornly high, despite large investments by the pharmaceutical industry.The cost of developing a new drug has been estimated at between $1.3 billion and upwards of $4 billion, if one takes into account the cost of failed development candidates'.

Borsook D, Hargreaves R, Bountra C, Porreca F. 2014. Lost but making progress--Where will new analgesic drugs come from? Sci Transl Med, 6 (249), pp. 249sr3. | Show Abstract | Read more

There is a critical need for effective new pharmacotherapies for pain. The paucity of new drugs successfully reaching the clinic calls for a reassessment of current analgesic drug discovery approaches. Many points early in the discovery process present significant hurdles, making it critical to exploit advances in pain neurobiology to increase the probability of success. In this review, we highlight approaches that are being pursued vigorously by the pain community for drug discovery, including innovative preclinical pain models, insights from genetics, mechanistic phenotyping of pain patients, development of biomarkers, and emerging insights into chronic pain as a disorder of both the periphery and the brain. Collaborative efforts between pharmaceutical, academic, and public entities to advance research in these areas promise to de-risk potential targets, stimulate investment, and speed evaluation and development of better pain therapies.

Rice AS, Dworkin RH, McCarthy TD, Anand P, Bountra C, McCloud PI, Hill J, Cutter G et al. 2014. EMA401, an orally administered highly selective angiotensin II type 2 receptor antagonist, as a novel treatment for postherpetic neuralgia: a randomised, double-blind, placebo-controlled phase 2 clinical trial. Lancet, 383 (9929), pp. 1637-1647. | Show Abstract | Read more

BACKGROUND: Existing treatments for postherpetic neuralgia, and for neuropathic pain in general, are limited by modest efficacy and unfavourable side-effects. The angiotensin II type 2 receptor (AT2R) is a new target for neuropathic pain. EMA401, a highly selective AT2R antagonist, is under development as a novel neuropathic pain therapeutic agent. We assessed the therapeutic potential of EMA401 in patients with postherpetic neuralgia. METHODS: In this multicentre, placebo-controlled, double-blind, randomised, phase 2 clinical trial, we enrolled patients (aged 22-89 years) with postherpetic neuralgia of at least 6 months' duration from 29 centres across six countries. We randomly allocated 183 participants to receive either oral EMA401 (100 mg twice daily) or placebo for 28 days. Randomisation was done according to a centralised randomisation schedule, blocked by study site, which was generated by an independent, unmasked statistician. Patients and staff at each site were masked to treatment assignment. We assessed the efficacy, safety, and pharmacokinetics of EMA401. The primary efficacy endpoint was change in mean pain intensity between baseline and the last week of dosing (days 22-28), measured on an 11-point numerical rating scale. The primary efficacy analysis was intention to treat. This trial is registered with the Australian New Zealand Clinical Trials Registry, number ACTRN12611000822987. FINDINGS: 92 patients were assigned to EMA401 and 91 were assigned to placebo. The patients given EMA401 reported significantly less pain compared with baseline values in the final week of treatment than did those given placebo (mean reductions in pain scores -2.29 [SD 1.75] vs -1.60 [1.66]; difference of adjusted least square means -0.69 [SE 0.25]; 95% CI -1.19 to -0.20; p=0.0066). No serious adverse events related to EMA401 occurred. Overall, 32 patients reported 56 treatment-emergent adverse events in the EMA401 group compared with 45 such events reported by 29 patients given placebo. INTERPRETATION: EMA401 (100 mg twice daily) provides superior relief of postherpetic neuralgia compared with placebo at the end of 28 days of treatment. EMA401 was well tolerated by patients. FUNDING: Spinifex Pharmaceuticals.

Picaud S, Wells C, Felletar I, Brotherton D, Martin S, Savitsky P, Diez-Dacal B, Philpott M et al. 2013. RVX-208, an inhibitor of BET transcriptional regulators with selectivity for the second bromodomain. Proc Natl Acad Sci U S A, 110 (49), pp. 19754-19759. | Show Abstract | Read more

Bromodomains have emerged as attractive candidates for the development of inhibitors targeting gene transcription. Inhibitors of the bromo and extraterminal (BET) family recently showed promising activity in diverse disease models. However, the pleiotropic nature of BET proteins regulating tissue-specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. Here, we report that RVX-208, a compound currently in phase II clinical trials, is a BET bromodomain inhibitor specific for second bromodomains (BD2s). Cocrystal structures revealed binding modes of RVX-208 and its synthetic precursor, and fluorescent recovery after photobleaching demonstrated that RVX-208 displaces BET proteins from chromatin. However, gene-expression data showed that BD2 inhibition only modestly affects BET-dependent gene transcription. Our data demonstrate the feasibility of specific targeting within the BET family resulting in different transcriptional outcomes and highlight the importance of BD1 in transcriptional regulation.

Newey PJ, Gorvin CM, Cleland SJ, Willberg CB, Bridge M, Azharuddin M, Drummond RS, van der Merwe PA, Klenerman P, Bountra C, Thakker RV. 2013. Mutant prolactin receptor and familial hyperprolactinemia. N Engl J Med, 369 (21), pp. 2012-2020. | Show Abstract | Read more

Hyperprolactinemia that is not associated with gestation or the puerperium is usually due to tumors in the anterior pituitary gland and occurs occasionally in hereditary multiple endocrine neoplasia syndromes. Here, we report data from three sisters with hyperprolactinemia, two of whom presented with oligomenorrhea and one with infertility. These symptoms were not associated with pituitary tumors or multiple endocrine neoplasia but were due to a heterozygous mutation in the prolactin receptor gene, PRLR, resulting in an amino acid change from histidine to arginine at codon 188 (His188Arg). This substitution disrupted the high-affinity ligand-binding interface of the prolactin receptor, resulting in a loss of downstream signaling by Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5). Thus, the familial hyperprolactinemia appears to be due to a germline, loss-of-function mutation in PRLR, resulting in prolactin insensitivity.

Shintre CA, Pike AC, Li Q, Kim JI, Barr AJ, Goubin S, Shrestha L, Yang J et al. 2013. Structures of ABCB10, a human ATP-binding cassette transporter in apo- and nucleotide-bound states. Proc Natl Acad Sci U S A, 110 (24), pp. 9710-9715. | Show Abstract | Read more

ABCB10 is one of the three ATP-binding cassette (ABC) transporters found in the inner membrane of mitochondria. In mammals ABCB10 is essential for erythropoiesis, and for protection of mitochondria against oxidative stress. ABCB10 is therefore a potential therapeutic target for diseases in which increased mitochondrial reactive oxygen species production and oxidative stress play a major role. The crystal structure of apo-ABCB10 shows a classic exporter fold ABC transporter structure, in an open-inwards conformation, ready to bind the substrate or nucleotide from the inner mitochondrial matrix or membrane. Unexpectedly, however, ABCB10 adopts an open-inwards conformation when complexed with nonhydrolysable ATP analogs, in contrast to other transporter structures which adopt an open-outwards conformation in complex with ATP. The three complexes of ABCB10/ATP analogs reported here showed varying degrees of opening of the transport substrate binding site, indicating that in this conformation there is some flexibility between the two halves of the protein. These structures suggest that the observed plasticity, together with a portal between two helices in the transmembrane region of ABCB10, assist transport substrate entry into the substrate binding cavity. These structures indicate that ABC transporters may exist in an open-inwards conformation when nucleotide is bound. We discuss ways in which this observation can be aligned with the current views on mechanisms of ABC transporters.

Quigley A, Dong YY, Pike AC, Dong L, Shrestha L, Berridge G, Stansfeld PJ, Sansom MS et al. 2013. The structural basis of ZMPSTE24-dependent laminopathies. Science, 339 (6127), pp. 1604-1607. | Show Abstract | Read more

Mutations in the nuclear membrane zinc metalloprotease ZMPSTE24 lead to diseases of lamin processing (laminopathies), such as the premature aging disease progeria and metabolic disorders. ZMPSTE24 processes prelamin A, a component of the nuclear lamina intermediate filaments, by cleaving it at two sites. Failure of this processing results in accumulation of farnesylated, membrane-associated prelamin A. The 3.4 angstrom crystal structure of human ZMPSTE24 has a seven transmembrane α-helical barrel structure, surrounding a large, water-filled, intramembrane chamber, capped by a zinc metalloprotease domain with the catalytic site facing into the chamber. The 3.8 angstrom structure of a complex with a CSIM tetrapeptide showed that the mode of binding of the substrate resembles that of an insect metalloprotease inhibitor in thermolysin. Laminopathy-associated mutations predicted to reduce ZMPSTE24 activity map to the zinc metalloprotease peptide-binding site and to the bottom of the chamber.

Anand U, Facer P, Yiangou Y, Sinisi M, Fox M, McCarthy T, Bountra C, Korchev YE, Anand P. 2013. Angiotensin II type 2 receptor (AT2 R) localization and antagonist-mediated inhibition of capsaicin responses and neurite outgrowth in human and rat sensory neurons. Eur J Pain, 17 (7), pp. 1012-1026. | Show Abstract | Read more

BACKGROUND: The angiotensin II (AngII) receptor subtype 2 (AT2 R) is expressed in sensory neurons and may play a role in nociception and neuronal regeneration. METHODS: We used immunostaining with characterized antibodies to study the localization of AT2 R in cultured human and rat dorsal root ganglion (DRG) neurons and a range of human tissues. The effects of AngII and AT2 R antagonist EMA401 on capsaicin responses in cultured human and rat (DRG) neurons were measured with calcium imaging, on neurite length and density with Gap43 immunostaining, and on cyclic adenosine monophosphate (cAMP) expression using immunofluorescence. RESULTS: AT2 R expression was localized in small-/medium-sized cultured neurons of human and rat DRG. Treatment with the AT2 R antagonist EMA401 resulted in dose-related functional inhibition of capsaicin responses (IC50  = 10 nmol/L), which was reversed by 8-bromo-cAMP, and reduced neurite length and density; AngII treatment significantly enhanced capsaicin responses, cAMP levels and neurite outgrowth. The AT1 R antagonist losartan had no effect on capsaicin responses. AT2 R was localized in sensory neurons of human DRG, and nerve fibres in peripheral nerves, skin, urinary bladder and bowel. A majority sub-population (60%) of small-/medium-diameter neuronal cells were immunopositive in both control post-mortem and avulsion-injured human DRG; some very small neurons appeared to be intensely immunoreactive, with TRPV1 co-localization. While AT2 R levels were reduced in human limb peripheral nerve segments proximal to injury, they were preserved in painful neuromas. CONCLUSIONS: AT2 R antagonists could be particularly useful in the treatment of chronic pain and hypersensitivity associated with abnormal nerve sprouting.

Kruidenier L, Chung CW, Cheng Z, Liddle J, Che K, Joberty G, Bantscheff M, Bountra C et al. 2012. A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. Nature, 488 (7411), pp. 404-408. | Show Abstract | Read more

The jumonji (JMJ) family of histone demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance, as well as in development, physiology and disease. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.

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Arrowsmith CH, Bountra C, Fish PV, Lee K, Schapira M. 2012. Epigenetic protein families: A new frontier for drug discovery Nature Reviews Drug Discovery, 11 (5), pp. 384-400. | Show Abstract | Read more

Epigenetic regulation of gene expression is a dynamic and reversible process that establishes normal cellular phenotypes but also contributes to human diseases. At the molecular level, epigenetic regulation involves hierarchical covalent modification of DNA and the proteins that package DNA, such as histones. Here, we review the key protein families that mediate epigenetic signalling through the acetylation and methylation of histones, including histone deacetylases, protein methyltransferases, lysine demethylases, bromodomain-containing proteins and proteins that bind to methylated histones. These protein families are emerging as druggable classes of enzymes and druggable classes of proteing-protein interaction domains. In this article, we discuss the known links with disease, basic molecular mechanisms of action and recent progress in the pharmacological modulation of each class of proteins. © 2012 Macmillan Publishers Limited. All rights reserved.

Norman TC, Bountra C, Edwards AM, Yamamoto KR, Friend SH. 2011. Leveraging crowdsourcing to facilitate the discovery of new medicines. Sci Transl Med, 3 (88), pp. 88mr1. | Show Abstract | Read more

Gloomy predictions about the future of pharma have forced the industry to investigate alternative models of drug discovery. Public-private partnerships (PPPs) have the potential to revitalize the discovery and development of first-in-class therapeutics. The new PPP Arch2POCM hopes to foster biomedical innovation through precompetitive validation of pioneer therapeutic targets for human diseases. In this meeting report, we capture insights garnered from the April 2011 Arch2POCM conference.

Norman T, Edwards A, Bountra C, Friend S. 2011. The precompetitive space: time to move the yardsticks. Sci Transl Med, 3 (76), pp. 76cm10. | Show Abstract | Read more

Industry, government, patient advocacy groups, public funders, and academic thought leaders met in Toronto, Canada, to set into motion an initiative that addresses some of the scientific and organizational challenges of modern therapeutics discovery. What emerged from the meeting was a public-private partnership that seeks to establish proof of clinical mechanism (POCM) for selected "pioneer" disease targets using lead compounds-all accomplished in the precompetitive space. The group will reconvene in April 2011 to create a business plan that specifies the generation of two positive POCM results per year.

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