Publications by authors named "James Sidaway"

25 Publications

  • Page 1 of 1

Editorial: COVID-19 in the tropics.

Authors:
James D Sidaway

Singap J Trop Geogr 2021 May 17;42(2):161-162. Epub 2021 May 17.

Department of Geography National University of Singapore Singapore.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/sjtg.12376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8206828PMC
May 2021

Label-Free Quantitative Proteomics and Substrate-Based Mass Spectrometry Imaging of Xenobiotic Metabolizing Enzymes in Ex Vivo Human Skin and a Human Living Skin Equivalent Model.

Drug Metab Dispos 2021 01 2;49(1):39-52. Epub 2020 Nov 2.

Department of Chemical and Biological Engineering (N.C., E.K.) and Sheffield Collaboratorium for Antimicrobial Resistance and Biofilms (SCARAB) (N.C., E.K.), University of Sheffield, Sheffield, United Kingdom; Centre for Applied Pharmacokinetic Research, University of Manchester, Manchester, United Kingdom (N.C., B.A., Z.M.A.-M., A.R.-H., J.B.); Centre for Mass Spectrometry Imaging, Biomolecular Sciences Research Centre, Sheffield Hallam University, Sheffield, United Kingdom (J.R.A.N., C.R., M.R.C.); Phenotox Ltd., Bollington, United Kingdom (J.S.); and Certara UK Limited (Simcyp Division), Sheffield, United Kingdom (A.R.-H.)

We report for the first time label-free quantification of xenobiotic metabolizing enzymes (XME), transporters, redox enzymes, proteases, and nucleases in six human skin explants and a three-dimensional living skin equivalent model from LabSkin. We aimed to evaluate the suitability of LabSkin as an alternative to animal testing for the development of topical formulations. More than 2000 proteins were identified and quantified from total cellular protein. Alcohol dehydrogenase 1C, the most abundant phase I XME in human skin, and glutathione S-transferase pi 1, the most abundant phase II XME in human skin, were present in similar abundance in LabSkin. Several esterases were quantified and esterase activity was confirmed in LabSkin using substrate-based mass spectrometry imaging. No cytochrome P450 (P450) activity was observed for the substrates tested, in agreement with the proteomics data, where the cognate P450s were absent in both human skin and LabSkin. Label-free protein quantification allowed insights into other related processes such as redox homeostasis and proteolysis. For example, the most abundant antioxidant enzymes were thioredoxin and peroxiredoxin-1. This systematic determination of functional equivalence between human skin and LabSkin is a key step toward the construction of a representative human in vitro skin model, which can be used as an alternative to current animal-based tests for chemical safety and for predicting dosage of topically administered drugs. SIGNIFICANCE STATEMENT: The use of label-free quantitative mass spectrometry to elucidate the abundance of xenobiotic metabolizing enzymes, transporters, redox enzymes, proteases, and nucleases in human skin enhance our understanding of the skin physiology and biotransformation of topical drugs and cosmetics. This will help to develop mathematical models to predict drug metabolism in human skin and to develop more robust in vitro engineered human skin tissue as alternatives to animal testing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1124/dmd.120.000168DOI Listing
January 2021

Analysis of β-catenin gene mutations and gene expression in liver tumours of C57BL/10J mice produced by chronic administration of sodium phenobarbital.

Toxicology 2020 01 10;430:152343. Epub 2019 Dec 10.

Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, UK.

In this study liver tumours produced in male and female mice of the low spontaneous liver tumour incidence C57BL/10J strain treated for 99 weeks with 1000 ppm in the diet with the model constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) were analysed for β-catenin mutations by Western immunoblotting and DNA/RNA analysis. Some gene array analysis was also performed to identify genes involved in CAR activation and in β-catenin and Hras gene mutations. Analysis of 8 male and 2 female NaPB-induced liver tumour samples (comprising 2 adenomas, 6 carcinomas and 2 samples containing separate adenomas and carcinomas) revealed truncated β-catenin forms in just 4 male liver tumour samples, with the presence of the truncated β-catenin forms being confirmed by β-catenin exon 1-3 mutation analysis. Microarray gene expression analysis was performed with three of the NaPB-induced male mouse liver tumour samples where β-catenin mutations had not been identified by Western immunoblotting and DNA/RNA analysis and with three liver samples from both NaPB-induced non-tumour tissue and control animals. Treatment with NaPB resulted in induction of Cyp2b subfamily gene expression in both NaPB-induced mouse liver tumours and in NaPB-treated non-tumour tissue. In addition, the gene expression analysis demonstrated that the β-catenin and Hras pathways were not modified in NaPB-induced mouse liver tumours not exhibiting truncated β-catenin forms. Overall, while chronic administration of the model CAR activator NaPB results in both hepatocellular adenoma and carcinoma in the low spontaneous liver tumour incidence C57BL/10J mouse strain, only 40 % of the liver tumours evaluated in this study had β-catenin mutations. These results are in agreement with previous studies with the CAR activator oxazepam and demonstrate that mouse liver tumours induced by nongenotoxic CAR activators in the absence of initiation with a genotoxic agent are due to a number of mechanisms, including those largely independent of either the Wnt/β-catenin signalling pathway or Hras oncogene mutations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tox.2019.152343DOI Listing
January 2020

Modeling of Bile Acid Processing by the Human Fecal Microbiota.

Front Microbiol 2018 5;9:1153. Epub 2018 Jun 5.

Division of Integrative Systems Medicine and Digestive Diseases, Imperial College London, London, United Kingdom.

Bile acids, the products of concerted host and gut bacterial metabolism, have important signaling functions within the mammalian metabolic system and a key role in digestion. Given the complexity of the mega-variate bacterial community residing in the gastrointestinal tract, studying associations between individual bacterial genera and bile acid processing remains a challenge. Here, we present a novel approach to determine the bacterial genera associated with the metabolism of different primary bile acids and their potential to contribute to inter-individual variation in this processing. Anaerobic, pH-controlled batch cultures were inoculated with human fecal microbiota and treated with individual conjugated primary bile acids (500 μg/ml) to serve as the sole substrate for 24 h. Samples were collected throughout the experiment (0, 5, 10, and 24 h) and the bacterial composition was determined by 16S rRNA gene sequencing and the bile acid signatures were characterized using a targeted ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) approach. Data fusion techniques were used to identify statistical bacterial-metabolic linkages. An increase in gut bacteria associated bile acids was observed over 24 h with variation in the rate of bile acid metabolism across the volunteers ( = 7). Correlation analysis identified a significant association between the genus and the deconjugation of glycine conjugated bile acids while the deconjugation of taurocholic acid was associated with bacteria from the and genera. A positive correlation between and deoxycholic acid production suggest a potential role for this genus in cholic acid dehydroxylation. A slower deconjugation of taurocholic acid was observed in individuals with a greater abundance of and . This work demonstrates the utility of integrating compositional (metataxonomics) and functional (metabonomics) systems biology approaches, coupled to model systems, to study the biochemical capabilities of bacteria within complex ecosystems. Characterizing the dynamic interactions between the gut microbiota and the bile acid pool enables a greater understanding of how variation in the gut microbiota influences host bile acid signatures, their associated functions and their implications for health.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2018.01153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5996868PMC
June 2018

Innovative organotypic in vitro models for safety assessment: aligning with regulatory requirements and understanding models of the heart, skin, and liver as paradigms.

Arch Toxicol 2018 Feb 23;92(2):557-569. Epub 2018 Jan 23.

Department of Molecular and Clinical Pharmacology, University of Liverpool, Liverpool, L69 3GE, UK.

The development of improved, innovative models for the detection of toxicity of drugs, chemicals, or chemicals in cosmetics is crucial to efficiently bring new products safely to market in a cost-effective and timely manner. In addition, improvement in models to detect toxicity may reduce the incidence of unexpected post-marketing toxicity and reduce or eliminate the need for animal testing. The safety of novel products of the pharmaceutical, chemical, or cosmetics industry must be assured; therefore, toxicological properties need to be assessed. Accepted methods for gathering the information required by law for approval of substances are often animal methods. To reduce, refine, and replace animal testing, innovative organotypic in vitro models have emerged. Such models appear at different levels of complexity ranging from simpler, self-organized three-dimensional (3D) cell cultures up to more advanced scaffold-based co-cultures consisting of multiple cell types. This review provides an overview of recent developments in the field of toxicity testing with in vitro models for three major organ types: heart, skin, and liver. This review also examines regulatory aspects of such models in Europe and the UK, and summarizes best practices to facilitate the acceptance and appropriate use of advanced in vitro models.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00204-018-2152-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818581PMC
February 2018

Preclinical imaging methods for assessing the safety and efficacy of regenerative medicine therapies.

NPJ Regen Med 2017 19;2:28. Epub 2017 Oct 19.

Institute of Veterinary Science, University of Liverpool, Liverpool, UK.

Regenerative medicine therapies hold enormous potential for a variety of currently incurable conditions with high unmet clinical need. Most progress in this field to date has been achieved with cell-based regenerative medicine therapies, with over a thousand clinical trials performed up to 2015. However, lack of adequate safety and efficacy data is currently limiting wider uptake of these therapies. To facilitate clinical translation, non-invasive in vivo imaging technologies that enable careful evaluation and characterisation of the administered cells and their effects on host tissues are critically required to evaluate their safety and efficacy in relevant preclinical models. This article reviews the most common imaging technologies available and how they can be applied to regenerative medicine research. We cover details of how each technology works, which cell labels are most appropriate for different applications, and the value of multi-modal imaging approaches to gain a comprehensive understanding of the responses to cell therapy in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41536-017-0029-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5677988PMC
October 2017

Statin regulated ERK5 stimulates tight junction formation and reduces permeability in human cardiac endothelial cells.

J Cell Physiol 2018 Jan 3;233(1):186-200. Epub 2017 Aug 3.

Department of Molecular and Clinical Pharmacology, MRC Centre for Drug Safety Science, University of Liverpool, Liverpool, UK.

The MEKK3/MEK5/ERK5 signaling axis is required for cardiovascular development in vivo. We analyzed the physiological role of ERK5 in cardiac endothelial cells and the consequence of activation of this kinase by the statin class of HMG Co-A reductase inhibitor drugs. We utilized human cardiac microvascular endothelial cells (HCMECs) and altered ERK5 expression using siRNA mediated gene silencing or overexpression of constitutively active MEK5 and ERK5 to reveal a role for ERK5 in regulating endothelial tight junction formation and cell permeability. Statin treatment of HCMECs stimulated activation of ERK5 and translocation to the plasma membrane resulting in co-localization with the tight junction protein ZO-1 and a concomitant reduction in endothelial cell permeability. Statin mediated activation of ERK5 was a consequence of reduced isoprenoid synthesis following HMG Co-A reductase inhibition. Statin pretreatment could overcome the effect of doxorubicin in reducing endothelial tight junction formation and prevent increased permeability. Our data provide the first evidence for the role of ERK5 in regulating endothelial tight junction formation and endothelial cell permeability. Statin mediated ERK5 activation and the resulting decrease in cardiac endothelial cell permeability may contribute to the cardioprotective effects of statins in reducing doxorubicin-induced cardiotoxicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jcp.26064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655747PMC
January 2018

Towards better models and mechanistic biomarkers for drug-induced gastrointestinal injury.

Pharmacol Ther 2017 Apr 27;172:181-194. Epub 2017 Jan 27.

MRC Centre for Drug Safety Science, Department of Clinical and Molecular Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK.

Adverse drug reactions affecting the gastrointestinal (GI) tract are a serious burden on patients, healthcare providers and the pharmaceutical industry. GI toxicity encompasses a range of pathologies in different parts of the GI tract. However, to date no specific mechanistic diagnostic/prognostic biomarkers or translatable pre-clinical models of GI toxicity exist. This review will cover the current knowledge of GI ADRs, existing biomarkers and models with potential application for toxicity screening/monitoring. We focus on the current gaps in our knowledge, the potential opportunities and recommend that a systematic approach is needed to identify mechanism-based GI biomarkers with potential for clinical translation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pharmthera.2017.01.002DOI Listing
April 2017

Cardiotoxic drugs Herceptin and doxorubicin inhibit cardiac microvascular endothelial cell barrier formation resulting in increased drug permeability.

Biol Open 2016 Oct 15;5(10):1362-1370. Epub 2016 Oct 15.

MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Sherrington Building, Ashton Street, The University of Liverpool, Liverpool, L69 3GE, UK

Cardiotoxicity induced by anti-cancer therapeutics is a severe, and potentially fatal, adverse reaction of the heart in response to certain drugs. Current in vitro approaches to assess cardiotoxicity have focused on analysing cardiomyocytes. More recently it has become apparent that non-cardiomyocyte cells of the heart can potentially contribute to cardiotoxicity. Herceptin and doxorubicin are known to induce cardiotoxicity in the clinic. The effect of these drugs on the endothelial tight junction barrier was tested by analysing tight junction formation and zona occludens-1 (ZO-1) levels, revealing that Herceptin and doxorubicin are able to induce barrier perturbment and decrease barrier function in human cardiac microvascular endothelial cells (HCMECs) leading to increased permeability. Herceptin treatment had no effect on the tight junction barrier function in human dermal and human brain microvascular endothelial cells. HCMECs showed detectable levels of HER2 compared with the other endothelial cells suggesting that Herceptin binding to HER2 in these cells may interfere with tight junction formation. Our data suggests that doxorubicin and Herceptin can affect tight junction formation in the cardiac microvasculature leading to increased drug permeability and adverse effects on the cardiac myocytes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/bio.020362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087671PMC
October 2016

Cardiac Non-myocyte Cells Show Enhanced Pharmacological Function Suggestive of Contractile Maturity in Stem Cell Derived Cardiomyocyte Microtissues.

Toxicol Sci 2016 07 28;152(1):99-112. Epub 2016 Apr 28.

*Department of Molecular and Clinical Pharmacology, MRC Centre for Drug Safety Science, Sherrington Building, the University of Liverpool, Ashton Street, L69 3GE, UK Safety and ADME Translational Sciences, AstraZeneca R&D, Cambridge, CB4 0WG, UK Safety and ADME Translational Sciences, AstraZeneca R&D, Cambridge, CB4 0WG, UK.

The immature phenotype of stem cell derived cardiomyocytes is a significant barrier to their use in translational medicine and pre-clinical in vitro drug toxicity and pharmacological analysis. Here we have assessed the contribution of non-myocyte cells on the contractile function of co-cultured human embryonic stem cell derived cardiomyocytes (hESC-CMs) in spheroid microtissue format. Microtissues were formed using a scaffold free 96-well cell suspension method from hESC-CM cultured alone (CM microtissues) or in combination with human primary cardiac microvascular endothelial cells and cardiac fibroblasts (CMEF microtissues). Contractility was characterized with fluorescence and video-based edge detection. CMEF microtissues displayed greater Ca(2+ )transient amplitudes, enhanced spontaneous contraction rate and remarkably enhanced contractile function in response to both positive and negative inotropic drugs, suggesting a more mature contractile phenotype than CM microtissues. In addition, for several drugs the enhanced contractile response was not apparent when endothelial cell or fibroblasts from a non-cardiac tissue were used as the ancillary cells. Further evidence of maturity for CMEF microtissues was shown with increased expression of genes that encode proteins critical in cardiac Ca(2+ )handling (S100A1), sarcomere assembly (telethonin/TCAP) and β-adrenergic receptor signalling. Our data shows that compared with single cell-type cardiomyocyte in vitro models, CMEF microtissues are superior at predicting the inotropic effects of drugs, demonstrating the critical contribution of cardiac non-myocyte cells in mediating functional cardiotoxicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/toxsci/kfw069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4922542PMC
July 2016

MicroRNA-122: a novel hepatocyte-enriched in vitro marker of drug-induced cellular toxicity.

Toxicol Sci 2015 Mar 18;144(1):173-85. Epub 2014 Dec 18.

MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Sherrington Buildings, Ashton Street, University of Liverpool, Liverpool L69 3GE, UK Stem Cells for Safer Medicines, 7th Floor, Southside, 105 Victoria Street, London SW1E 6QT, UK Faculty of Life Sciences, University of Manchester, Manchester M13 9PL, UK School of Medicine and Dentistry, University of Central Lancashire, Preston PR1 2HE, UK Centre for Endocrinology and Diabetes, Institute of Human Development, Faculty of Medical and Human Sciences, Manchester Academic Health Science Centre, University of Manchester, Manchester M13 9PT, UK Endocrinology Department, Central Manchester University Hospitals NHS Foundation Trust, Oxford Road, Manchester M13 9PT, UK Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK, Takara Bio Europe AB (former Cellartis), Arvid Wallgrens Backe 20, 413 46 Göteborg, Sweden, School of Life Sciences, The Systems Biology Research Centre, University of Skövde, Box 408, 541 28 Skövde, Sweden, MRC Centre for Regenerative Medicine, SCRM Building, The University of Edinburgh, Edinburgh Bioquarter, 5 Little France Drive, Edinburgh EH16 4UU, UK, Newcastle University, Institute of Cellular Medicine, The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK Drug Safety and Metabolism, AstraZeneca R&D, Alderley Park, Cheshire SK10 4TG, UK and North Western Hepatobiliary Unit, Aintree University Hospital NHS Foundation Trust, Longmoor Lane, Liverpool L9 7AL, UK MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Sherrington Buildings, Ashton Street, University of Liverpool, Liverpool L69 3GE, UK Stem Cells for Safer Medicines, 7th Floor, Southside, 105 Victoria Street, London SW1E 6QT, UK Faculty of Life Sciences, University of Manchester, Manchester M13 9PL, UK School of Medicine and Dentistry, University of Central Lancashire, Preston PR1 2HE, UK Centre fo

Emerging hepatic models for the study of drug-induced toxicity include pluripotent stem cell-derived hepatocyte-like cells (HLCs) and complex hepatocyte-non-parenchymal cellular coculture to mimic the complex multicellular interactions that recapitulate the niche environment in the human liver. However, a specific marker of hepatocyte perturbation, required to discriminate hepatocyte damage from non-specific cellular toxicity contributed by non-hepatocyte cell types or immature differentiated cells is currently lacking, as the cytotoxicity assays routinely used in in vitro toxicology research depend on intracellular molecules which are ubiquitously present in all eukaryotic cell types. In this study, we demonstrate that microRNA-122 (miR-122) detection in cell culture media can be used as a hepatocyte-enriched in vitro marker of drug-induced toxicity in homogeneous cultures of hepatic cells, and a cell-specific marker of toxicity of hepatic cells in heterogeneous cultures such as HLCs generated from various differentiation protocols and pluripotent stem cell lines, where conventional cytotoxicity assays using generic cellular markers may not be appropriate. We show that the sensitivity of the miR-122 cytotoxicity assay is similar to conventional assays that measure lactate dehydrogenase activity and intracellular adenosine triphosphate when applied in hepatic models with high levels of intracellular miR-122, and can be multiplexed with other assays. MiR-122 as a biomarker also has the potential to bridge results in in vitro experiments to in vivo animal models and human samples using the same assay, and to link findings from clinical studies in determining the relevance of in vitro models being developed for the study of drug-induced liver injury.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/toxsci/kfu269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4349141PMC
March 2015

Probiotic administration attenuates myocardial hypertrophy and heart failure after myocardial infarction in the rat.

Circ Heart Fail 2014 May 13;7(3):491-9. Epub 2014 Mar 13.

From the Departments of Physiology and Pharmacology (X.T.G., C.X.H., J.V.H., V.R., M.K.), Microbiology and Immunology (G.E., J.P.B., G.R.), Surgery (J.P.B., G.R.), and Biochemistry (G.B.G.), Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada; Canadian Research and Development Centre for Probiotics, Lawson Health Research Institute, London, Ontario, Canada (G.E., J.P.B., G.B.G., G.R.); and Department of Food and Nutritional Sciences, School of Chemistry, Food and Pharmacy, University of Reading, Whiteknights, Reading, United Kingdom (J.E.S., G.M., J.R.S.).

Background: Probiotics are extensively used to promote gastrointestinal health, and emerging evidence suggests that their beneficial properties can extend beyond the local environment of the gut. Here, we determined whether oral probiotic administration can alter the progression of postinfarction heart failure.

Methods And Results: Rats were subjected to 6 weeks of sustained coronary artery occlusion and administered the probiotic Lactobacillus rhamnosus GR-1 or placebo in the drinking water ad libitum. Culture and 16s rRNA sequencing showed no evidence of GR-1 colonization or a significant shift in the composition of the cecal microbiome. However, animals administered GR-1 exhibited a significant attenuation of left ventricular hypertrophy based on tissue weight assessment and gene expression of atrial natriuretic peptide. Moreover, these animals demonstrated improved hemodynamic parameters reflecting both improved systolic and diastolic left ventricular function. Serial echocardiography revealed significantly improved left ventricular parameters throughout the 6-week follow-up period including a marked preservation of left ventricular ejection fraction and fractional shortening. Beneficial effects of GR-1 were still evident in those animals in which GR-1 was withdrawn at 4 weeks, suggesting persistence of the GR-1 effects after cessation of therapy. Investigation of mechanisms showed a significant increase in the leptin:adiponectin plasma concentration ratio in rats subjected to coronary ligation, which was abrogated by GR-1. Metabonomic analysis showed differences between sham control and coronary artery ligated hearts particularly with respect to preservation of myocardial taurine levels.

Conclusions: The study suggests that probiotics offer promise as a potential therapy for the attenuation of heart failure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCHEARTFAILURE.113.000978DOI Listing
May 2014

Characterization of the adenosine pharmacology of ticagrelor reveals therapeutically relevant inhibition of equilibrative nucleoside transporter 1.

J Cardiovasc Pharmacol Ther 2014 Mar 10;19(2):209-19. Epub 2014 Jan 10.

1Safety Pharmacology, Global Safety Assessment, AstraZeneca, Alderley Park, Macclesfield, Cheshire, United Kingdom.

Introduction: Studies have shown that ticagrelor has a further adenosine-mediated mechanism of action in addition to its potent inhibition of the P2Y12 receptor, which may explain some of ticagrelor's clinical characteristics. This study aimed to further characterize the adenosine pharmacology of ticagrelor, its major metabolites, and other P2Y12 receptor antagonists.

Methods: Inhibition of nucleoside transporter-mediated [(3)H]adenosine uptake by ticagrelor, its major metabolites, and alternative P2Y12 antagonists was examined in recombinant Madin-Darby canine kidney (MDCK) cells. The pharmacology of ticagrelor and its major metabolites at adenosine A1, A2A, A2B, and A3 receptor subtypes was examined using in vitro radioligand binding and functional assays and ex vivo C-fiber experiments in rat and guinea pig vagus nerves.

Results: Ticagrelor (and less effectively its metabolites) and the main cangrelor metabolite inhibited [(3)H]adenosine uptake in equilibrative nucleoside transporter (ENT) 1-expressing MDCK cells, whereas cangrelor and the active metabolites of prasugrel or clopidogrel had no effect. No significant inhibitory activity was observed in MDCK cells expressing ENT2 or concentrative nucleoside transporters 2/3. Ticagrelor demonstrated high affinity (inhibition constant [Ki] = 41 nmol/L) for ENT1. In adenosine receptor-binding experiments, ticagrelor and its major circulating metabolite, AR-C124910XX, had low affinity (Ki > 6 µmol/L) for each of the adenosine A1, A2A, and A2B receptors, whereas ticagrelor had a submicromolar (Ki = 190 nmol/L) affinity for the adenosine A3 receptor. However, in functional assays, at high concentrations (10 µmol/L) ticagrelor only partially inhibited 3 mmol/L adenosine-induced depolarizations in the guinea pig and rat vagus nerve preparations (by 35% and 49%, respectively).

Conclusions: Ticagrelor inhibits cellular adenosine uptake selectively via ENT1 inhibition at concentrations of clinical relevance. However, the low-binding affinity and functional inhibition of adenosine receptors observed with ticagrelor or its metabolites indicate that they possess a negligible adenosine-like activity at clinically relevant concentrations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1074248413511693DOI Listing
March 2014

Safety pharmacology--current and emerging concepts.

Toxicol Appl Pharmacol 2013 Dec 1;273(2):229-41. Epub 2013 Jun 1.

MRC Centre for Drug Safety Science, University of Liverpool, UK.

Safety pharmacology (SP) is an essential part of the drug development process that aims to identify and predict adverse effects prior to clinical trials. SP studies are described in the International Conference on Harmonisation (ICH) S7A and S7B guidelines. The core battery and supplemental SP studies evaluate effects of a new chemical entity (NCE) at both anticipated therapeutic and supra-therapeutic exposures on major organ systems, including cardiovascular, central nervous, respiratory, renal and gastrointestinal. This review outlines the current practices and emerging concepts in SP studies including frontloading, parallel assessment of core battery studies, use of non-standard species, biomarkers, and combining toxicology and SP assessments. Integration of the newer approaches to routine SP studies may significantly enhance the scope of SP by refining and providing mechanistic insight to potential adverse effects associated with test compounds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.taap.2013.04.039DOI Listing
December 2013

Phenotypic profiling of structural cardiotoxins in vitro reveals dependency on multiple mechanisms of toxicity.

Toxicol Sci 2013 Apr 12;132(2):317-26. Epub 2013 Jan 12.

Molecular Toxicology, Global Safety Assessment UK, Innovative Medicines, AstraZeneca R&D, Macclesfield, SK10 4TG, UK.

Morphological damage to cardiomyocytes or loss of viability (structural cardiotoxicity) is a common cause of attrition in preclinical and clinical drug development. Currently, no predictive in vitro approaches are available to detect this liability early in drug discovery, and knowledge of the mechanisms involved is limited. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and the rat myoblastic H9c2 cell lines were used to phenotypically profile a panel of structural cardiotoxins by live-cell fluorescent imaging of mitochondrial membrane potential, endoplasmic reticulum integrity, Ca(2+) mobilization, and membrane permeability combined with an assessment of cell viability (ATP depletion). Assay results were normalized to known therapeutically relevant concentrations. By comparing the outcome of each assay to the known in vivo effects, hESC-CMs offered an improved model over H9c2 cells for the detection of structural cardiotoxicity at therapeutically relevant concentrations. Inhibition of the spontaneously beating phenotype, a feature of stem cell-derived cardiomyocytes, revealed some degree of cardioprotection following 10 out of 13 structural cardiotoxins, illustrating the intricate relationship between the function and structure of cardiomyocytes. Classification of structural cardiotoxins into mechanistic themes revealed mitochondria and calcium mobilization to be major distal targets, with only 4 out of 15 compounds affecting contractile function in freshly isolated canine cardiomyocytes at therapeutically relevant concentrations. Our data demonstrate the utility of hESC-CMs during drug development to support structural cardiotoxicity hazard identification and to gain insight into the intricate mechanisms implicated in structural cardiotoxicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/toxsci/kft005DOI Listing
April 2013

Pharmacological activation of the pyruvate dehydrogenase complex reduces statin-mediated upregulation of FOXO gene targets and protects against statin myopathy in rodents.

J Physiol 2012 Dec 8;590(24):6389-402. Epub 2012 Oct 8.

MRC/Arthritis Research UK Centre for Musculoskeletal Ageing Research, School of Biomedical Sciences, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK.

We previously reported that statin myopathy is associated with impaired carbohydrate (CHO) oxidation in fast-twitch rodent skeletal muscle, which we hypothesised occurred as a result of forkhead box protein O1 (FOXO1) mediated upregulation of pyruvate dehydrogenase kinase-4 (PDK4) gene transcription. Upregulation of FOXO gene targets known to regulate proteasomal and lysosomal muscle protein breakdown was also evident. We hypothesised that increasing CHO oxidation in vivo, using the pyruvate dehydrogenase complex (PDC) activator, dichloroacetate (DCA), would blunt activation of FOXO gene targets and reduce statin myopathy. Female Wistar Hanover rats were dosed daily for 12 days (oral gavage) with either vehicle (control, 0.5% w/v hydroxypropyl-methylcellulose 0.1% w/v polysorbate-80; n = 9), 88 mg( )kg(-1) day(-1) simvastatin (n = 8), 88 mg( )kg(-1) day(-1) simvastatin + 30 mg kg(-1) day(-1) DCA (n = 9) or 88 mg kg(-1) day(-1) simvastatin + 40 mg kg(-1) day(-1) DCA (n = 9). Compared with control, simvastatin reduced body mass gain and food intake, increased muscle fibre necrosis, plasma creatine kinase levels, muscle PDK4, muscle atrophy F-box (MAFbx) and cathepsin-L mRNA expression, increased PDK4 protein expression, and proteasome and cathepsin-L activity, and reduced muscle PDC activity. Simvastatin with DCA maintained body mass gain and food intake, abrogated the myopathy, decreased muscle PDK4 mRNA and protein, MAFbx and cathepsin-L mRNA, increased activity of PDC and reduced proteasome activity compared with simvastatin. PDC activation abolished statin myopathy in rodent skeletal muscle, which occurred at least in part via inhibition of FOXO-mediated transcription of genes regulating muscle CHO utilisation and protein breakdown.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1113/jphysiol.2012.238022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3533200PMC
December 2012

Ticagrelor inhibits adenosine uptake in vitro and enhances adenosine-mediated hyperemia responses in a canine model.

J Cardiovasc Pharmacol Ther 2012 Jun 22;17(2):164-72. Epub 2011 Jun 22.

Department of BioScience, AstraZeneca R&D, Mölndal, Sweden.

Aims: A routine secondary pharmacology screen indicated that reversibly binding oral P2Y(12) receptor antagonist ticagrelor could inhibit adenosine uptake in human erythrocytes, suggesting that ticagrelor may potentiate adenosine-mediated responses in vivo. The aim of this study was to further characterize the adenosine uptake inhibition in vitro and study possible physiological consequences of adenosine uptake inhibition by ticagrelor in an anesthetized dog model of coronary blood flow compared to dipyridamole.

Methods And Results: We measured [2-3H]adenosine uptake in purified human erythrocytes and several cell lines in the presence of ticagrelor or the known uptake inhibitor dipyridamole as a comparator. Using an open-chest dog model (beagles), we measured the left anterior descending (LAD) coronary artery blood flow during reactive hyperemia after 1 minute occlusion or intracoronary infusion of adenosine before and after administration of vehicle, ticagrelor, or dipyridamole (each n = 8). Ticagrelor concentration-dependently inhibited adenosine uptake in human erythrocytes and in cell lines of rat, canine, or human origin. In the dog model, ticagrelor and dipyridamole dose-dependently augmented reactive hyperemia after LAD occlusion, as assessed by percentage repayment of flow debt relative to control (both Ps < .05). Ticagrelor and dipyridamole also dose-dependently augmented intracoronary adenosine-induced increases in LAD blood flow relative to control (both Ps < .05).

Conclusion: Ticagrelor inhibits adenosine uptake in vitro and subsequently augments cardiac blood flow in a canine model of reactive hypoxia- or adenosine-induced blood flow increases. These findings suggest that ticagrelor may have additional benefits in patients with acute coronary syndrome beyond inhibition of platelet aggregation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1074248411410883DOI Listing
June 2012

Variation in antibiotic-induced microbial recolonization impacts on the host metabolic phenotypes of rats.

J Proteome Res 2011 Aug 29;10(8):3590-603. Epub 2011 Jun 29.

Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, South Kensington, London SW7 2AZ, United Kingdom.

The interaction between the gut microbiota and their mammalian host is known to have far-reaching consequences with respect to metabolism and health. We investigated the effects of eight days of oral antibiotic exposure (penicillin and streptomycin sulfate) on gut microbial composition and host metabolic phenotype in male Han-Wistar rats (n = 6) compared to matched controls. Early recolonization was assessed in a third group exposed to antibiotics for four days followed by four days recovery (n = 6). Fluorescence in situ hybridization analysis of the intestinal contents collected at eight days showed a significant reduction in all bacterial groups measured (control, 10(10.7) cells/g feces; antibiotic-treated, 10(8.4)). Bacterial suppression reduced the excretion of mammalian-microbial urinary cometabolites including hippurate, phenylpropionic acid, phenylacetylglycine and indoxyl-sulfate whereas taurine, glycine, citrate, 2-oxoglutarate, and fumarate excretion was elevated. While total bacterial counts remained notably lower in the recolonized animals (10(9.1) cells/g faeces) compared to the controls, two cage-dependent subgroups emerged with Lactobacillus/Enterococcus probe counts dominant in one subgroup. This dichotomous profile manifested in the metabolic phenotypes with subgroup differences in tricarboxylic acid cycle metabolites and indoxyl-sulfate excretion. Fecal short chain fatty acids were diminished in all treated animals. Antibiotic treatment induced a profound effect on the microbiome structure, which was reflected in the metabotype. Moreover, the recolonization process was sensitive to the microenvironment, which may impact on understanding downstream consequences of antibiotic consumption in human populations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/pr200243tDOI Listing
August 2011

Systemic gut microbial modulation of bile acid metabolism in host tissue compartments.

Proc Natl Acad Sci U S A 2011 Mar 13;108 Suppl 1:4523-30. Epub 2010 Sep 13.

Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College, London SW7 2AZ, United Kingdom.

We elucidate the detailed effects of gut microbial depletion on the bile acid sub-metabolome of multiple body compartments (liver, kidney, heart, and blood plasma) in rats. We use a targeted ultra-performance liquid chromatography with time of flight mass-spectrometry assay to characterize the differential primary and secondary bile acid profiles in each tissue and show a major increase in the proportion of taurine-conjugated bile acids in germ-free (GF) and antibiotic (streptomycin/penicillin)-treated rats. Although conjugated bile acids dominate the hepatic profile (97.0 ± 1.5%) of conventional animals, unconjugated bile acids comprise the largest proportion of the total measured bile acid profile in kidney (60.0 ± 10.4%) and heart (53.0 ± 18.5%) tissues. In contrast, in the GF animal, taurine-conjugated bile acids (especially taurocholic acid and tauro-β-muricholic acid) dominated the bile acid profiles (liver: 96.0 ± 14.5%; kidney: 96 ± 1%; heart: 93 ± 1%; plasma: 93.0 ± 2.3%), with unconjugated and glycine-conjugated species representing a small proportion of the profile. Higher free taurine levels were found in GF livers compared with the conventional liver (5.1-fold; P < 0.001). Bile acid diversity was also lower in GF and antibiotic-treated tissues compared with conventional animals. Because bile acids perform important signaling functions, it is clear that these chemical communication networks are strongly influenced by microbial activities or modulation, as evidenced by farnesoid X receptor-regulated pathway transcripts. The presence of specific microbial bile acid co-metabolite patterns in peripheral tissues (including heart and kidney) implies a broader signaling role for these compounds and emphasizes the extent of symbiotic microbial influences in mammalian homeostasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1006734107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3063584PMC
March 2011

Gut microbiome modulates the toxicity of hydrazine: a metabonomic study.

Mol Biosyst 2009 Apr 23;5(4):351-5. Epub 2009 Jan 23.

Department of Biomolecular Medicine, Division of Surgery, Oncology, Reproductive Biology and Anaesthetics (SORA), Faculty of Medicine, Imperial College London, South Kensington Campus, London SW72AZ, UK.

The influence of gut microbiota on the toxicity and metabolism of hydrazine has been investigated in germ-free and 'conventional' Sprague Dawley rats using 1H NMR based metabonomic analysis of urine and plasma. Toxicity was more severe in germ-free rats compared with conventional rats for equivalent exposures indicating that bacterial presence altered the nature or extent of response to hydrazine and that the toxic response can vary markedly in the absence of a functional microbiome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/b811468dDOI Listing
April 2009

Blunted Akt/FOXO signalling and activation of genes controlling atrophy and fuel use in statin myopathy.

J Physiol 2009 Jan 10;587(1):219-30. Epub 2008 Nov 10.

Centre for Integrated Systems Biology and Medicine, School of Biomedical Sciences, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK.

Statins are used clinically for cholesterol reduction, but statin therapy is associated with myopathic changes through a poorly defined mechanism. We used an in vivo model of statin myopathy to determine whether statins up-regulate genes associated with proteasomal- and lysosomal-mediated proteolysis and whether PDK gene expression is simultaneously up-regulated leading to the impairment of muscle carbohydrate oxidation. Animals were dosed daily with 80 mg kg(-1) day(-1) simvastatin for 4 (n = 6) and 12 days (n = 5), 88 mg kg(-1) day(-1) simvastatin for 12 days (n = 4), or vehicle (0.5% w/v hydroxypropyl-methylcellulose and 0.1% w/v polysorbate 80; Control, n = 6) for 12 days by oral gavage. We found, in biceps femoris muscle, decreased Akt(Ser473), FOXO1(Ser253) and FOXO3a(Ser253) phosphorylation in the cytosol (P < 0.05, P < 0.05, P < 0.001, respectively) and decreased phosphorylation of FOXO1 in the nucleus after 12 days simvastatin when compared to Control (P < 0.05). This was paralleled by a marked increase in the transcription of downstream targets of FOXO, i.e. MAFbx (P < 0.001), MuRF-1 (P < 0.001), cathepsin-L (P < 0.05), PDK2 (P < 0.05) and PDK4 (P < 0.05). These changes were accompanied by increased PPARalpha (P < 0.05), TNFalpha (P < 0.01), IL6 (P < 0.01), Mt1A (P < 0.01) mRNA and increased muscle glycogen (P < 0.05) compared to Control. RhoA activity decreased after 4 days simvastatin (P < 0.05); however, activity was no different from Control after 12 days. Simvastatin down-regulated PI3k/Akt signalling, independently of RhoA, and up-regulated FOXO transcription factors and downstream gene targets known to be implicated in proteasomal- and lysosomal-mediated muscle proteolysis, carbohydrate oxidation, oxidative stress and inflammation in an in vivo model of statin-induced myopathy. These changes occurred in the main before evidence of extensive myopathy or a decline in the muscle protein to DNA ratio.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1113/jphysiol.2008.164699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2670035PMC
January 2009

Statins but not thiazolidinediones attenuate albumin-mediated chemokine production by proximal tubular cells independently of endocytosis.

Am J Nephrol 2008 6;28(5):823-30. Epub 2008 Jun 6.

Department of Infection, Faculty of Medicine and Biological Sciences, University of Leicester, Leicester, UK.

Background: Proximal tubular epithelial cells (PTEC) secrete chemokines under proteinuric conditions. Both statins and thiazolidinediones (TZDs) possess pleiotropic anti-inflammatory effects. This study examined the ability of statins and TZDs and the natural peroxisome proliferator activated receptor-gamma (PPARgamma) agonist 15-deoxy-Delta(12,14)-prostaglandin J(2) (PGJ(2)) to attenuate the proteinuria-induced pro-inflammatory phenotype of PTEC.

Methods: Mouse PTEC were treated with statins, TZDs and PGJ(2 )and effects on uptake and binding of FITC-albumin determined. PTEC were incubated with fatty acid free bovine serum albumin with or without statins/TZDs/PGJ(2), and the release of MCP-1 and RANTES measured.

Results: Statins and TZDs significantly inhibited PTEC albumin endocytosis. PGJ(2 )had no effect. Incubation of PTEC with albumin significantly stimulated production of MCP-1 and RANTES. Co-treatment with statins and PGJ(2) significantly reduced albumin-stimulated chemokine production, an effect reversed by the addition of mevalonate and geranylgeranyl pyrophosphate. In contrast, TZDs had no effect on albumin-mediated chemokine production.

Conclusion: Statins and PGJ(2), but not TZDs, prevent the development of a PTEC pro-inflammatory phenotype in response to albumin. Albumin endocytosis is not a prerequisite for PTEC chemokine production, and inhibition of albumin endocytosis alone is insufficient to attenuate chemokine production. These studies suggest a therapeutic role for statins and some PPARgamma ligands in proteinuric renal disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000137682DOI Listing
October 2008

Systems toxicology: integrated genomic, proteomic and metabonomic analysis of methapyrilene induced hepatotoxicity in the rat.

J Proteome Res 2006 Jul;5(7):1586-601

Biological Chemistry, Biomedical Sciences Division, Faculty of Life Sciences, Imperial College London, Sir Alexander Fleming Building, South Kensington, London SW7 2AZ, United Kingdom.

Administration of high doses of the histamine antagonist methapyrilene to rats causes periportal liver necrosis. The mechanism of toxicity is ill-defined and here we have utilized an integrated systems approach to understanding the toxic mechanisms by combining proteomics, metabonomics by 1H NMR spectroscopy and genomics by microarray gene expression profiling. Male rats were dosed with methapyrilene for 3 days at 150 mg/kg/day, which was sufficient to induce liver necrosis, or a subtoxic dose of 50 mg/kg/day. Urine was collected over 24 h each day, while blood and liver tissues were obtained at 2 h after the final dose. The resulting data further define the changes that occur in signal transduction and metabolic pathways during methapyrilene hepatotoxicity, revealing modification of expression levels of genes and proteins associated with oxidative stress and a change in energy usage that is reflected in both gene/protein expression patterns and metabolites. The difficulties of combining and interpreting multiomic data are considered.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/pr0503376DOI Listing
July 2006

Inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase reduce receptor-mediated endocytosis in opossum kidney cells.

J Am Soc Nephrol 2004 Sep;15(9):2258-65

AstraZeneca, Safety Assessment, 23S37-69, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK.

Renal proximal tubule cells are responsible for the reabsorption of proteins that are present in the tubular lumen. This occurs by receptor-mediated endocytosis, a process that has a requirement for some GTP-binding proteins. Statins are inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase used for the therapeutic reduction of cholesterol-containing plasma lipoproteins. However, they can also reduce intracellular levels of isoprenoid pyrophosphates that are derived from the product of the enzyme, mevalonate, and are required for the prenylation and normal function of GTP-binding proteins. The hypothesis that inhibition of HMG-CoA reductase in renal proximal tubule cells could reduce receptor mediated-endocytosis was therefore tested. Five different statins inhibited the uptake of FITC-labeled albumin by the proximal tubule-derived opossum kidney cell line in a dose-dependent manner and in the absence of cytotoxicity. The reduction in albumin uptake was related to the degree of inhibition of HMG-CoA reductase. Simvastatin (e.g., statin) inhibited receptor-mediated endocytosis of both FITC-albumin and FITC-beta(2)-microglobulin to similar extents but without altering the binding of albumin to the cell surface. The effect on albumin endocytosis was prevented by mevalonate and by the isoprenoid geranylgeranyl pyrophosphate but not by cholesterol. Finally, evidence that the inhibitory effect of statins on endocytosis of proteins may be caused by reduced prenylation and thereby decreased function of one or more GTP-binding proteins is provided. These data establish the possibility in principle that inhibition of HMG-CoA reductase by statins in proximal tubule cells may reduce tubular protein reabsorption.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/01.ASN.0000138236.82706.EEDOI Listing
September 2004

Pregnane X receptor-dependent and -independent effects of 2-acetylaminofluorene on cytochrome P450 3A23 expression and liver cell proliferation.

Biochem Biophys Res Commun 2003 Jan;300(2):278-84

INSERM U456, Faculté de Pharmacie, 2 Avenue du Pr Leon Bernard, Rennes 35043, France.

The arylamide 2-acetylaminofluorene (AAF) is a powerful carcinogen displaying a marked promoting activity, also known to regulate expression of liver detoxifying proteins. In this study we identified CYP3A23, a major inducible cytochrome P-450 (CYP) isoform, as an AAF target in hepatocytes. Indeed, exposure to AAF of primary rat hepatocytes resulted in a marked up-regulation of CYP3A23 expression at both mRNA and protein levels. Using CYP3A23 reporter gene constructs, we further demonstrated that AAF activated the CYP3A23 Direct Repeat 3 (DR3) promoter element interacting with the nuclear pregnane X receptor (PXR). Moreover, the PXR antagonist ecteinascidin-743 fully suppressed AAF-related CYP3A23 induction. Low doses of AAF inhibiting DNA synthesis in hepatocytes however failed to trigger PXR-related CYP3A23 induction and PXR-negative epithelial liver cells remained sensitive to the mito-inhibitory effects of AAF. Such data indicate that AAF up-regulates CYP3A23 through PXR activation but does not require PXR for exerting its carcinogenic promoting properties based on inhibition of cell growth.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s0006-291x(02)02847-4DOI Listing
January 2003
-->