The commutability of enzyme linked immunosorbent assays for the quantification of serum eosinophil-derived neurotoxin (EDN).

Eosinophil-derived neurotoxin (EDN) is a surrogate biomarker of eosinophil activation and has considerable potential as a precision medicine biomarker in diseases where eosinophils may play a causative role. Clinical data for EDN have been generated using different quantitative immunoassays, but comparisons between these individual data sets are challenging as no internationally recognised EDN standards or orthogonal methods exist. In this study we aimed to compare commercial EDN assays from ALPCO, MBL, LSBio and CUSABIO for sample commutability. Firstly, we analytically validated the ALPCO enzyme linked immunosorbent assay (ELISA) and demonstrated appropriate analytical characteristics, including an intra/inter-assay precision coefficient-of-variation of between 1.9 and 6.8%. EDN purified from blood proved to be a good quality control material, whereas recombinant EDN, expressed in E.coli, did not react in the ALPCO immunoassay. Using healthy and asthma patient serum samples we confirmed that the ALPCO assay correlated well with the MBL assay, with a coefficient of determination (R2) of 0.92. However, the results from LSBio and CUSABIO assays were not commutable to the other assays.

A novel automated SARS-CoV-2 saliva PCR test protects a global asymptomatic workforce.

Regular PCR testing of nasopharyngeal swabs from symptomatic individuals for SARS-CoV-2 virus has become the established method by which health services are managing the COVID-19 pandemic. Businesses such as AstraZeneca have also prioritised voluntary asymptomatic testing to keep workplaces safe and maintain supply of essential medicines to patients. We describe the development of an internal automated SARS-CoV-2 testing programme including the transformative introduction of saliva as an alternative sample type.

Eosinophil-derived neurotoxin: A biologically and analytically attractive asthma biomarker.

There is a growing body of evidence for the utility of eosinophil-derived neurotoxin (EDN) as a biomarker in asthma, including association with eosinophilic airway inflammation, assessment of disease severity and potential for predicting pathogenic risks, including exacerbations. However, to interpret any biomarker data with confidence, it is first important to understand the preanalytical factors and biological variation that may affect its reliable measurement and results interpretation. In this study we defined the healthy serum EDN reference range for men and women as 1.98 to 26.10 ng/mL, with no significant gender differences. Smoking did not impact the mean EDN levels and no circadian rhythm was identified for EDN, unlike blood eosinophils (EOS) where levels peaked at 00:00h. EDN expression in different cell types was investigated and shown to occur primarily in eosinophils, indicating they are likely to be the main cellular repository for EDN. We also confirm that the quantification of serum EDN is not influenced by the type of storage tube used, and it is stable at ambient temperature or when refrigerated for at least 7 days and for up to one year when frozen at -20°C or -80°C. In summary, EDN is a stable biomarker that may prove useful in precision medicine approaches by enabling the identification of a subpopulation of asthma patients with activated eosinophils and a more severe form of the disease.

Highly sensitive and specific LC-MS/MS method to determine endogenous leukotriene B4 levels in human plasma.

Aim: To develop a high sensitivity and specific analytical method to measure endogenous levels of leukotriene B4 (LTB4) in human plasma. Methodology: LC-MS/MS and ELISA. Results: An LC-MS/MS method was developed with a sensitivity of 1.0 pg/ml, and within and between batch precision of <16% and <13% RSD, respectively. Conclusion: We have developed a sensitive LC-MS/MS method that can detect endogenous LTB4 in human plasma. The LC-MS/MS method displayed correlation with a commercial LTB4 ELISA when analyzing in ex vivo ionophore-stimulated blood samples. For untreated plasma this correlation was lost. Endogenous LTB4 was shown to be unstable in plasma during storage at -20°C and subject to stereoisomer formation. Neither of the assays could quantify endogenous plasma LTB4 in samples stored for long term.

Impact of a five-dimensional framework on R&D productivity at AstraZeneca.

In 2011, AstraZeneca embarked on a major revision of its research and development (R&D) strategy with the aim of improving R&D productivity, which was below industry averages in 2005-2010. A cornerstone of the revised strategy was to focus decision-making on five technical determinants (the right target, right tissue, right safety, right patient and right commercial potential). In this article, we describe the progress made using this '5R framework' in the hope that our experience could be useful to other companies tackling R&D productivity issues. We focus on the evolution of our approach to target validation, hit and lead optimization, pharmacokinetic/pharmacodynamic modelling and drug safety testing, which have helped improve the quality of candidate drug nomination, as well as the development of the right culture, where 'truth seeking' is encouraged by more rigorous and quantitative decision-making. We also discuss where the approach has failed and the lessons learned. Overall, the continued evolution and application of the 5R framework are beginning to have an impact, with success rates from candidate drug nomination to phase III completion improving from 4% in 2005-2010 to 19% in 2012-2016.

Determining myeloperoxidase activity and protein concentration in a single assay: Utility in biomarker and therapeutic studies.

Myeloperoxidase (MPO) is predominantly expressed by neutrophils and is an important enzyme used by the immune system for the neutralisation of bacteria and other microorganisms. The strong oxidative activity of MPO has been linked to pro-inflammatory responses in surrounding cells and tissues with implication in the pathophysiology of cardiovascular, neuroscience and inflammatory diseases. This broad disease association has made MPO an attractive biomarker and therapeutic target. Here we describe the construction and validation of a single combined MPO activity and protein concentration assay using commercially available reagents. This method offers the investigative laboratory the ability to generate results from blood plasma samples in a single analytical run using the same sample aliquot.

Cerebrospinal fluid kynurenines in multiple sclerosis; relation to disease course and neurocognitive symptoms.

Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative disease of the central nervous system, with a high rate of neurocognitive symptoms for which the molecular background is still uncertain. There is accumulating evidence for dysregulation of the kynurenine pathway (KP) in different psychiatric and neurodegenerative conditions. We here report the first comprehensive analysis of cerebrospinal fluid (CSF) kynurenine metabolites in MS patients of different disease stages and in relation to neurocognitive symptoms. Levels of tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYNA) and quinolinic acid (QUIN) were determined with liquid chromatography mass spectrometry in cell-free CSF. At the group level MS patients (cohort 1; n=71) did not differ in absolute levels of TRP, KYN, KYNA or QUIN as compared to non-inflammatory neurological disease controls (n=20). Stratification of patients into different disease courses revealed that both absolute QUIN levels and the QUIN/KYN ratio were increased in relapsing-remitting MS (RRMS) patients in relapse. Interestingly, secondary progressive MS (SPMS) displayed a trend for lower TRP and KYNA, while primary progressive (PPMS) patients displayed increased levels of all metabolites, similar to a group of inflammatory neurological disease controls (n=13). In the second cohort (n=48), MS patients with active disease and short disease duration were prospectively evaluated for neuropsychiatric symptoms. In a supervised multivariate analysis using orthogonal projection to latent structures (OPLS-DA) depressed patients displayed higher KYNA/TRP and KYN/TRP ratios, mainly due to low TRP levels. Still, this model had low predictive value and could not completely separate the clinically depressed patients from the non-depressed MS patients. No correlation was evident for other neurocognitive measures. Taken together these results demonstrate that clinical disease activity and differences in disease courses are reflected by changes in KP metabolites. Increased QUIN levels of RRMS patients in relapse and generally decreased levels of TRP in SPMS may relate to neurotoxicity and failure of remyelination, respectively. In contrast, PPMS patients displayed a more divergent pattern more resembling inflammatory conditions such as systemic lupus erythematosus. The pattern of KP metabolites in RRMS patients could not predict neurocognitive symptoms.

Macrophage migration and invasion is regulated by MMP10 expression.

This study was designed to identify metalloproteinase determinants of macrophage migration and led to the specific hypothesis that matrix metalloproteinase 10 (MMP10/stromelysin-2) facilitates macrophage migration. We first profiled expression of all MMPs in LPS-stimulated primary murine bone marrow-derived macrophages and Raw264.7 cells and found that MMP10 was stimulated early (3 h) and down-regulated later (24 h). Based on this pattern of expression, we speculated that MMP10 plays a role in macrophage responses, such as migration. Indeed, using time lapse microscopy, we found that RNAi silencing of MMP10 in primary macrophages resulted in markedly reduced migration, which was reversed with exogenous active MMP10 protein. Mmp10 (-/-) bone marrow-derived macrophages displayed significantly reduced migration over a two-dimensional fibronectin matrix. Invasion of primary wild-type macrophages into Matrigel supplemented with fibronectin was also markedly impaired in Mmp10 (-/-) cells. MMP10 expression in macrophages thus emerges as an important moderator of cell migration and invasion. These findings support the hypothesis that MMP10 promotes macrophage movement and may have implications in understanding the control of macrophages in several pathologies, including the abnormal wound healing response associated with pro-inflammatory conditions.

Preclinical studies of PF-04849285, an interferon-α8 fusion protein for the treatment of HCV.

BACKGROUND: This study presents preclinical data of a novel interferon (IFN)-α8 fusion protein, PF-04849285, and compares it with IFN-α2 and pegylated IFN-α2; the latter being the current standard of care for HCV. METHODS: The antiviral properties were evaluated in vitro using the HCV replication assay (replicon) and the general encephalomyocarditis virus assay. The binding affinity to both IFNR-subunits was assessed using surface plasmon resonance. Ex vivo experiments using cynomolgus monkey and human blood were used for the evaluation of induction of IFN-inducible biomarkers (interferon inducible protein 10 [IP-10], 2'-5'-oligoadenylate synthetase [OAS2] and interleukin-6 [IL-6]). The molecule was tested intravenously and subcutaneously in cynomolgus monkey in a single dose study for two weeks at 0.01, 1, 5 and 20 mg/kg. Each route and dose combination was given to a single male animal, blood samples were collected for evaluation of biomarkers and pharmacokinetics. The compound was also tested in cynomolgus monkey in a multiple dose study for four weeks, with a twice-a-week dosing prior to a three-week wash-out period for toxicokinetics, pharmacokinetics, and biomarker evaluation at 20, 50 or 100 mg/kg subcutaneously and 20 mg/kg intravenously. RESULTS: The molecule is 10× more potent than the pegylated IFN-α2a, with potency similar to the unmodified IFN-α2a. No unanticipated findings were observed in cynomolgus monkey when dosed up to 20 mg/kg, >10,000-fold margin over the anticipated efficacious human dose. CONCLUSIONS: The biomarker and toxicological findings were consistent with a potent IFN molecule. The potency and pharmacokinetic properties of the molecule are consistent with dosing at least every two weeks with the potential for monthly dosing' and not 'at least twice daily' as presented in the original [corrected].

Therapy with TLR7 agonists induces lymphopenia: correlating pharmacology to mechanism in a mouse model.

BACKGROUND AND OBJECTIVE: Synthetic TLR7 agonists have been proposed as oral replacements for interferonα (IFNα) therapy in the treatment of hepatitis C virus infection. However, adverse effects, such as lymphopenia and cardiovascular irregularities, have been observed in the clinical following treatment with TLR7 agonists. We wished to understand and characterise the relationship between TLR7 agonism and adverse effects. METHODS: We compared responses to two prototypic TLR7 agonists (Resiquimod: R-848; and PF-04878691) in a mouse model and compared the responses to treatment with IFNα. We measured clinically relevant adverse effects such as lymphopenia and cardiovascular irregularities and related them to plasma drug levels and clinically relevant efficacy biomarkers such as the pro-inflammatory cytokine IP-10, 2'5'OAS and TLR7 receptor expression. RESULTS: By 2 h post dose all agents had induced a dose-dependent transient lymphopenia. IFNα increased heart rate immediately following dosing, persisting for 5 h, whilst PF-04878691 induced significant reductions in blood pressure. Lymphopenia co-incided with maximum plasma drug levels, raised levels of IP-10 and the auto-induction of TLR7 expression in the blood and lymph nodes. Peak levels of 2'5'OAS occurred at 24 h post-dose and only at doses which also induced lymphopenia. CONCLUSIONS: We conclude that systemic delivery of TLR7 agonists or IFNα induces similar exaggerated pharmacology, consistent with there being a narrow therapeutic window between efficacy and safety. This clinically validated mouse model will help to investigate whether more potent agonists or optimised dosing schedules, will be successful strategies for targeting TLR7 in patients.

Quantitative UPLC-MS/MS analysis of the gut microbial co-metabolites phenylacetylglutamine, 4-cresyl sulphate and hippurate in human urine: INTERMAP Study.

The role of the gut microbiome in human health, and non-invasive measurement of gut dysbiosis are of increasing clinical interest. New high-throughput methods are required for the rapid measurement of gut microbial metabolites and to establish reference ranges in human populations. We used ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) -- positive and negative electrospray ionization modes, multiple reaction monitoring transitions -- to simultaneously measure three urinary metabolites (phenylacetylglutamine, 4-cresyl sulphate and hippurate) that are potential biomarkers of gut function, among multi-ethnic US men and women aged 40-59 from the INTERMAP epidemiologic study (n = 2000, two timed 24-hr urine collections/person). Metabolite concentrations were quantified via stable isotope labeled internal standards. The assay was linear in the ranges 1ng/mL (lower limit of quantification) to 1000ng/mL (phenylacetylglutamine and 4-cresyl sulfate) and 3ng/mL to 3000ng/mL (hippurate). These quantitative data provide new urinary reference ranges for population-based human samples: mean (standard deviation) 24-hr urinary excretion for phenylacetylglutamine was: 1283.0 (751.7) µmol/24-hr (men), 1145.9 (635.5) µmol/24-hr (women); for 4-cresyl sulphate, 1002.5 (737.1) µmol/24-hr (men), 1031.8 (687.9) µmol/24-hr (women); for hippurate, 6284.6 (4008.1) µmol/24-hr (men), 4793.0 (3293.3) µmol/24-hr (women). Metabolic profiling by UPLC-MS/MS in a large sample of free-living individuals has provided new data on urinary reference ranges for three urinary microbial co-metabolites, and demonstrates the applicability of this approach to epidemiological investigations.

Chemical genomics identifies compounds affecting Xenopus laevis pigment cell development.

A forward chemical genomic screen was carried out using Xenopus laevis embryos to identify compounds disrupting pigmented cell development, including the retinal pigment epithelial (RPE) layer of the eye and the melanophores (melanocytes). Phenotypes showing changes in cell migration, morphology and pigmentation were observed. The screen also identified compounds affecting other aspects of Xenopus development including general patterning and morphogenesis, eye development and edema formation. Evidence is presented for the molecular targets of three of the compounds identified. Xenopus melanophore and human melanoma cell lines were also utilised in follow-up cell morphology assays. Chemical genomic screens of this type have an important role to play in the identification of novel compounds providing new molecular tools, and biological information, along with identification of new protein targets and leads for potential therapeutic agents.

A chemical genomic approach identifies matrix metalloproteinases as playing an essential and specific role in Xenopus melanophore migration.

To dissect the function of matrix metalloproteinases (MMPs) involved in cellular migration in vivo, we undertook both a forward chemical genomic screen and a functional approach to discover modulators of melanophore (pigment cell) migration in Xenopus laevis. We identified the 8-quinolinol derivative NSC 84093 as affecting melanophore migration in the developing embryo and have shown it to act as a MMP inhibitor. Potential targets of NSC 84093 investigated include MMP-14 and MMP-2. MMP-14 is expressed in migrating neural crest cells from which melanophores are derived. MMP-2 is expressed at the relevant time of development and in a pattern that suggests it contributes to melanophore migration. Morpholino-mediated knockdown of both MMPs demonstrates they play a key role in melanophore migration and partially phenocopy the effect of NSC 84093.

Anti-inflammatory modulation of chronic airway inflammation in the murine house dust mite model.

Asthma affects 300 million people worldwide and continues to be a major cause of morbidity and mortality. Disease relevant animal models of asthma are required for benchmarking of novel therapeutic mechanisms in comparison to established clinical approaches. We demonstrate that chronic exposure of mice to house dust mite (HDM) extract results in allergic airway inflammation, that can be significantly attenuated by therapeutic intervention with phosphodiesterase 4 inhibition and corticosteroid treatment. Female BALB/c mice were administered intranasally with HDM (Dermatophagoides pteronyssinus) extract daily for five weeks, and therapeutic intervention with anti-inflammatory treatment (dexamethasone 1 mg/kg subcutaneous once daily, prednisolone 10mg/kg orally twice daily, fluticasone 3, 10 and 30 microg intranasally twice daily, roflumilast 10 mg/kg orally twice daily and intranasally 10 and 30 microg twice daily) was initiated after three weeks of exposure. Chronic HDM extract exposure resulted in significant airway inflammation, demonstrated by bronchoalveolar lavage cell infiltration and lung tissue inflammatory gene expression by TaqMan low density array. Chronic steroid treatment significantly inhibited these parameters. In addition, roflumilast caused a significant reduction in airway inflammatory cell infiltration. We have demonstrated that chronic HDM-induced allergic inflammation can be significantly ameliorated by steroid treatment, and that phosphodiesterase 4 inhibition modulates inflammatory cell infiltration. Therefore, the murine HDM model may be a useful tool for evaluating new targets for the treatment of asthma.

Allosteric effects of antagonists on signalling by the chemokine receptor CCR5.

Antagonists of the chemokine receptor, CCR5, may provide important new drugs for the treatment of HIV-1. In this study we have examined the mechanism of action of two functional antagonists of the chemokine receptor CCR5 (UK-396,794, UK-438,235) in signalling and internalisation assays using CHO cells expressing CCR5. Both compounds were potent inverse agonists versus agonist-independent [(35)S]GTPgammaS binding to membranes of CHO cells expressing CCR5. Both compounds also acted as allosteric inhibitors of CCL5 (RANTES) and CCL8 (MCP-2)-stimulated [(35)S]GTPgammaS binding to CHO-CCR5 membranes, reducing the potency and maximal effects of the two chemokines. The data are consistent with effects of the allosteric inhibitors on both the binding and signalling of the chemokines. Both compounds inhibited CCR5 internalisation triggered by chemokines. When CHO-CCR5 cells were treated with either of the two compounds for prolonged periods of time (24 h) an increase (approximately 15%) in cell surface CCR5 was detected.

Novel snake venom ligand dendroaspis natriuretic peptide is selective for natriuretic peptide receptor-A in human heart: downregulation of natriuretic peptide receptor-A in heart failure.

The natriuretic peptides are considered to be cardioprotective; however, their receptors have not been identified in human myocardium using radiolabeled analogs. Dendroaspis natriuretic peptide (DNP) has been recently identified as a new member of this peptide family and is thought to be less susceptible to enzymatic degradation. Therefore, we have developed the novel radiolabeled analog [125I]-DNP and used this to localize high-affinity (K(D)=0.2 nmol/L), saturable, specific binding sites in adult human heart (n=6) and coronary artery (n=8). In competition binding experiments, atrial natriuretic peptide and brain type natriuretic peptide had greater affinity for [125I]-DNP binding sites than C-type natriuretic peptide and the natriuretic peptide receptor (NPR)-C ligand, cANF. This rank order of potency suggested binding of [125I]-DNP was specific to NPR-A. Messenger RNA encoding NPR-A was identified in left ventricle and coronary artery smooth muscle, and expression was confirmed by immunocytochemical studies at the protein level. In addition, fluorescence dual labeling immunocytochemistry localized NPR-A protein to cardiomyocytes, endocardial endothelial cells, and smooth muscle of intramyocardial vessels. Importantly, we demonstrated a significant downregulation in the density of NPR-A in heart and coronary artery of patients with ischemic heart disease that may explain, in part, the attenuated natriuretic peptide response reported in this patient group.

Interactions between the Mas-related receptors MrgD and MrgE alter signalling and trafficking of MrgD.

When expressed via an inducible promoter in human embryonic kidney 293 cells, the rat Mas-related gene D (rMrgD) receptor responded to beta-alanine but not L-alanine by elevating intracellular [Ca(2+)], stimulating phosphorylation of the mitogenactivated protein kinases known as extracellular signal-regulated kinase (ERK) 1 and ERK2 and translocating from the plasma membrane to punctate intracellular vesicles. By contrast, the related rat Mas-related gene E (rMrgE) receptor did not respond to beta-alanine. Coexpression of rMrgD with rMrgE, which occurs in peripheral nociceptive neurons, allowed coimmunoprecipitation of the two receptors and resulted in the detection of cell surface rMrgD-rMrgE heterodimers via timeresolved fluorescence resonance energy transfer. These interactions increased the potency of beta-alanine to phosphorylate ERK1 and ERK2 as well as maintaining the capacity of beta-alanine to elevate intracellular [Ca(2+)], which was reduced in magnitude and slowed in response with increasing times of expression of rMrgD in isolation. Associated with these effects, the presence of rMrgE restricted beta-alanine-induced internalization of rMrgD. This is the first report of heterodimeric interactions between members of the Mas-related gene (Mrg) receptor family and indicates that interactions between rMrgD and rMrgE modulate the function of rMrgD. Because the Mrg receptors are potential therapeutic targets in pain, these results suggest that efforts to understand the function and regulation of individual Mrg family receptors may require coexpression of relevant pairs.

Identification and cellular localisation of NPW1 (GPR7) receptors for the novel neuropeptide W-23 by [125I]-NPW radioligand binding and immunocytochemistry.

Using a novel radioligand, we have identified high-affinity binding sites (K(D)=0.44+/-0.13 nM) for the recently discovered peptide, neuropeptide W (NPW), in rat brain. Binding density was highest in the amygdala (B(max)=149.9+/-13.8 fmol/mg protein), thalamic, and hypothalamic nuclei. A similar distribution was observed in mouse brain. We have confirmed the identity of these binding sites as the G-protein-coupled receptor, NPW(1) (previously designated orphan receptor GPR7), using site-directed antisera that revealed receptors were expressed by neuronal cell bodies and processes. Additionally, we have demonstrated the presence of NPW-like immunoreactivity in neuronal cell bodies in areas projecting to the amygdala, such as the dorsal raphe nucleus and ventral tegmental area, providing evidence for an emerging new transmitter system.