Publications by authors named "Andrew Lockhart"

23 Publications

  • Page 1 of 1

Rash developing after cessation of Daclizumab for relapsing remitting MS; a case series.

Mult Scler Relat Disord 2019 Oct 5;35:239-240. Epub 2019 Aug 5.

St Vincent's University Hospital, Dublin, Ireland. Electronic address:

Daclizumab, a monoclonal antibody directed against CD25, a subunit of the high-affinity IL-2 receptor, was licensed as a disease modifying therapy (DMT) for relapsing remitting multiple sclerosis in 2017. Interference with IL-2 signalling is hypothesised to modulate T cell function. For example it results in a preferential shift of innate lymphoid cell (ILC) into CD56 natural killer cells and a decrease in regulatory T Cells. We present three patients who developed urticarial papulovesicular rashes at a median of 3 months after discontinuation of Daclizumab. We propose an unexpected T cell mediated immune reaction as the cause.
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http://dx.doi.org/10.1016/j.msard.2019.08.008DOI Listing
October 2019

New versus old: Implications of evolving diagnostic criteria for relapsing-remitting multiple sclerosis.

Mult Scler 2019 05 12;25(6):867-870. Epub 2018 Apr 12.

Department of Neurology, St. Vincent's University Hospital, Dublin, Ireland/School of Medicine & Medical Science, University College Dublin, Dublin, Ireland.

The International Panel on Diagnosis of Multiple Sclerosis (MS) recently revised the 2010 McDonald criteria and made recommendations for revision, allowing for the earliest possible, accurate diagnosis of MS. For relapsing-remitting MS, positive, unmatched cerebrospinal fluid oligoclonal bands may substitute for dissemination in time. Symptomatic lesions, including brainstem and spinal cord, may demonstrate dissemination in space or in time if enhancing (with the exception of the optic nerve). Cortical and juxtacortical lesions are equivalent. In this retrospective analysis, we applied revised criteria to 250 patients previously diagnosed with relapsing-remitting MS according to 2010 criteria and assessed for change in diagnostic times. There was a significant improvement in time to diagnosis between 2010 and 2017 groups ( p < 0.01). Median time to diagnosis according to McDonald 2010 was 7.4 months, compared with 2.3 months for McDonald 2017. Use of cerebrospinal fluid results most frequently resulted in a reduction in time to diagnosis. Symptomatic gadolinium-enhancing lesions led to earliest diagnostic times.
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http://dx.doi.org/10.1177/1352458518770088DOI Listing
May 2019

Mechanisms of vascular disease in dementia: what does industry want to know?

Clin Sci (Lond) 2017 May;131(9):799-802

Neurosciences, GlaxoSmithKline, Clinical Unit Cambridge, Addenbrooke's Hospital, Cambridge CB2 0GG, U.K.

Despite recent advances in basic and clinical science, dementia remains an area of high unmet medical need. The role of cerebrovascular mechanisms in the pathogenesis and progression of cognitive and functional impairment in dementia is being revived. In order to facilitate the development of therapeutic approaches, it is critical that a number of fundamental elements are integrated into research strategies investigating cerebrovascular pathologies as these will maximize the opportunity of bringing medicines to patients in a timely manner.
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http://dx.doi.org/10.1042/CS20160724DOI Listing
May 2017

Evaluation of the safety, pharmacokinetics, pharmacodynamics, and drug-drug interaction potential of a selective Lp-PLA2 inhibitor (GSK2647544) in healthy volunteers
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Int J Clin Pharmacol Ther 2016 Dec;54(12):935-949

Objective: To evaluate in healthy volunteers the safety, pharmacokinetics (PK), pharmacodynamics (PD), and drug-drug interaction (DDI) potential of GSK2647544, (a selective lipoprotein-associated phospholipase A2 (Lp-PLA2) inhibitor).

Methods: Study 1 was a single-blind, randomized, placebo-controlled, crossover study with healthy male volunteers randomized to receive single escalating oral doses (0.5 - 750 mg) of GSK2647544. Study 2 was a single-blind, randomized, placebo-controlled study with healthy volunteers randomized to receive repeat doses (80 mg) of GSK2647544. The drug-drug interaction of GSK2647544 with simvastatin was also evaluated in study 2.

Results: Across both studies GSK2647544 doses were generally well tolerated with no GSK2647544-related clinically significant findings. GSK2647544 was readily absorbed and its plasma concentration declined bi-exponentially with a terminal half-life ranging from 8 to 16 hours. Plasma exposure of GSK2647544 increased approximately dose-proportionally. There was GSK2647544 dose-dependent inhibition of plasma Lp-PLA2 activity, with a trough inhibition (12 hours after dose) of 85.6% after 7-day twice daily dosing. The administration of simvastatin concomitantly with GSK2647544 increased the overall exposure (area under the plasma concentration-time curve and maximum plasma concentration) of simvastatin and simvastatin acid by 3.6- to 4.3-fold and 1.5- to 3.1-fold, respectively.

Conclusions: GSK2647544 was generally well tolerated and had a reasonable PK-PD profile. The clinically significant drug-drug interaction led to an early termination of study 2.
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http://dx.doi.org/10.5414/CP202565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5299597PMC
December 2016

Investigation of the Brain Biodistribution of the Lipoprotein-Associated Phospholipase A (Lp-PLA) Inhibitor [F]GSK2647544 in Healthy Male Subjects.

Mol Imaging Biol 2017 02;19(1):153-161

GlaxoSmithKline, Neurosciences, Clinical Unit Cambridge, Addenbrooke's Hospital, Cambridge, CB2 0GG, UK.

Purpose: GSK2647544 is a potent and specific inhibitor of lipoprotein-associated phospholipase A (Lp-PLA), which was in development as a potential treatment for Alzheimer's disease (AD). In order to refine therapeutic dose predictions and confirm brain penetration, a radiolabelled form of the inhibitor, [F]GSK2647544, was manufactured for use in a positron emission tomography (PET) biodistribution study.

Procedures: [F]GSK2647544 was produced using a novel, copper iodide (Cu(I)) mediated, [F]trifluoromethylation methodology. Healthy male subjects (n = 4, age range 34-42) received an oral dose of unlabelled GSK2647544 (100 mg) and after 2 h an intravenous (iv) injection of [F]GSK2647544 (average injected activity and mass were 106 ± 47 MBq and 179 ± 55 μg, respectively) followed by dynamic PET scans for 120 min. Defined regions of interest (ROI) throughout the brain were used to obtain regional time-activity curves (TACs) and compartmental modelling analysis used to estimate the primary outcome measure, whole brain volume of distribution (V). Secondary PK and safety endpoints were also recorded.

Results: PET dynamic data were successfully obtained from all four subjects and there were no clinically significant variations of the safety endpoints. Inspection of the TACs indicated a relatively homogenous uptake of [F]GSK2647544 across all the ROIs examined. The mean whole brain V was 0.56 (95 % CI, 0.41-0.72). Secondary PK parameters, C (geometric mean) and T (median), were 354 ng/ml and 1.4 h, respectively. Metabolism of GSK2647544 was relatively consistent across subjects, with 20-40 % of the parent compound [F]GSK2647544 present after 120 min.

Conclusions: The study provides evidence that GSK2647544 is able to cross the blood brain barrier in healthy male subjects leading to a measurable brain exposure. The administered doses of GSK2647544 were well tolerated. Exploratory modelling suggested that a twice-daily dose of 102 mg, at steady state, would provide ~80 % trough inhibition of brain Lp-PLA activity.

Trial Registration: Clintrials.gov: NCT01924858.
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http://dx.doi.org/10.1007/s11307-016-0982-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5209404PMC
February 2017

A 24-week study to evaluate the effect of rilapladib on cognition and cerebrospinal fluid biomarkers of Alzheimer's disease.

Alzheimers Dement (N Y) 2015 Sep 30;1(2):131-140. Epub 2015 Jun 30.

Neurosciences, GlaxoSmithKline, Clinical Unit Cambridge, Addenbrooke's Hospital, Cambridge, UK.

Background: The lipoprotein-associated phospholipase A inhibitor (Lp-PLA), rilapladib (SB659032), is being evaluated as a potential treatment to slow the progression of Alzheimer's disease (AD).

Methods: One hundred twenty-four subjects with possible mild AD and with neuroimaging evidence of cerebrovascular disease were randomized to placebo or 250-mg rilapladib once daily, for 24 weeks, in addition to stable background acetylcholinesterase inhibitor and/or memantine. The study assessed the safety and tolerability of rilapladib and its effects on cognition, mechanistic, and disease-related biomarkers. Although the overall intent behind the study was to take a broad exploratory view of the data, two primary end points of interest (cerebrospinal fluid [CSF] amyloid beta peptide 1-42 [Aβ] and CogState executive function/working memory [EF/WM] composite score at week 24) were prespecified in the analysis plan for inferential statistical analysis.

Results: Rilapladib was well tolerated with no significant safety concerns. A significant difference from placebo was observed for rilapladib on change from baseline in EF/WM (effect size, 0.45;  = .026). There was no significant difference between groups on the change from baseline in CSF Aβ ( = .133). Preliminary evidence of effects was detected on other mechanistic (albumin quotient) and disease-related biomarkers (tau/P-tau and neurofilament light chain).

Conclusion: These data provide initial evidence supporting Lp-PLA inhibition as a novel treatment for dementia.

Clinical Trial Registration: Clinicaltrials.gov identifier: NCT01428453.
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http://dx.doi.org/10.1016/j.trci.2015.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5975052PMC
September 2015

Imaging as a biomarker in drug discovery for Alzheimer's disease: is MRI a suitable technology?

Alzheimers Res Ther 2014 30;6(4):51. Epub 2014 Jul 30.

National Institute of Mental Health, 6001 Executive Boulevard, BG NSC RM 7209, Rockville, MD 20892, USA.

This review provides perspectives on the utility of magnetic resonance imaging (MRI) as a neuroimaging approach in the development of novel treatments for Alzheimer's disease. These considerations were generated in a roundtable at a recent Wellcome Trust meeting that included experts from academia and industry. It was agreed that MRI, either structural or functional, could be used as a diagnostic, for assessing worsening of disease status, for monitoring vascular pathology, and for stratifying clinical trial populations. It was agreed also that MRI implementation is in its infancy, requiring more evidence of association with the disease states, test-retest data, better standardization across multiple clinical sites, and application in multimodal approaches which include other imaging technologies, such as positron emission tomography, electroencephalography, and magnetoencephalography.
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http://dx.doi.org/10.1186/alzrt276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4255417PMC
December 2014

Cholestenoic acids regulate motor neuron survival via liver X receptors.

J Clin Invest 2014 Nov 1;124(11):4829-42. Epub 2014 Oct 1.

Cholestenoic acids are formed as intermediates in metabolism of cholesterol to bile acids, and the biosynthetic enzymes that generate cholestenoic acids are expressed in the mammalian CNS. Here, we evaluated the cholestenoic acid profile of mammalian cerebrospinal fluid (CSF) and determined that specific cholestenoic acids activate the liver X receptors (LXRs), enhance islet-1 expression in zebrafish, and increase the number of oculomotor neurons in the developing mouse in vitro and in vivo. While 3β,7α-dihydroxycholest-5-en-26-oic acid (3β,7α-diHCA) promoted motor neuron survival in an LXR-dependent manner, 3β-hydroxy-7-oxocholest-5-en-26-oic acid (3βH,7O-CA) promoted maturation of precursors into islet-1+ cells. Unlike 3β,7α-diHCA and 3βH,7O-CA, 3β-hydroxycholest-5-en-26-oic acid (3β-HCA) caused motor neuron cell loss in mice. Mutations in CYP7B1 or CYP27A1, which encode enzymes involved in cholestenoic acid metabolism, result in different neurological diseases, hereditary spastic paresis type 5 (SPG5) and cerebrotendinous xanthomatosis (CTX), respectively. SPG5 is characterized by spastic paresis, and similar symptoms may occur in CTX. Analysis of CSF and plasma from patients with SPG5 revealed an excess of the toxic LXR ligand, 3β-HCA, while patients with CTX and SPG5 exhibited low levels of the survival-promoting LXR ligand 3β,7α-diHCA. Moreover, 3β,7α-diHCA prevented the loss of motor neurons induced by 3β-HCA in the developing mouse midbrain in vivo.Our results indicate that specific cholestenoic acids selectively work on motor neurons, via LXR, to regulate the balance between survival and death.
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http://dx.doi.org/10.1172/JCI68506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4347238PMC
November 2014

Plasma lipoprotein-associated phospholipase A2 activity in Alzheimer's disease, amnestic mild cognitive impairment, and cognitively healthy elderly subjects: a cross-sectional study.

Alzheimers Res Ther 2012 7;4(6):51. Epub 2012 Dec 7.

Worldwide Epidemiology, GlaxoSmithKline R&D, 5 Moore Drive, Research Triangle Park, NC 27709, USA.

Introduction: Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a circulating enzyme with pro-inflammatory and oxidative activities associated with cardiovascular disease and ischemic stroke. While high plasma Lp-PLA2 activity was reported as a risk factor for dementia in the Rotterdam study, no association between Lp-PLA2 mass and dementia or Alzheimer's disease (AD) was detected in the Framingham study. The objectives of the current study were to explore the relationship of plasma Lp-PLA2 activity with cognitive diagnoses (AD, amnestic mild cognitive impairment (aMCI), and cognitively healthy subjects), cardiovascular markers, cerebrospinal fluid (CSF) markers of AD, and apolipoprotein E (APOE) genotype.

Methods: Subjects with mild AD (n = 78) and aMCI (n = 59) were recruited from the Memory Clinic, University Hospital, Basel, Switzerland; cognitively healthy subjects (n = 66) were recruited from the community. Subjects underwent standardised medical, neurological, neuropsychological, imaging, genetic, blood and CSF evaluation. Differences in Lp-PLA2 activity between the cognitive diagnosis groups were tested with ANOVA and in multiple linear regression models with adjustment for covariates. Associations between Lp-PLA2 and markers of cardiovascular disease and AD were explored with Spearman's correlation coefficients.

Results: There was no significant difference in plasma Lp-PLA2 activity between AD (197.1 (standard deviation, SD 38.4) nmol/min/ml) and controls (195.4 (SD 41.9)). Gender, statin use and low-density lipoprotein cholesterol (LDL) were independently associated with Lp-PLA2 activity in multiple regression models. Lp-PLA2 activity was correlated with LDL and inversely correlated with high-density lipoprotein (HDL). AD subjects with APOE-ε4 had higher Lp-PLA2 activity (207.9 (SD 41.2)) than AD subjects lacking APOE-ε4 (181.6 (SD 26.0), P = 0.003) although this was attenuated by adjustment for LDL (P = 0.09). No strong correlations were detected for Lp-PLA2 activity and CSF markers of AD.

Conclusion: Plasma Lp-PLA2 was not associated with a diagnosis of AD or aMCI in this cross-sectional study. The main clinical correlates of Lp-PLA2 activity in AD, aMCI and cognitively healthy subjects were variables associated with lipid metabolism.
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http://dx.doi.org/10.1186/alzrt154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3580460PMC
May 2014

Reference measurement procedures for Alzheimer's disease cerebrospinal fluid biomarkers: definitions and approaches with focus on amyloid β42.

Biomark Med 2012 Aug;6(4):409-17

Clinical Neurochemistry Laboratory, Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, the Sahlgrenska Academy at University of Gothenburg, Sahlgrenska University Hospital, Mölndal, Sweden.

Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) are increasingly used in clinical settings, research and drug trials. However, their broad-scale use on different technology platforms is hampered by the lack of standardization at the level of sample handling, determination of concentrations of analytes and the absence of well-defined performance criteria for in vitro diagnostic or companion diagnostic assays, which influences the apparent concentration of the analytes measured and the subsequent interpretation of the data. There is a need for harmonization of CSF AD biomarker assays that can reliably, across centers, quantitate CSF biomarkers with high analytical precision, selectivity and stability over long time periods. In this position paper, we discuss reference procedures for the measurement of CSF AD biomarkers, especially amyloid β42 and tau. We describe possible technical approaches, focusing on a selected reaction monitoring mass spectrometry assay as a candidate reference method for quantification of CSF amyloid β42.
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http://dx.doi.org/10.2217/bmm.12.39DOI Listing
August 2012

Cerebrospinal fluid steroidomics: are bioactive bile acids present in brain?

J Biol Chem 2010 Feb 7;285(7):4666-79. Epub 2009 Dec 7.

Institute of Mass Spectrometry, School of Medicine, Grove Building, Swansea University, Singleton Park, Swansea SA2 8PP, United Kingdom.

In this study we have profiled the free sterol content of cerebrospinal fluid by a combination of charge tagging and liquid chromatography-tandem mass spectrometry. Surprisingly, the most abundant cholesterol metabolites were found to be C(27) and C(24) intermediates of the bile acid biosynthetic pathways with structures corresponding to 7alpha-hydroxy-3-oxocholest-4-en-26-oic acid (7.170 +/- 2.826 ng/ml, mean +/- S.D., six subjects), 3beta-hydroxycholest-5-en-26-oic acid (0.416 +/- 0.193 ng/ml), 7alpha,x-dihydroxy-3-oxocholest-4-en-26-oic acid (1.330 +/- 0.543 ng/ml), and 7alpha-hydroxy-3-oxochol-4-en-24-oic acid (0.172 +/- 0.085 ng/ml), and the C(26) sterol 7alpha-hydroxy-26-norcholest-4-ene-3,x-dione (0.204 +/- 0.083 ng/ml), where x is an oxygen atom either on the CD rings or more likely on the C-17 side chain. The ability of intermediates of the bile acid biosynthetic pathways to activate the liver X receptors (LXRs) and the farnesoid X receptor was also evaluated. The acidic cholesterol metabolites 3beta-hydroxycholest-5-en-26-oic acid and 3beta,7alpha-dihydroxycholest-5-en-26-oic acid were found to activate LXR in a luciferase assay, but the major metabolite identified in this study, i.e. 7alpha-hydroxy-3-oxocholest-4-en-26-oic acid, was not an LXR ligand. 7Alpha-hydroxy-3-oxocholest-4-en-26-oic acid is formed from 3beta,7alpha-dihydroxycholest-5-en-26-oic acid in a reaction catalyzed by 3beta-hydroxy-Delta(5)-C(27)-steroid dehydrogenase (HSD3B7), which may thus represent a deactivation pathway of LXR ligands in brain. Significantly, LXR activation has been found to reduce the symptoms of Alzheimer disease (Fan, J., Donkin, J., and Wellington C. (2009) Biofactors 35, 239-248); thus, cholesterol metabolites may play an important role in the etiology of Alzheimer disease.
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http://dx.doi.org/10.1074/jbc.M109.086678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2836072PMC
February 2010

Interaction of the amyloid imaging tracer FDDNP with hallmark Alzheimer's disease pathologies.

J Neurochem 2009 Apr 13;109(2):623-30. Epub 2009 Feb 13.

GlaxoSmithKline, R&D China, UK-Hub, Addenbrookes Hospital, Cambridge, UK.

The distinctive cortical uptake of the tracer (18)F-FDDNP (2-(1-{6-[(2-fluoroethyl(methyl)amino]-2-naphthyl}ethylidene)malononitrile) in Alzheimer's disease (AD) is believed to be because of its binding to both neurofibrillary tangles (NFTs) and highly fibrillar senile plaques. We therefore investigated the binding of a tracer concentration of (3)H-FDDNP to brain sections containing AD hallmark pathologies. Semi-adjacent sections were labelled with (3)H-PIB (Pittsburgh compound-B, 2-[4'-(methylamino)phenyl]-6-hydroxybenzothiazole) and (14)C-SB13 (4-N-methylamino-4'-hydroxystilbene) for comparison. Neocortical sections containing widespread senile plaques and cerebrovascular amyloid angiopathy, produced a sparse and weak labelling following incubation with (3)H-FDDNP. Furthermore, in sections containing NFTs, there was no overt labelling of the pathology by (3)H-FDDNP. In contrast, sections labelled with (3)H-PIB displayed extensive labelling of diffuse plaques, classical plaques, cerebrovascular amyloid angiopathy and NFTs. (14)C-SB13 produced a broadly similar binding pattern to PIB. Radioligand binding assays employing in vitro generated amyloid-beta peptide fibrils demonstrated a approximately 10-fold reduced affinity for (3)H-FDDNP (85.0 +/- 2.0 nM) compared with (3)H-PIB (8.5 +/- 1.3 nM). These data provide an alternative mechanistic explanation for the observed low cortical uptake of (18)F-FDDNP in AD; in that the ligand is only weakly retained by the hallmark neuropathology because of its low affinity for amyloid structures.
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http://dx.doi.org/10.1111/j.1471-4159.2009.05996.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2933070PMC
April 2009

Monitoring the amyloid beta-peptide in vivo--caveat emptor.

Drug Discov Today 2009 Mar 20;14(5-6):241-51. Epub 2009 Jan 20.

GlaxoSmithKline, R&D China, UK Hub, Cambridge, UK.

As a wave of 'disease modifying' (DM) therapies for Alzheimer's disease (AD) progresses towards the later stages of clinical development, an evaluation of our ability to measure relevant pharmacodynamic effects of such therapies is warranted. Reducing accumulation of amyloid beta (Abeta)-peptide in the brain parenchyma is the primary objective of most current DM approaches. Although a number of methods are available to measure Abeta in blood, cerebrospinal fluid (CSF) and the cerebrum, putative DM-induced changes in the levels of the peptides may not be fully captured, and the reasons for any such changes are not fully understood. Additional candidate biofluid (tau and isoprostanes) and imaging (MRI, FDG-PET) measures may provide alternative supporting evidence of drug activity and subsequent clinical efficacy in patient populations.
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http://dx.doi.org/10.1016/j.drudis.2008.12.004DOI Listing
March 2009

Discovering oxysterols in plasma: a window on the metabolome.

J Proteome Res 2008 Aug 8;7(8):3602-12. Epub 2008 Jul 8.

Institute of Mass Spectrometry, School of Medicine, Grove Building, Swansea University, Singleton Park, Swansea, UK.

While the proteome defines the expressed gene products, the metabolome results from reactions controlled by such gene products. Plasma represents an accessible "window" to the metabolome both in regard of availability and content. The wide range of the plasma metabolome, in terms of molecular diversity and abundance, makes its comprehensive analysis challenging. Here we demonstrate an analytical method designed to target one region of the metabolome, that is, oxysterols. Since the discovery of their biological activity as ligands to nuclear receptors there has been a reawakening of interest in oxysterols and their analysis. In addition, the oxysterols, 24S- and 27-hydroxycholesterol, are currently under investigation as potential biomarkers associated with neurodegenerative disorders such as Alzheimer's disease and multiple sclerosis; widespread analysis of these lipids in clinical studies will require the development of robust, sensitive and rapid analytical techniques. In this communication we present results of an investigation of the oxysterols content of human plasma using a newly developed high-performance liquid chromatography-mass spectrometry (HPLC-MS) method incorporating charge-tagging and high-resolution MS. The method has allowed the identification in plasma of monohydroxylated cholesterol molecules, 7alpha-, 24S-, and 27-hydroxycholesterol; the cholestenetriol 7alpha,27-dihydroxycholesterol; and 3beta-hydroxycholest-5-en-27-oic acid and its metabolite 3beta,7alpha-dihydroxycholest-5-en-27-oic acid. The methodology described is also applicable for the analysis of other sterols in plasma, that is, cholesterol, 7-dehydrocholesterol, and desmosterol, as well as cholesterol 5,6- seco-sterols and steroid hormones. Although involving derivatization, sample preparation is straightforward and chromatographic analysis rapid (17 min), while the MS method offers high sensitivity (ng/mL of sterol in plasma, or pg on-column) and specificity. The methodology is suitable for targeted metabolomic analysis of sterols, oxysterols, and steroid hormones opening a "window" to view this region of the metabolome.
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http://dx.doi.org/10.1021/pr8001639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2567817PMC
August 2008

In vitro high affinity alpha-synuclein binding sites for the amyloid imaging agent PIB are not matched by binding to Lewy bodies in postmortem human brain.

J Neurochem 2008 May 21;105(4):1428-37. Epub 2008 Jan 21.

GlaxoSmithKline, Clinical Science and Technology, Neurology DM, New Frontiers Science Park, Harlow, UK.

Amyloid containing deposits are a defining neuropathological feature of a wide range of dementias and movement disorders. The positron emission tomography tracer PIB (Pittsburgh Compound-B, 2-[4'-(methylamino)phenyl]-6-hydroxybenzothiazole) was developed to target senile plaques, an amyloid containing pathological hallmark of Alzheimer's disease, formed from the amyloid-beta peptide. Despite the fact that PIB was developed from the pan-amyloid staining dye thioflavin T, no detailed characterisation of its interaction with other amyloid structures has been reported. In this study, we demonstrate the presence of a high affinity binding site (K(d) approximately 4 nM) for benzothiazole derivatives, including [3H]-PIB, on alpha-synuclein (AS) filaments generated in vitro, and further characterise this binding site through the use of radioligand displacement assays employing 4-N-methylamino-4'-hydroxystilbene (SB13) (K(i) = 87 nM) and 2-(1-{6-[(2-fluoroethyl(methyl)amino]-2-naphthyl}ethylidene)malononitrile (FDDNP) (K(i) = 210 nM). Despite the presence of a high-affinity binding site on AS filaments, no discernible interaction of [3H]-PIB was detected with amygdala sections from Parkinson's disease cases containing frequent AS-immunoreactive Lewy bodies and related neurities. These findings suggest that the density and/or accessibility of AS binding sites in vivo are significantly less than those associated with amyloid-beta peptide lesions. Lewy bodies pathology is therefore unlikely to contribute significantly to the retention of PIB in positron emission tomography imaging studies.
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http://dx.doi.org/10.1111/j.1471-4159.2008.05245.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2408655PMC
May 2008

Imaging Alzheimer's disease pathology: one target, many ligands.

Authors:
Andrew Lockhart

Drug Discov Today 2006 Dec 30;11(23-24):1093-9. Epub 2006 Oct 30.

GlaxoSmithKline, Addenbrooke's Centre for Clinical Investigation (ACCI), Box No. 128, Addenbrookes Hospital, Hills Road, Cambridge CB2 2GG, UK.

Over the past five years there has been a surge of interest in using positron emission tomography (PET) to determine the in vivo density of the senile plaque, a key pathological feature of Alzheimer's disease. The development of the tracers [(11)C]-PIB, [(11)C]-SB13 and [(18)F]-FDDNP has coincided with drug strategies aimed at altering the brain metabolism of amyloid-beta peptides. The evolution of these novel ligands serves not only as an excellent example of how rapidly imaging technologies can progress but also as a reminder that the fundamental biological knowledge, which is necessary to fully interpret the PET data, can be left trailing behind.
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http://dx.doi.org/10.1016/j.drudis.2006.10.008DOI Listing
December 2006

Characterisation of the binding of amyloid imaging tracers to rodent Abeta fibrils and rodent-human Abeta co-polymers.

Biochem Biophys Res Commun 2006 Sep 30;347(3):669-77. Epub 2006 Jun 30.

GlaxoSmithKline Research and Development, Translational Medicine and Genetics, Addenbrookes Hospital, Cambridge, UK.

Despite the application of amyloid imaging agents such as PIB, SB13, and FDDNP in Alzheimer's disease (AD) patients, the successful use of these agents in transgenic mice models of AD has not been reported to date. As a first step in understanding the behaviour of these ligands in transgenic models of AD, we have investigated in a series of in vitro ligand binding assays the interaction of selected agents, including PIB, FDDNP, SB13, and BSB, with amyloid fibrils produced from rodent Abeta(1-40) (roAbeta) peptide. The data indicate that the ligand binding affinities together with the pattern and number of binding sites on the roAbeta fibrils are broadly conserved with that reported previously for human Abeta(1-40) (huAbeta) fibrils. However, characterisation of huAbeta fibrils formed in the presence of increasing amounts of roAbeta (1, 5, 10% w/w) demonstrated a dose-dependent reduction in the number of high affinity [(3)H]Me-BTA-1 binding sites such that at the highest amount of roAbeta the specific signal was reduced by approximately 95%. These studies suggest that (i) the presence of small amounts of roAbeta in huAbeta fibrils has the potential to cause subtle ultrastructural alterations in the polymers and (ii) the weak binding signal observed in vivo in the transgenic mouse models of AD may in part be due to the decreased number of high affinity binding sites on the Abeta fibrils.
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http://dx.doi.org/10.1016/j.bbrc.2006.06.126DOI Listing
September 2006

Fluorinase mediated C-(18)F bond formation, an enzymatic tool for PET labelling.

Chem Commun (Camb) 2006 Feb 18(6):652-4. Epub 2006 Jan 18.

University of St Andrews, School of Chemistry and Centre for Biomolecular Sciences, North Haugh, St Andrews, Fife, UK KY16 9ST.

The fluorinase enzyme from S. cattleya is applied as a catalyst for the efficient incorporation of [18F]-fluoride into [18F]-5'-fluoro-5'-deoxyadenosine, [18F]-5'-fluoro-5'-deoxyinosine and [18F]-5-fluoro-5-deoxyribose for positron emission tomography (PET) applications.
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http://dx.doi.org/10.1039/b516861aDOI Listing
February 2006

Delineation of positron emission tomography imaging agent binding sites on beta-amyloid peptide fibrils.

J Biol Chem 2005 Jun 26;280(25):23599-604. Epub 2005 Apr 26.

Translational Medicine and Genetics, GlaxoSmithKline Research and Development, Addenbrookes Hospital, Cambridge, CB2 2GG, United Kingdom.

A range of imaging agents for use in the positron emission tomography of Alzheimer's disease is currently under development. Each of the main compound classes, derived from thioflavin T (PIB), Congo Red (BSB), and aminonaphthalene (FDDNP) are believed to bind to mutually exclusive sites on the beta-amyloid (Abeta) peptide fibrils. We recently reported the presence of three classes of binding sites (BS1, BS2, BS3) on the Abeta fibrils for thioflavin T derivatives and now extend these findings to demonstrate that these sites are also able to accommodate ligands from the other chemotype classes. The results from competition assays using [3H]Me-BTA-1 (BS3 probe) indicated that both PIB and FDDNP were able to displace the radioligand with Ki values of 25 and 42 nM, respectively. BSB was unable to displace the radioligand tracer from the Abeta fibrils. In contrast, each of the compounds examined were able to displace thioflavin T (BS1 probe) from the Abeta fibrils when evaluated in a fluorescence competition assay with Ki values for PIB, FDDNP, and BSB of 1865, 335, and 600 nM, respectively. Finally, the Kd values for FDDNP and BSB binding to Abeta fibrils were directly determined by monitoring the increases in the ligand intrinsic fluorescence, which were 290 and 104 nM, respectively. The results from these assays indicate that (i) the three classes of thioflavin T binding sites are able to accommodate a wide range of chemotype structures, (ii) BSB binds to two sites on the Abeta fibrils, one of which is BS2, and the other is distinct from the thioflavin T derivative binding sites, and (iii) there is no independent binding site on the fibrils for FDDNP, and the ligand binds to both the BS1 and BS3 sites with significantly lower affinities than previously reported.
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http://dx.doi.org/10.1074/jbc.M501285200DOI Listing
June 2005

Evidence for the presence of three distinct binding sites for the thioflavin T class of Alzheimer's disease PET imaging agents on beta-amyloid peptide fibrils.

J Biol Chem 2005 Mar 21;280(9):7677-84. Epub 2004 Dec 21.

Translational Medicine and Technology, GlaxoSmithKline Research and Development, Addenbrookes Hospital, Cambridge CB2 2GG, United Kingdom.

Imaging the progression of Alzheimer's disease would greatly facilitate the discovery of therapeutics, and a wide range of ligands are currently under development for the detection of beta-amyloid peptide (Abeta)-containing plaques by using positron emission tomography. Here we report an in-depth characterization of the binding of seven previously described ligands to in vitro generated Abeta-(1-40) polymers. All of the compounds were derived from the benzothiazole compound thioflavin T and include 2-[4'-(methylamino)phenyl]benzothiazole and 2-(4'-dimethylamino-)phenyl-imidazo[1,2-a]-pyridine derivatives, 2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole and 2-[4'-(4''-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, and a benzofuran compound (5-bromo-2-(4-dimethylaminophenyl)benzofuran). By using a range of fluorescent and radioligand binding assays, we find that these compounds display a more complex binding pattern than described previously and are consistent with three classes of binding sites on the Abeta fibrils. All of the compounds bound with very high affinity (low nm K(d)) to a low capacity site (BS3) (1 ligand-binding site per approximately 300 Abeta-(1-40) monomers) consistent with the previously recognized binding site for these compounds on the fibrils. However, the compounds also bound with high affinity (K(d) approximately 100 nm) to either one of two additional binding sites on the Abeta-(1-40) polymer. The properties of these sites, BS1 and BS2, suggest they are adjacent or partially overlapping and have a higher capacity than BS3, occurring every approximately 35 or every approximately 4 monomers of Abeta-(1-40)-peptide, respectively. Compounds appear to display selectivity for BS2 based on the presence of a halogen substitution (2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole, 2-[4'-(4''-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, and 5-bromo-2-(4-dimethylaminophenyl)benzofuran) on their aromatic ring system. The presence of additional ligand-binding sites presents potential new targets for ligand development and may allow a more complete modeling of the current positron emission tomography data.
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http://dx.doi.org/10.1074/jbc.M412056200DOI Listing
March 2005

Kinesin heads fused to hinge-free myosin tails drive efficient motility.

FEBS Lett 2004 Jul;569(1-3):54-8

Molecular Motors Group, Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 0TL, UK.

The rat kinesin motor domain was fused at residues 433, 411, 376 or 367, respectively, to the C-terminal 1185, 1187, 1197 or 1185 residues of the brush border myosin tail. In motility assays, K433myt and K411myt, which preserve the head-proximal kinesin hinge, and K367myt, which deletes it, drove rapid microtubule sliding ( approximately 0.6 microms(-1)) that was optimal when the head-pairs were spaced apart by adding 1:1 headless myosin tails. K376myt, which partially deletes the head-proximal hinge, showed poor motility in sliding assays but wild type processivity, velocity and stall force in single molecule optical trapping. Accordingly, the head-proximal kinesin hinge is functionally dispensable.
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http://dx.doi.org/10.1016/j.febslet.2004.05.050DOI Listing
July 2004

PPARgamma activation enhances cell surface ENaCalpha via up-regulation of SGK1 in human collecting duct cells.

FASEB J 2003 Oct 15;17(13):1966-8. Epub 2003 Aug 15.

Translational Medicine and Technology, GlaxoSmithKline, ACCI, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2GG, UK.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-dependent transcription factor that belongs to the nuclear receptor family that plays a critical role in adipocyte differentiation and lipid metabolism. Here we report for the first time that PPARgamma is expressed in human renal cortical collecting ducts (CCD), segments of the nephor involved in regulation of sodium and water homeostasis via action of the epithelial sodium channel (ENaC). ENaC activity is regulated by the hormones aldosterone and insulin, primarily through co-ordinate actions on serum and glucocorticoid regulated kinase 1 (SGK1). We show that SGK1 activity is stimulated by treatment of a human CCD cell line with PPARgamma agonists, paralleled by an increase in SGK1 mRNA that is abolished by pretreatment with a specific PPARgamma antagonist, and that this leads to increased levels of cell surface ENaCalpha. Electrophoretic mobility shift assays suggest that these effects are caused by binding of PPARgamma to a specific response element in the SGK1 promoter. Our results identify SGK1 as a target for PPARgamma and suggest a novel role for PPARgamma in regulation of sodium re-absorption in the CCD via stimulation of ENaC activity. This pathway may play a role in sodium retention caused by activation of PPARgamma in man.
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http://dx.doi.org/10.1096/fj.03-0181fjeDOI Listing
October 2003

The peripheral benzodiazepine receptor ligand PK11195 binds with high affinity to the acute phase reactant alpha1-acid glycoprotein: implications for the use of the ligand as a CNS inflammatory marker.

Nucl Med Biol 2003 Feb;30(2):199-206

GlaxoSmithKline, Translational Medicine and Technology, Addenbrooke's Centre for Clinical Investigation, Addenbrooke's Hospital, Cambridge CB2 2GG, UK.

The peripheral benzodiazepine receptor ligand PK11195 has been used as an in vivo marker of neuroinflammation in positron emission tomography studies in man. One of the methodological issues surrounding the use of the ligand in these studies is the highly variable kinetic behavior of [(11)C]PK11195 in plasma. We therefore undertook a study to measure the binding of [(3)H]PK11195 to whole human blood and found a low level of binding to blood cells but extensive binding to plasma proteins. Binding assays using [(3)H]PK11195 and purified human plasma proteins demonstrated a strong binding to alpha1-acid glycoprotein (AGP) and a much weaker interaction with albumin. Immunodepletion of AGP from plasma resulted in the loss of plasma [(3)H]PK11195 binding demonstrating: (i) the specificity of the interaction and (ii) that AGP is the major plasma protein to which PK11195 binds with high affinity. PK11195 was able to displace fluorescein-dexamethasone from AGP with IC(50) of <1.2 microM, consistent with a high affinity interaction. These findings are important for understanding the behavior of the ligand in positron emission tomography studies for three reasons. Firstly, AGP is an acute phase protein and its levels will vary during infection and pathological inflammatory diseases such as multiple sclerosis. This could significantly alter the free plasma concentrations of the ligand and contribute to its variable kinetic behavior. Secondly, AGP and AGP-bound ligand may contribute to the access of [(11)C]PK11195 to the brain parenchyma in diseases with blood brain barrier breakdown. Finally, local synthesis of AGP at the site of brain injury may contribute the pattern of [(11)C]PK11195 binding observed in neuroinflammatory diseases.
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http://dx.doi.org/10.1016/s0969-8051(02)00410-9DOI Listing
February 2003