Animal Models Abstract Booklet 20110915

8/17/2019 Animal Models Abstract Booklet 20110915

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Scientific Organizing Committee

Maria Gabriela Belvisi, BSc, PhDImperial College London

Garret A. FitzGerald, MDUniversity of Pennsylvania School of Medicine

Susan D. Brain, BSc, PhD, FBPharmacoIS

King's College London

Brooke Grindlinger, PhD

The New York Academy of Sciences

Julia C. Buckingham, BSc, PhD, DSc, FBPharmacolSImperial College London

Ray Hill, PhD, DSc, (Hon), FMedSciImperial College London

Sandra J. Engle, PhDPfizer Inc.

Kerstin Hofmeyer, PhDThe New York Academy of Sciences

Simon Howell, PhDKing's College London

Poster Sessions 1 and 2 are Generously Sponsored By


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Travel Fellowships have been awarded on a competitive basis to support the attendance andparticipation as a poster presenter of the following junior investigators:

Elizabeth J. Cartwright, PhDUniversity of Manchester

Hannah C. Greenwood, BScImperial College London

Ian F. Harrison, BSc (Hons), MResImperial College London

Neil Hill, BMBS Imperial College London

Paul HollowayImperial College London

Christina Nesarajah, BScImperial College London

Emily S. Sena, PhDUniversity of Edinburgh

Anna Starr, PhDKing’s College London

The aforementioned travel fellowships were made possible by the generous support of the:

Aisah A. Aubdool, BSc (Hons) Pharmacology, MResKing’s College London

Jennifer V. Bodkin, BSc, MResKings College London

Paul Holloway Imperial College London

Emma S.J. Robinson, BSc(Hons), PhDUniversity of Bristol

The aforementioned travel fellowships were made possible by the generous support of the:

PPOOSSTTEERR PPRREESSEENNTTAATTIIOONN PPRRIIZZEE

A certificate and prize of US$250.00 will each be awarded for the two best posters presented at theconference. Posters will be judged on the basis of scientific merit by members of the ScientificOrganizing Committee. Poster prizes will be awarded during Joint Session VIII on the afternoonof Friday, September 16, 2011.

Poster Presentation Prizes were made by possible by the generous support of the:


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PS1 - 1 Anjali A. AminPS1 - 2 Aisah Aubdool

PS1 - 3 Martin Auger

PS1 - 4 Prabal Banerjee

PS1 - 5 Katherine Banks

PS1 - 6 Richard Beger

PS1 - 7 Jennifer V. Bodkin

PS1 - 8 Benedikt Bosbach

PS1 - 9 Omer I. Butt

PS1 - 10 James Cao

PS1 - 11 Elizabeth J. Cartwright

PS1 - 12 D. Kemp Covington

PS1 - 13 Mahmoud Danab

PS1 - 14 Michael Emerson

PS1 - 15 Michael Emerson

PS1 - 16 Paul Ernsberger

PS1 - 17 Isabel Gracia

PS1 - 18 Ray GreekPS1 - 19 Hannah C. Greenwood

PS1 - 20 Robert Haberzettl

PS1 - 21 Ian F. Harrison

PS1 - 22 Neil Hill

PS1 - 23 Paul Holloway

PS1 - 24 Laura Howe

PS1 - 25 Brian Karolewski

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1. THE USE OF IN VIVO MODELS IN STUDYING DIABETES

Anjali Amin, MBBS, BSc, MRCP, Stephen R. Bloom, MA, MD, DSc, FRCPath, FRCP, Kevin G.Murphy, PhD

Section of Investigative Medicine, Imperial College London, London, United Kingdom

Diabetes is a complex chronic condition, associated with significant morbidity and mortality, affecting

approximately 347 million people worldwide. Diabetes-related deaths are predicted to doublebetween 2005 and 2030, driving the need for urgent and effective treatments in the management ofthis condition. Treatments for type 2 diabetes remain suboptimal, with marked side-effects, andtreatment for the majority of patients with type 1 diabetes remains life-long subcutaneous insulintherapy. The complex interplay of genetic and environmental factors which influence the nature ofdiabetes make it difficult to use in vitro models to investigate the specific molecular mechanismsresponsible. In vivo models allow the investigation of these mechanisms under physiological andpath physiological conditions. Animal models of diabetes can be generated spontaneously, orinduced by chemicals such as streptozotocin, by dietary or surgical manipulations, or by acombination of these techniques. The development of transgenic models of diabetes has allowedfurther insight into the molecular mechanisms involving autoimmunity against β-cells. Thedevelopment of new technologies such as transcriptomics, proteomics and metabolomics will aid theidentification of new diabetes susceptibility genes. While rodent models do not identically mimic

human diabetes, these models are vital tools for the study of the genetic, endocrine, metabolic andunderlying aetiological mechanisms which cause diabetes. The use of rodent models is thusessential to the development of new treatments for both type 1 and type 2 diabetes.

2. SERIAL ASSESSMENT OF CARDIOVASCULAR HAEMODYNAMICS IN HYPERTENSION

Aisah A. Aubdool1, 2, Jennifer V. Bodkin1, Stuart Bevan3 and Susan D. Brain1, 2

1Cardiovascular Division, School of Medicine, King’s College London, United Kingdom2Centre of Integrative Biomedicine, King’s College London, United Kingdom3Wolfson CARD Centre, King’s College London, United Kingdom

Hypertension is associated with secondary conditions that include peripheral vascular and micro

vascular disease. Recent studies suggest that transient receptor potential ankyrin 1 (TRPA1) maypotentially regulate blood vessel tone, as activation of TRPA1 in the peripheral vasculature leads tothe release of the neuropeptide CGRP. It is well established that CGRP possesses potentvasodilator and cardiovascular properties. We investigated the role of TRPA1 in cardiovascularhaemodynamics and peripheral blood flow in vivo using the Angiotensin II−induced experimentalmodel of hypertension in transgenic mice. The use of subcutaneous osmotic mini-pumps tochronically deliver Angiotensin-II potentially reduced animal stress by eliminating chronic repeateddaily manual infusions. Using the non-surgical tail-cuff method, 5 different blood pressureparameters; systolic, diastolic and mean blood pressure, tail blood volume and flow were assessedat baseline and several time-points following infusion of Angiotensin-II or saline (control) in eachrestrained, conscious mouse. Heart rate, perfusion index and oxygen saturation were also monitorednon-invasively using the MouseSTAT pulse oximeter. Furthermore, peripheral blood flow wasrecorded in both ears, paws, thighs and tail at baseline and several time-points after the induction of

experimental hypertension in mice anaesthetised under isoflurane for 5-10 min using a Full-FieldLaser Perfusion Imager. At Day 14, plasma and multiple tissues such as ears, brain, aorta, heart andpaws were collected for microarray and qRT-PCR experiments to map genes for hypertension. Thisstudy design allowed each individual mouse to be serially used to assess multiple parameters over 2weeks to study cardiovascular pathophysiology and hence, reduced the total number of animalsrequired for the study.


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3. ANALYSIS OF MULTIPLE ENDPOINTS IN THE MOUSE BLEOMYCIN MODEL OF LUNGFIBROSIS

Martin Auger1, Steve Underwood1, Nick Vitali1, Maria Mauricio1, Sandra Dinocca1, Ling-Min Lu1,Linping Wang2, Anne Minnich1, Jingbo Gao1, Diann Burtis1, Jennifer Sebalusky1, Rong Chen1, GeoffVarty1, Rachel Yabkowitz1

1 Fibrosis and Wound Repair Therapeutic Strategy Unit, Sanofi U.S., Bridgewater, New Jersey2

Molecular Innovative Therapeutics, Sanofi U.S., Bridgewater, New Jersey

Idiopathic pulmonary fibrosis (IPF) is a debilitating disease with limited treatment options. Prognosisis poor, and patients have a median survival time of less than five years. The pathology of IPF islikely driven by repeated damage to the airway epithelium that leads to dysregulated repairmechanisms, fibroblastic foci in which myofibroblasts deposit extracellular matrix components suchas collagen and a progressive decline in lung function. Ideally, an animal model of IPF should allowthe effects of potential new therapies to be assessed within a reasonably short timeframe, andshould predict clinical efficacy. Bleomycin is often used to induce lung fibrosis in mice, and to profilepotential new therapies. However, the clinical predictability of this model has been questioned. Ourgoal has been to establish a protocol and methods that will maximize the clinical predictability of thismodel. We demonstrate that bleomycin causes deposition of collagen in lungs that can bequantified by histopathology or assays of tissue homogenates, increased expression of the

myofibroblast marker alpha smooth muscle actin in lungs, increased serum titers of non-invasiveputative IPF biomarkers, impaired oxygen saturation of blood, and changes in lung mechanics thatmodel those associated with restrictive changes in IPF.

4. HUMANIZED NOG MOUSE: A NOVEL MODEL FOR STUDYING MYCOBACTERIUMTUBERCULOSIS PATHOGENESIS AND HOST-IMMUNE RESPONSE AGAINSTINFECTION

Angelo Izzo1, Gerold Feuer2, 3 and Prabal Banerjee, PhD3

1Department of Microbiology, Immunology & Pathology, Colorado State University, Colorado2Department of Microbiology and Immunology, SUNY Upstate Medical University, Syracuse, NewYork3

HuMurine Technologies, Inc., La Verne, California

Humanized mice are a recent breakthrough with the potential to circumvent the implicit obstacles ofconventional animal models. The technology used to develop and generate humanized mice hasimproved significantly in recent years due to the development of the humanized NOD/SCID IL-2rgc

null (NOG) mice that develop a complete lineage of human cells of the innate and adaptive immunesystem including monocytes/macrophages, plasmacytoid and myeloid DC, NK cells and T and Blymphocytes, post reconstitution with human hematopoietic stem cells. We have characterized andstandardized the parameters of humanization and the long-term multilineage hematopoiesis in NOGmice thereby leading to the development of a humanized mouse model that supports a robust andcomplete pattern of human hematopoiesis. These humanized mice provide a unique opportunity togain insight into pathogenesis of human-specific pathogens including intracellular pathogenicbacterial infections such as tuberculosis. We have recently developed a humanized mouse model for

tuberculosis in which the human cells in the mice respond to infection and there is formation ofpulmonary granulomas when these mice are infected with a low dose aerosol infection of virulentMycobacterium tuberculosis H37Rv. Specifically, after infection, both human CD4+ and CD8+ T cellsexpand in response to infection and produce IFN-γ , TNF-α and IL-2, such that these cytokines aresignificantly elevated by day 34 PI. In addition, engrafted mice produce lesions in the lungs thatresemble granulomas, suggesting that these mice may provide a suitable model to investigate theinteraction between the human host cells and the pathogen in an in vivo environment.


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5. USING IN VIVO MODELS TO INVESTIGATE THE ROLE OF REWARD IN FOOD INTAKEAND OBESITY

Katherine Banks, BSc, Kylie E. Beale, PhD, Stephen R. Bloom, MA, MD, DSc, FRCPath, FRCP andKevin G. Murphy, PhD

Imperial College London, United Kingdom

The brain pleasure-reward system is evolutionarily important in reinforcing food seeking behaviour.The mesocorticolimbic dopaminergic systemis a critical component. Dopamineneurons arising in theventral tegmental area (VTA) signal to the nucleus accumbens and pre-frontal cortex to activatereward pathways and associate food intake with pleasure. The VTA is heavily implicated in non-homeostatic eating; the continued ingestion of food when already satiated. The ready availability ofconvenient high-fat and high-sugar foods in the Western diet may trigger non-homeostatic eatingdue to their high reward value and thus contribute to the current obesity pandemic. Hypothalamicneuronal pathways responsible for homeostatic regulation of food intake communicate with the VTA,which integrates these neural signals with circulating signals to modulate the motivation to eat. Invivo models can be used to investigate the neuronal circuitry involved in the pleasure-rewardsystem. Hormones such as leptin, released from adipose tissue, and ghrelin, released from thestomach, signal to the VTA to influence food intake. Our current work focuses on how suchcirculating signals modulate neural feeding impulses sent to dopaminergic VTA neurons, and how

they differentially regulate reward pathways depending on nutritional state. Altered indices of rewardin genetic and environmental models of obesity can also aid our understanding of the role of themesocorticolimbic system in food intake. The use of in vivo models to investigate reward and foodintake may identify new therapeutic targets for the treatment of obesity.

6. AN INTEGRATED METABOLOMICS STUDY OF GENTAMICIN-INDUCEDNEPHROTOXICITY

Richard Beger, PhD1, Laura Schnackenberg, PhD1, Jinchun Sun, PhD1, Sudeepa Bhattacharyya,PhD1, Yosuke Ando, PhD1,2

1National Center for Toxicological Research, US FDA, Jefferson, Arkansas2Daiichi Sankyo Co., Ltd., Tokyo, Japan

Gentamicin is an aminoglycoside antibiotic used in the treatment of bacterial infections. However, in10-25% of patients, there is an increase in blood urea nitrogen and a reduction in glomerularfiltration rate. In this study, LC/MS- and NMR-based global metabolomics analyses of urine wereemployed to discover metabolite biomarkers of kidney injury and recovery from toxic insult. MaleSprague Dawley rats were divided into four groups that were injected with gentamicin sulfate (0, 75,100, or 300 mg/kg/day dissolved in 0.4 mL saline) for one, two or three consecutive days starting onday 0. Four animals from each group were sacrificed on days 1, 2, 3, 7, 10, 15, 18, 22, 29, 36, and44. The kidneys were harvested and serum collected at sacrifice. Sixteen hours prior to sacrifice,rats were placed in metabolism cages and urine samples collected in 6 hr intervals. Severalmetabolites including amino acids and hydroxyproline were closely associated with thehistopathology during the injury and recovery periods. Glucosuria was noted prior to the increase inBUN and serum creatinine and may represent an early general marker of renal injury. LC/MS

analyses detected significant increases in homovanillic acid sulfate and homoveratric acid sulfatethat may be indicators of renal adaptive response prior to gentamicin-induced injury. Biomarkersrelated to the efficacy of gentamicin were also detected. Specifically, gut-microflora-relatedcompounds including hippurate, indole derivatives, and phenyl derivatives were decreased on days2-10. An integrated LC/MS and NMR-based metabolomics approach identified potential biomarkersof gentamicin-induced renal injury and efficacy.


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7. APPLYING THE 3R’S TO MURINE CARDIOVASCULAR PHENOTYPING

Jennifer V. Bodkin BSc, MRes, Aisah A. Aubdool, and Susan D. Brain

King’s College London, Cardiovascular Division, School of Medicine, London, United Kingdom

Transient Receptor Potential Ankyrin 1 (TRPA1) channels are membrane expressed, non-selectivecation channels, activated by noxious compounds. These include exogenous agonists such as

mustard oil, cinnamaldehyde and components of air pollution. In vivo , TRPA1 channels aresuggested to be activated by reactive oxygen species (ROS) and products of oxidative stress. Agrowing number of publications have demonstrated TRPA1 agonists to mediate vasodilationresponses. We were the first to demonstrate the role of TRPA1 in this response by using TRPA1KO mice. As peripheral blood vessel tone is an important component of peripheral resistance andblood pressure control, we have gone on to conduct a novel cardiovascular characterization studyusing TRPA1 wild-type (WT) and -KO mice. To do this we have adopted 3Rs strategies, refining ourtechniques to maximize data output and reducing animal numbers. Our mice are profiled at baselineby telemetry, showing conscious blood pressures, heart rate and activity levels. Their heartmorphology and function is also assessed non-invasively by echocardiography. They then undergoexperimental hypertension induction using subcutaneously implanted osmotic minipump infusion ofangiotensin II. This additional pathological stress is designed to exacerbate the effect of removingTRPA1-mediated dilation in KO mice. Mice are characterized throughout a 14-day infusion by

telemetry, before final cardiac examination with echocardiography. Organs from these mice are thencollected and analyzed using a variety of methods, measuring remodeling and inflammatorymarkers.

8. IMATINIB-RESISTANCE AND MICROCYTIC ERYTHROCYTOSIS IN A MOUSE MODELOF GASTROINTESTINAL STROMAL TUMOR (GIST)

Benedikt Bosbach1, Shayu Deshpande1, Ferdinand Rossi1, Cristina Antonescu2, Peter Besmer1 1Developmental Biology Program, Sloan-Kettering Institute, New York, New York2Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York

Most GISTs harbor KIT receptor gain-of-function mutations. In GIST patients treated with thetyrosine kinase inhibitor imatinib tumor clones frequently grow out with second-site KIT mutationsthat are thought to disrupt binding of the inhibitor. We had previously generated a mouse model ofGIST by introducing the Kit V558∆ mutation found in a case of familial GIST into the Kit gene. Now, toinvestigate the consequences of second-site KIT mutations on imatinib-susceptibility and GISTdevelopment, we generated a mouse model introducing into the endogenous Kit locussimultaneously the V558∆ and the kinase “gatekeeper” mutation T669I (human T670I) found inimatinib-resistant GIST. Invariably, these Kit V558∆;T669I/+ mice developed pronounced interstitial cell ofCajal (ICC) hyperplasia in the stomach and colon, and cecal GIST. Treatment of single-mutantKit V558∆ /+ mice with imatinib, dasatinib, sunitinib or sorafenib significantly reduced GIST signaltransduction and cell proliferation. In contrast, treatment of double-mutant mice with imatinib ordasatinib did not inhibit GIST growth. However, the resistance mediated by the gatekeeper mutationcould be overcome by treatment with sunitinib and sorafenib.Interestingly, the Kit V558∆;T669I/+ mice developed a pronounced microcytic erythrocytosis and this is in

contrast to the known macrocytic anemia observed in Kit loss-of-function mutant mice.This mouse model should be useful for the development of therapeutic strategies designed toovercome gatekeeper-mediated imatinib-resistance in GIST and in the investigation of theconsequences of different levels of oncogenic KIT signaling in diverse KIT-dependent cell lineages.


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9. MARKERS OF OXIDATIVE STRESS AND APOPTOSIS FOR PRECLINICALEVALUATION OF HEMOGLOBIN-BASED OXYGEN THERAPEUTICS INNONASCORBATE-PRODUCING SPECIES

Omer I. Butt, PhD, Paul W. Buehler, PhD, Felice D’Agnillo, PhD

Division of Hematology, CBER/FDA, Bethesda, Maryland

Hemoglobin-induced oxidative stress and apoptosis may contribute to some of the unresolvedtoxicities of hemoglobin-based therapeutics. Guinea pigs may be a useful species for examininghemoglobin oxidative toxicity because, similar to humans, they lack the ability to produce ascorbate,which is a powerful reductant capable of controlling intravascular hemoglobin oxidation. Here, weexamined sensitive and specific biomarkers of oxidative stress and apoptosis. Using a 50%exchange transfusion model with polymerized bovine hemoglobin in guinea pigs, we analyzedNRF2, a potent marker of oxidative stress; 4-hydroxynonenal (4-HNE)-modified protein adducts, anindex of lipid peroxidation; 8-hydroxy-2-deoxyguanosine (8-OHdG), a marker of oxidative DNAdamage; and cleaved caspase 3, a marker of apoptosis. Immunohistochemical and western blotanalyses revealed translocation of NRF2 from the cytoplasm to the nucleus. There was also

enhanced accumulation of 4-HNE−modified protein adducts, along with increased nuclear 8-OHdGimmunoreactivity in renal proximal tubules and glomeruli. Cleaved caspase 3 was detectable bywestern blot in the kidneys but was not associated with significant increases in serum creatinine orother common histopathological indices. Cleaved caspase 3 levels were also elevated in hearts andlungs. Clinical trial results with some hemoglobin-based therapeutics have led to suggestions thatpreclinical testing may not have been sufficiently predictive of safety in humans. The present findingssuggest that sensitive and specific markers of oxidative stress and apoptosis in a guinea pig animalmodel may be useful in preclinical studies designed to evaluate the safety of these products.

10. HIGHLY-SENSITIVE IN VIVO PHOTONIC IMAGING OF APOPTOTIC CELLS BYUTILIZATION OF TRANSGENIC MOUSE MODELS

James Cao, Derek Adler, Xiaoyou Ying

Biomarker, Bioimaging and Biological Assays (BBB), Disposition, Safety & Animal Research (DSAR)US Operational Center, Sanofi U.S., Bridgewater, New Jersey

A highly sensitive photonic imaging method has been developed for in vivo apoptosis visualizationand analysis in live mice. This method is based on a caspase-3/7 activatable substrate (cagedluciferin, Z-DEVD-Aminoluciferin) and two luciferase-expressing transgenic models: 1) Taconicubiquitous luciferase mouse model (β-actin-luc), which can be used for any mouse organ apoptosisimaging, 2) Sanofi’s myelin basic protein-luciferase mouse model, which can be used for mousebrain and intestine apoptosis imaging. The method has been used to detect cadmium-induced liverapoptosis and radiation-induced intestinal apoptosis in live mice. Our data demonstrated that theluminescent apoptosis probe was highly sensitive in vivo to detect apoptosis that was induced byionizing radiation and cadmium. The apoptosis imaging window is consistent withimmunohistochemical data in the literature. Our results indicated that this apoptosis imaging methodcould be used to study apoptosis-related mouse disease mechanisms and to profile compounds invivo.


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11. TREATMENT OF CARDIAC HYPERTROPHY WITH A NOVEL PHARMACOLOGICALINHIBITOR OF PLASMA MEMBRANE CALCIUM ATPASE 4 (PMCA4) MIMICS THEPHENOTYPE OF THE PMCA4 KNOCKOUT MOUSE

Elizabeth J. Cartwright, PhD, T. Mohamed, D. Oceandy, R. Abou-Leisa, F. Baudoin, M. Zi, S.Prehar, and L. Neyses

Manchester Academic Health Science Centre, University of Manchester, Manchester, United

Kingdom

One of most prevalent causes of morbidity and mortality worldwide is heart failure (HF); with 5million people in the USA currently affected by this disease it is essential that we increase ourunderstanding of its mechanistic basis to develop effective treatment strategies. Pathologicalhypertrophy, resulting from high blood pressure, myocardial infarction and aortic stenosis, is a pre-requisite for heart failure and thus reduction in cardiac hypertrophy is a potent treatment strategy.Using a gene knockout model we have identified that plasma membrane calcium ATPase isoform 4(PMCA4) is a regulator of pathological cardiac hypertrophy and have identified a pharmacologicalinhibitor whose action mimics the phenotype of the knockout mouse. Gene deletion of PMCA4(PMCA4KO) resulted in the attenuation of pathological hypertrophy induced by pressure overload,leading us screen a chemical library to identify a novel specific inhibitor of PMCA4. AP2 wasidentified, which inhibited PMCA4 activity with high affinity (IC50=150nM) but not other related

ATPases expressed in the heart. Testing the action of AP2 on hypertrophy we found that in vitro andin vivo AP2 significantly prevented the response to hypertrophic stimuli. Importantly AP2 was able toeffectively reverse established hypertrophy, which is a more realistic clinical scenario. Whole bodyphenotyping of PMCA4KO mice indicated no abnormalities in 58 parameters tested, predicting few,if any, on-target side effects when using PMCA4 as an anti-hypertrophic target. In conclusion,PMCA4 has a key role in the development of pathological cardiac hypertrophy and is a novel andeffective target for its treatment.

12. THE FUTURE OF ANIMALS IN RESEARCH PROJECT: AN INNOVATIVE APPROACH TOBUILDING NEW RESEARCH STRATEGIES AT GLAXOSMITHKLINE

D. Kemp Covington, DVM1, Sarah Hawthorne, BEng (Hons) MIChemE2, Julie Huxley-Jones, PhD3,Ghislaine Poirier, PhD, DVM3

1GlaxoSmithKline, Research Triangle Park, North Carolina2GlaxoSmithKline, Brentford, United Kingdom3GlaxoSmithKline, Stevenage, United Kingdom

Animal research represents a small but vital role in the process of discovering medicines. Due tolimitations in scientific knowledge and technology, as well as the complexity of disease mechanisms,there have been no broadly applicable alternatives found at this time. As part of our commitment todelivering the best science while reducing reliance on animals, GlaxoSmithKline recently chartered aglobal project to investigate the way we approach and conduct animal research, specifically in drugefficacy models. The project, The Future of Animals in Research, was commissioned by GSK’sHead of Research and Development to challenge current processes and drive novel thinking incompany drug discovery efforts. Over a three month diagnostic phase, the team engaged over one

hundred forty internal and external experts to understand the current needs for in vivo research,examine existing ways of working, and explore opportunities for improvements. Specific objectivesincluded evaluating the company’s application of the 3Rs (replacement, refinement, reduction),improving internal and external communications regarding animal research, determining whereinvestments in innovative solutions make sense for the practice of best science, and partnering withpublic stakeholders in the animal welfare community to examine how animals contribute to drugdiscovery. The project has identified key opportunities to optimize efficacy-based animal research atGSK, in a “traditional 3Rs” approach, and through enhanced consideration of animal modelrelevance, robustness, and reproducibility. We will present an overview of how effective


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communication and engagement, scientific peer review, and maximizing the value of data canpositively impact animal research.

13. ANTI- INFLAMMATORY AND CYTOTOXIC ACTIVITY OF THE PLANT CANNABISSATIVA (L) PERTROLIUM ETHER EXTRACT IN ALBINO RATS

M.M. Dahab1, I.A. Musa2, E.A. Osman1, M.A. Jah Elnabi3, and E.L. Badwi S.M.4

1Department of Microbiology, Faculty of Pure and Applied Sciences, International University ofAfrica, Khartoum, Sudan2Department of Biochemistry, Nutrition, Toxicology and Pharmacology Central of VeterinaryResearch laboratory, Khartoum, Sudan3National Ribat University, Khartoum, Sudan4Department of Medicine, Pharmacology and Toxicology, University of Khartoum, Sudan

In this study, the plant Cannabis sativa seeds petroleum oil extract was investigated for anti-inflammatory activity and explore the toxicity on albino rats. All extracts showed no significantcytotoxic activity on the Vero cell line. The inflammation was firstly obtained by using carrageenansuspension 0.1 ml of 10% saline injected at the sub – plantar region of the left limb for inducing alocal acute oedema. A decrease in oedema size was reported after 24 hours for the rats pretreatedwith carrageenan 30 minutes before injection with suspension (4.56, 0.59 and 0.93 for control,1ml/kg per day and 0.5ml/kg per day groups given C. sativa seed extracts respectively.), comparedto Indomethacin (standard anti-inflammatory drug), which reported a decrease in oedema sizediameter to 0.55mm, which indicated an increase inhibition percentages were reported for thedifferent pretreated groups 0.00, 87.03, 79.56 and 87.91 including the comparative Indomethacintreated groups of rats respectively. On the other hand, the post-treated groups of rats (given C.sativa oil extract after 30 minutes of injection of suspension) showed similar results for maximumconcentration 1 ml/day of C. sativa oil extract in comparison to the standard drug. Hence, suchresults recommend the prospectice focus for the preventive medical use of the extract.

14. ELUCIDATION OF THE ROLE OF ENDOGENOUS NO AND ENDOTHELIAL NOSYNTHASE IN REGULATING PLATELET FUNCTION IN VIVO

Christopher Moore, PhD, Michael Emerson, PhD

Platelet Biology Group, National Heart and Lung Institute, Imperial College London, London, UnitedKingdom

Nitric oxide (NO) regulates vessel tone and platelet function in vitro . The role of endogenous NO andthe eNOS (endothelial nitric oxide synthase) isoform in regulating platelets in vivo remains unclearsince conflicting reports have been published concerning the thrombotic phenotype of eNOS-/- miceand models for assessing platelet function in vivo in the mouse have not been available. Wedeveloped and employed a mouse model for measuring platelet aggregation in vivo as theaccumulation of radiolabelled platelets in the pulmonary vasculature via external scintillation probes.Collagen- and thrombin-induced in vivo platelet aggregation were enhanced by NOS inhibitors. Incontrast, vasoconstriction with phenylephrine had no effect on platelet aggregation. Platelet

aggregation in vitro was not modified by NOS inhibitors and eNOS was not detected in platelets byWestern blotting. The duration of platelet aggregation in eNOS-/- mice was potentiated followingmoderate thrombotic stimulation. NOS inhibition enhanced platelet aggregation in wild-type mice buthad no effect on aggregation responses in eNOS -/- mice. In addition, iNOS and nNOS inhibitors wereineffective in both WT and eNOS-/- mice. Endogenous NO therefore negatively regulates platelets invivo through a direct action on platelets and not a secondary vascular effect. The source of bioactiveNO in vivo is exclusively eNOS, with a negligible contribution by other NOS isoforms. Platelets areregulated primarily by NO originating from the vascular endothelium rather than the platelet itself.Thus, eNOS in the vascular endothelium is a key modulator of platelet function and a potentialtherapeutic target for platelet-driven disorders.


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15. MOUSE AND HUMANIZED MOUSE MODELS FOR EVALUATING THROMBOTICPLATELET RESPONSES IN VIVO

Lisa-Marie Holbrook, PhD, Charalambos Tymvios, PhD, Christopher Moore, PhD, Michael Emerson,PhD

Platelet Biology Group, National Heart and Lung Institute, Imperial College London, London, UnitedKingdom

In vitro platelet aggregation assays poorly predict platelet function and thrombus formation in vivo partly due to the critical role of the vascular endothelium in regulating platelet activity. We thereforedeveloped methods for measuring platelet aggregation in situ in the mouse. Platelets were isolatedfrom donor mice following cardiac puncture or human volunteers following venepuncture,radiolabelled with 111Indium Oxine and infused into anaesthetized C57Bl/6 or platelet depleted NOD-scid recipients respectively. Circulating radiolabelled platelets were monitored via external 1cmdiameter scintillation probes over the pulmonary region. The platelet agonists ADP, thrombin andcollagen induced platelet aggregation responses, detected as rapid and transient increases inradioactive counts as platelets aggregated and became trapped in the pulmonary region. Countsthen returned to baseline in a variable time frame. The peak responses and area under curvemeasurements indicated dose-dependency. There were no detectable changes in counts uponinjection of maximal doses of agonists when erythrocytes were labeled rather than platelets

indicating a platelet specific effect. In addition, platelet aggregates could be detected histologically inlungs from mice exposed to platelet agonists. Administration of aspirin significantly reduced plateletaggregation in response to collagen. We therefore present a novel method for evaluating human andmouse platelet aggregation responses in vivo which may find applications in the identification ofnovel anti-thrombotic targets as well as in drug evaluation and safety profiling.

16. SHROB RAT MODEL OF METABOLIC SYNDROME REVEALS UNEXPECTED BENEFITSAND OFF-TARGET EFFECTS FOR MULTIPLE AGENTS

Paul Ernsberger, PhD and Richard J. KoletskyDepartment of Nutrition, Case Western Reserve University School of Medicine, Cleveland, Ohio

SHROB rats are a substrain of spontaneously hypertensive rat (SHR) with a naturally occurring

knockout of the leptin receptor. SHROB rats exhibit multiple abnormalities characteristic of humanmetabolic syndrome. We hypothesized that antidiabetic and antihypertensive agents may have aspectrum of therapeutic activity on components of metabolic syndrome. Antidiabetic agents: Allagents tested normalized glucose tolerance. PPAR-gamma agents also correctedhypertriglyceridemia, and ameliorated steatohepatitis and hypertension while increasingsubcutaneous fat. Glyburide did not affect body fat but worsened left ventricular hypertrophy.Sitagliptin normalized elevated glucagon levels and reduced visceral fat without affecting overalladiposity. Antihypertensive agents: All agents tested normalized blood pressure. Hydralazine andalpha-methyldopa worsened glucose tolerance and insulin resistance, whereas both were improvedby another sympathoinhibitor, moxonidine. Captopril worsened glucose tolerance but loweredtriglycerides, LDL cholesterol and proteinuria. Allylmercaptocaptoril, with an allyl moiety from garlic,improved glucose tolerance. Relative to captopril, it induced greater reductions in blood pressureand proteinuria. Both agents reduced fatty acid turnover in isolated abdominal adipocytes. Anothergarlic compound, diallyl sulfide, was effective in lowering blood glucose during an oxidative stresschallenge. Thus, preclinical drug trials in the SHROB model parallel those in humans and suggestunexpected benefits of new agents and possible risks of older agents. The SHROB model mayidentify useful agents for treating metabolic syndrome.


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17. EVALUATION OF THE PRECLINICAL EFFICACY AND SAFETY IN NONHUMANPRIMATE OF A RECOMBINANT VACCINE AGAINST HEPATITIS E

Mabel Tinoco, DVM1, Isabel Gracia, MSc1, Braulio Hernández, Phys. Ant.2, Alejandra Ibañez, Biol2,Jorge Revilla, Dr,3, Aarón Molina, MSc3, Laura Cobos, PhD

1National Autonomous Universitiy of Mexico, Mexico City, Mexico2Proyecto Camina A.C. Mexico3Probiomed S.A. de C.V. Mexico

Infection with hepatitis E virus (EHV) is one of the most common liver infections in developingcountries. It is transmitted through the gastrointestinal tract by ingestion of contaminated water withthe virus. Like hepatitis A, EHV is a short-live entity that can cause liver failure in rare cases. Majorepidemics have been observed in countries in Asia and South America where sanitary measures arenot implemented properly. The development of a recombinant DNA vaccine, produced in Mexico, willprovide a therapeutic option for this disease, increasing in numbers, and in our country isunderestimated. The advantages of this new vaccine are the development of a broad spectrum ofprotective antibodies, and achieving an early and complete immunization as the viral structure iscontained in this recombinant formulation. Based on the above, we decided to perform preclinicalstudies to evaluate the safety and efficacy of a new vaccine modified in Mexico against hepatitis Evirus in healthy rhesus monkeys (Macaca mulatta ) treated with different doses, performinganthropometric studies, ultrasounds, clinical analysis and immunological studies.

18. MODELING COMPLEX SYSTEMS IN LIGHT OF EVOLUTION

Ray Greek, MD

Americans for Medical Advancement, Goleta, California

Animals are routinely used to model humans in order to predict drug efficacy and toxicity. There isimmense empirical evidence calling this practice into question. An oft-overlooked consideration ofusing animals to predict human response is the fact that animals and humans are examples ofcomplex systems that have different evolutionary histories. Complex systems exhibit the propertiesof emergence, nonlinearity, robustness, and modularity. Perhaps the most important characteristicsof complex systems as they pertain to efficacy and toxicity are: 1. complex systems are verydependent upon initial conditions; 2. perturbations to the system have effects that are nonlinear.Large perturbations may result in no change while small perturbations may cause havoc; and 3. thewhole is greater than the sum of the parts. The reason evolution, as manifest by changes in allelefrequency over time as well as new genes and gene functions, must be considered can perhaps bestbe illustrated by the fact of intra -species differences. Personalized medicine is based on the fact thateven individual humans may not respond similarly to the same drug. Inter -species differences shouldbe even more profound and indeed this has been revealed to be the case. To put all this in thecontext of using animals in efficacy and toxicity evaluation, very small differences in the geneticmakeup of two otherwise similar complex systems / species can result in very different responses.The evolution of complex systems should be expected to result in profound differences toperturbations such as drugs.


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19. THE USE OF ANIMAL MODELS TO STUDY ENERGY HOMEOSTASIS AND THERAPIESFOR OBESITY

Hannah C. Greenwood, BSc, Anne McGavigan, BSc, Kylie E. Beale, BSc, Mohammad A. Ghatei,PhD, Stephen R. Bloom, MB, BChir, and Kevin G. Murphy, PhD

Department of Medicine, Imperial College London, London, United Kingdom

Obesity rates are increasing throughout the world, with yearly obesity-related deaths in Americabeing second only to tobacco-related deaths. This has driven great research interest in thephysiological systems that regulate body weight and identifying potential targets for the treatment ofobesity. Animal models are invaluable to indentify the signaling molecules that regulate food intakeand energy expenditure. However, a reduction in food intake following administration of a novelagent does not necessarily reflect the activation of a physiological satiety pathway. It can also resultfrom non-specific toxic or behavioural effects. It is therefore crucial to establish robust protocols toestablish the specificity of an anorectic effect before human studies can commence. Rodent modelsare widely used to study energy homeostasis. However, as rats and mice are unable tocommunicate feelings of discomfort or to vomit, it is critical to use other techniques to determinewhether anorectic agents have non-specific effects. We have studied the effects of specificmicronutrients on food intake in rodent models, and used behavioural analysis, conditioned tasteaversion protocols and neuronal activation studies to assess the specificity of their anorectic effects.

Our studies suggest a combination of animal studies is required to determine whether the effects ofanorectic agent on food intake are likely to reflect a specific or a non-specific effect. Such studiesshould be performed early in the pre-clinical assessment of potential therapies for obesity.

20. THE IMPACT OF THE 5-HT1A-RECEPTOR ON THE MURINE 5-HT-SYNDROME

Robert Haberzettl, Bettina Bert, PhD, Jan Brosda, PhD, Heidrun Fink, PhD

Institute of Pharmacology and Toxicology, Freie Universität Berlin, Berlin, Germany

The incidence of the serotonin (5-HT)-syndrome in humans has increased over the last decade,most likely due to a higher prescription rate of serotonergic drugs. The 5-HT-syndrome can beevoked by serotonergic drugs in high doses. It is characterized by severe autonomic, neuromuscular

and mental symptoms. It is also possible to elicit a 5-HT-syndrome in mice, which is linked to theoccurrence of the Straub tail response. We revealed in male NMRI mice five core responses(hindlimb abduction, low body posture, tremor, piloerection, decrease of rearing) that reliably occurand dose-dependently increase in occurrence and intensity after the treatment with fluoxetine, 5-HTP, and tranylcypromine as well as their combinations. Here, we investigated which signs of the 5-HT-syndrome are mediated by the 5-HT1A-receptor. We administered a full 5-HT1A-receptor agonistand a partial 5-HT1A-receptor agonist at different doses to evaluate the effect of 5-HT1A-receptoractivation on the occurrence of 15 behavioral and physiological responses including bodytemperature. Both agonists produced all five core responses. The full 5-HT1A-agonist induced oneadditional response, the Straub tail, which was not evoked by any other tested serotonergic agonist.In summary, 5-HT1A-receptor activation elicits the core responses of the murine 5-HT-syndrome.However, the Straub tail response was only provoked by the full agonist. Based on the presynaptic5-HT1A-receptor reserve and the higher intrinsic activity of the full agonist, the Straub tail response

seems to be associated to postsynaptic 5-HT1A-receptor activation. Therefore, the Straub tailresponse is not a general parameter for describing the 5-HT-syndrome in mice.


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21. MAGNETIC RESONANCE IMAGING AS A TOOL FOR LONGITUDINAL MONITORING INTHE LACTACYSTIN MODEL OF PARKINSON’S DISEASE AND STUDYING THENEUROPROTECTIVE EFFECTS OF VALPROATE

Ian F. Harrison, BSc (Hons) MRes, David T. Dexter, BSc (Hons), PhD

Imperial College London, London, United Kingdom

Parkinson’s disease (PD) is the second most common neurodegenerative disease, with cardinalclinical symptoms of rigidity, tremor and bradykinesia, resulting from degeneration of thedopaminergic nigrostriatal pathway. Dopaminergic replacement strategies are the primary point oftherapy for PD; however such drugs do not stop the progressing neurodegeneration and are onlyable to provide temporary symptomatic relief. Hence novel neuroprotective agents are sought.Recent evidence has highlighted a pathological imbalance of the epigenetic acetylation apparatus infavor of histone deacetylation, in neurodegenerative conditions such as PD. This imbalance hasbeen shown to induce apoptosis and neurodegeneration following excessive chromatincondensation. Therefore it is theorized that this imbalance can be corrected with histonedeacetylase inhibitors (HDACIs) and the neurodegeneration avoided. The HDACI Valproate (VPA) isthe most commonly prescribed anti-epileptic drug. Recent in vitro evidence has suggested that VPAis neuroprotective towards dopaminergic neurons. Similarly, in vivo , VPA treatment in the Rotenonerat model of PD resulted in a significant reduction in nigrostriatal dopaminergic cell death. We are

currently testing the longitudinal efficacy of VPA in the progressive Lactacystinrat model of PD, bycombining behavioral analysis, and immunohistochemistry of the nigrostriatal pathway, with therelatively novel technique of preclinical structural magnetic resonance imaging (MRI).Lactacystinlesioned animals are treated daily with either VPA or saline, for 28 days, starting 7 daysafter lesioning. By using volumetric and T2 signal intensity analysis the use of MRI provides us withlongitudinal information of the neuropathological progression in the disease model alongsidebehavioral information.

22. DEVELOPMENT OF A LONG-TERM RODENT MODEL OF CRITICAL ILLNESS

Neil Hill1, Kevin Murphy1, Stephen Brett2, Duncan Wilson3, Gary Frost1, Waljit Dhillo1, MohammadGhatei1, Steve Bloom1, Mervyn Singer4

1 Section of Investigative Medicine, Imperial College London, London, United Kingdom2 Centre for Peri-operative Medicine and Critical Care Research, Imperial College Healthcare NHSTrust, London, United Kingdom3 Academic Department of Military Medicine, Royal Centre for Defence Medicine, Birmingham,United Kingdom4 Wolfson Institute for Biomedical Research, University College London, London, United Kingdom

Patients with critical illness develop a catabolic state of negative energy balance resulting in rapidloss of lean body mass, which can take months to recover from. Reducing the period of rehabilitationand the time to return to normal function in this population is challenging. Evidence suggests theorexigenic gastric hormone ghrelin will be useful in recovery and rehabilitation from critical illness.Most animal models of critical illness are acute, severe short-term models used to investigate

mortality and physiological response. Muscle wasting can be induced by denervation of specificmuscle groups but does not reflect whole-body allostatic adaptations to critical illness. Traumamodels focus on specific injury patterns e.g. spinal cord injury, haemorrhage; and are not designedto cause cachexia. Burns models do cause cachexia but the principals of 3R means that they areinfrequently used. Cancer cachexia is an effective model of muscle wasting but does not induce thesame metabolic response as critical illness, thus limiting its applicability. Sepsis models are well-established and include injection of live bacteria into the bloodstream, pneumonia models andadministration of lipopolysaccharide. Intra-peritoneal sepsis is considered the gold standard insepsis research. We have developed a long-term rodent model of critical illness cachexia usinginjection of intra-peritoneal faecal slurry in which acute sepsis is followed by recovery of food intake


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and body mass occur. This will enable the novel investigation of the effects of ghrelin on recovery ofbody mass, food intake, muscle strength, and protein turnover after critical illness.

23. THE MELANOCORTIN RECEPTOR SYSTEM AS AN ANTI-INFLAMMATORYTHERAPEUTIC TARGET FOR STROKE

Paul Holloway1, Stephen Getting2, Felicity N.E. Gavins1

1Wolfson Neuroscience Laboratories, Imperial College London, London, United Kingdom2School of Life Sciences, University of Westminster, London, United Kingdom

A disproportionate inflammatory response has been shown to play a major role in the pathogenesisof a wide variety of disorders and is increasingly being recognised as a significant factor in thepathophysiology of stroke. Therefore targeting the inflammatory response in stroke may dramaticallyincrease the time window for therapeutic intervention, improving functional outcome in thisdebilitating disease. The melanocortin receptor system activates potent neuro-protective and anti-inflammatory circuits and is rapidly becoming acknowledged as an exciting pharmacological targetfor a number of diseases. This project utilises the bilateral common carotid artery occlusion (BCCAo)mouse model of global stroke to evaluate the anti-inflammatory therapeutic potential of themelanocortin receptor system. Intravital microscopy has been employed to quantify the cerebral

inflammatory response through a real time in vivo visualisation of the rolling and adhesion ofleukocytes in the cerebral microcirculation. BCCAo (5 minutes ischemia, 40 minutes reperfusion)induced a considerable increase in leukocyte rolling and adhesion. Treatment with the pan receptoragonist, α-MSH, dramatically reduced ischemia reperfusion induced leukocyte rolling (69%) andadhesion (81%). Combination treatment with α-MSH and SHU9119 (MC3 /MC4 antagonist) failed toabrogate the anti-inflammatory effects of α-MSH, suggesting anti-inflammatory circuits independentof MC3 and MC4. These preliminary results suggest an important role for MC1 in mediating theprotective anti-inflammatory effects of the melanocortins following BCCAo.

24. PDE INHIBITORS: A TREATMENT FOR MOTHERS AND BABIES AT RISK OFPRETERM DELIVERY?

Laura Howe, BSc (Hons), Johann Malawana, MBBS, Bronwen Herbert, PhD, Mark Johnson, MBBS

PhD MRCP MRCOG

Institute of Reproductive and Developmental Biology, Faculty of Medicine, Imperial College London,United Kingdom

Premature delivery is the most important problem in obstetrics causing over 70% of neonatal deathand handicap. Although some treatments reduce the risk of preterm labour (PTL), none have beenshown to improve neonatal outcomes. Cyclic adenosine monophosphate (cAMP) has been shown todown-regulate the oxytocin receptor and is also known to be an immunomodulator, inhibiting bothNFkB-driven transcription and mitogen activated kinase (MAPK) activation, suggesting that cAMPagonists may be ideal agents for the prevention of PTL.

Some data support the role of Phosphodiesterase 4 (PDE) inhibitors in the management of PTL, but

the role of PDE3 has not been assessed. PDE3 is abundant in embryonic neuroepithelium, andPDE3 inhibitors have been found to have neuroprotective effects in cultured neurons. Weadministered 10g of LPS into the right horn of the uterus, at laparotomy, performed on E16 ofgestation in CD1 outbred mice. 2 hours prior to this we administered 2mg/kg of Milrinone (PDE3inhibitor) or control via intraperitoneal injection. The Milrinone group of mice showed no delay indelivery time compared with the control group, but an improvement in pup survival (=0.02).

These data suggest that PDE3 inhibition may have a neuroprotective effect in our model of PTL.Further studies will establish the mechanisms involved and define whether a combined approach ofPDE3 and 4 inhibition is a better combination for neuroprotection and PTL prevention than eitheralone.


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25. THE RABBIT IN ABDOMINAL ADHESION DRUG DEVELOPMENT

Milinda Kowalik, BS, SRT1, Dave MacGeorge, BS1, Mayra Fernandez, BA1, Christina Blaney, BS,MS1 Robert Resnick, BS2, Christopher VanDeusen, PhD2 and Brian Karolewski, VMD, PhD,DACLAM 1

1 Disposition Safety and Animal Research, Sanofi US, Bridgewater, New Jersey 2

Fibrosis and Wound Repair, Sanofi US, Bridgewater New Jersey

The development of discovery and preclinical animal models of postoperative abdominal adhesionsare a critical component of the drug discovery process. Abdominal adhesions are bands of fibroustissue that form abnormal attachments between abdominal structures and are considered to be amajor source of post-operative morbidity and mortality. Abdominal adhesions can lead to chronicpelvic pain, bowel obstruction and infertility, resulting in significant health care costs. The rabbit is acommonly used pharmacology model for studying surgical adhesions in the abdomen, andantiadhesive treatments. One commonly used surgical model is the sidewall and cecal abrasionprocedure. This model is used for profiling barrier and non-barrier antiadhesive agents, studyingadhesions in the formation and reformation mode, and investigating adhesion formation withminimally invasive surgical approaches (example: laparoscopy). This animal model producesconsistent abdominal adhesions to the intestines and is easily modified to study adhesions to the

reproductive organs. The rabbit is a critical pharmacology model for advancing antiadhesivetreatments.


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PPRREESSEENNTTIINNGG AAUUTTHHOORR

PPOOSSTTEERR SSEESSSSIIOONN 22 FFRRIIDDAAYY,, SSEEPPTTEEMMBBEERR 1166,, 22001111

11::1155PPMM – – 22::4455PPMM

PS2 - 1 Deborah Iglesias-BregnaPS2 - 2 Nicholas S. Kirkby

PS2 - 3 Holger Kissel

PS2 - 4 Johann Malawana

PS2 - 5 Johann Malawana

PS2 - 6 Megan MacBride

PS2 - 7 Anne McGavigan

PS2 - 8 Jennifer Moffat

PS2 - 9 Manasi Nandi

PS2 - 10 Christina Nesarajah

PS2 - 11 Egberanmwen Ode

PS2 - 12 Vilma Petrikaite

PS2 - 13 Stephen Previs

PS2 - 14 Marisol Rivera

PS2 - 15 Emma S. J. Robinson

PS2 - 16 Avi Rosenstrauch

PS2 - 17 Sara Tobias Savage

PS2 - 18 Nico ScheerPS2 - 19 Emily S. Sena

PS2 - 20 Anna Starr

PS2 - 21 Paritosh Suman

PS2 - 22 Mabel Tinoco

PS2 - 23 Eric M. Walters

PS2 - 24 Edward Weinstein


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1. TRANSCRANIAL MAGNETIC STIMULATION IS A USEFUL METHOD TO ASSESSMOTOR FUNCTION IN THE DA-RAT MODEL OF EAE

Deborah Iglesias-Bregna1, MS, Zhongqi Ji1, MS, Margaret Petty1, PhD, Li Liu2, PhD, DonghuiZhang2, PhD, Kathleen McMonagle-Strucko1, MBA, and Susan Hanak1, PhD

1Immuno-inflammation Therapeutic Strategy Unit, Sanofi-aventis, Bridgewater, New Jersey2Department of Statistics, Sanofi-aventis, Bridgewater, New Jersey

Transcranial magnetic stimulation (TMS) is a technique that uses head-mounted wire coils that sendstrong but short magnetic pulses directly into specific brain regions, thus safely and painlesslyinducing eddy currents which cause brain cells to fire. TMS is currently being evaluated in humansand in several animal models for the study of various diseases, including depression, epilepsy,chronic pain, Parkinson’s dystonia, spinal cord injury and Multiple Sclerosis (MS). In MS patients,TMS-motor evoked potentials (tcMMEPs) have longer conduction times and significantly reducedamplitudes. Teriflunomide, an oral immunomodulatory compound currently in Phase III studies forthe treatment of MS, has been reported to delay onset and progression of experimental autoimmuneencephalomyelitis (EAE) in the Dark Agouti (DA) rat model of MS and electrophysiologically, hasdemonstrated preservation of the somatosensory pathway. In this study, functional assessment ofthe descending motor spinal tracts was determined using tcMMEPs in the presence of therapeutictreatment of teriflunomide. During acute attack, remission, and relapse-remitting phase of EAE, there

was a significant increase in the latency of tcMMEPs in EAE vehicle-treated animals compared tosham animals and to EAE-teriflunomide-treated animals. This methodology provides a functionalassessment of the nervous system that translates well between the human condition and animalmodels.

2. EVALUATION OF ASPIRIN-TRIGGERED 15-EPI-LIPOXIN A4 AS A BIOMARKER OFCOX2 EXPRESSION IN VIVO

Nicholas S. Kirkby, PhD1,2, Martina H. Lundberg, BSc1, William Edmands3, PhD, Timothy D. Warner,PhD2, Jane A. Mitchell, PhD1

1National Heart & Lung Institute, Imperial College London, London, United Kingdom2William Harvey Research Institute, Barts & the London School of Medicine, London, United

Kingdom3Department of Surgery & Cancer, Imperial College London, London, United Kingdom

The inducible cyclo-oxygenase isoform, COX2, has been implicated in the pathogenesis of severaldiseases but the benefits of COX2 inhibition are limited by cardiovascular toxicity. COX2 ordinarilyconverts arachidonic acid to prostaglandin H2, but when acetylated by aspirin, conversion is divertedto 15-R-HETE. This undergoes further metabolism by leucocyte 15-lipoxygenases to form 15-epi-lipoxin A4. We investigated whether the aspirin-triggered formation of 15-epi-lipoxin A4 might providea novel biomarker for COX2 expression in vivo . Methods: WT, COX1-/- and COX2-/- mice weretreated with LPS (10mg/kg) to induce COX2, or vehicle. After 4 hours, they received aspirin(10mg/kg) or vehicle, and were killed 30 mins later. Plasma 15-epi-lipoxin A4 concentration wasdetermined by ELISA. Vascular COX2 expression was determined by en face aorticimmunohistochemistry. COX activity was determined as TXB2 formation in A23187-stimulated blood,

and PGE2 formation in lung homogenates. Results: Aortic COX2 immunoreactivity was weak inuntreated mice and significantly increased after LPS administration. TXB2 formation in whole bloodand PGE2 production by lung homogenates were strongly inhibited by aspirin administration. Plasmalevels of 15-epi-lipoxin A4, however, were not altered by any combination of LPS and aspirin, in anygenotype of mice. Similar results were obtained when the aspirin dose was increased 10-fold.Conclusions: Plasma levels of 15-epi-lipoxin A4 were not detectably altered by aspirinadministration in vivo , despite confirmed vascular COX2 expression and inhibition of prostanoidsynthesis by aspirin. As such, these data suggest that aspirin-triggered 15-epi-lipoxin A4 cannot beused as a biomarker of COX2 expression in vivo .


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3. INNOVATIVE TOOLS TO EVALUATE GENETIC TARGETS AND THE EFFICACY OFNOVEL CANCER THERAPEUTICS

Yan Xu1, Marina Tugusheva1, Jost Seibler1, Holger Kissel1, Jos Jonkers2, David Grass1 and OlesiaBuiakova1

1Taconic Farms Inc., Hudson, New York2Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands

More predictive small animal models for compound and genetic target assessment are needed toimprove development of more efficacious drugs. For assessment of novel cancer therapeutics wehave developed an in vivo and ex vivo bioluminescent imaging platform to evaluate compoundefficacy. In these mouse models orthotopic tumor grafts are being used that are more relevant withrespect to host-tumor interactions, display metastatic potential and allow the evaluation of responsesto novel therapeutics. These models closely mimic the progression of human disease and allowquantitative analysis and high-throughput in vivo assessment of potential anti-tumor activity. Inaddition we will present an oncology platform using mouse models for breast cancer. The Brca1/p53breast cancer model resembles hormone receptor- and ERBB2-negative (‘‘triple-negative’’)mammary carcinomas and shows responses to therapies similar to humans, including theemergence of drug resistance. We will present an orthotopic allograft system using primary tumorsfrom Brca1/p53-deficient mice to allow the generation of cohorts developing breast cancer. These

grafted mice can readily be used for testing of novel therapeutics in a tumor model resemblinghuman disease. Finally, we will present a transgenic RNAi system that can be used for studying theloss-of-function of target genes. We will present how this system can be used to develop noveldisease models and how potentially this model can be combined with disease models to study thegenetic loss of a drug target in an established disease state.

4. IMMUNOMODULATION IN PRE TERM LABOUR: AN ANSWER TO IN UTERONEUROLOGICAL INSULT

Johann Malawana, L. Howe, B. Herbert, R. Hua, P.R. Bennett, and M.R. Johnson,

Institute of Reproductive and Developmental Biology, Imperial College London, United Kingdom

Rates of preterm delivery are increasing across the developed world and range between 8-13%.Overall, preterm delivery is the most important cause of perinatal morbidity and mortality; with mostproblems occurring in babies born before 32 weeks. In this group, preterm labour (PTL) is mostcommonly caused by infection/inflammation, prompting the search for agents that can modulate thematernal inflammatory response, thereby reducing rates of PTL. We use a proven mouse model ofinflammatory PTL based on the administration of lipopolysaccharide (LPS), a major cell-wallcomponent of gram-negative bacteria, which activates the innate immune system via the toll-likereceptor type 4. In this study, we used Rolipram, a phospodiesterase (PDE) 4 inhibitor to modulatethe maternal immune system in an attempt to improve rates of pup survival and prolong pregnancy.We administered 10ug of LPS into the right horn of the uterus, at laparotomy, performed on E16 ofgestation in CD1 outbred mice. 2 hours prior to this we administered 2mg/kg of Rolipram or DMSOvehicle diluted in PBS (control) via intraperitoneal injection. The Rolipram group of mice showed asignificant delay in delivery time compared with the control group, with a p value of 0.0253. However,

pup survival was not significantly improved. These data suggest that inhibition of PDE4 may prolongpregnancy, but that it does not appear to improve fetal outcomes. Further studies will define themechanisms by which rolipram delays parturition and define whether this approach might beclinically useful.


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5. PDE INHIBITORS: A TREATMENT FOR MOTHERS AND BABIES AT RISK OFPRETERM DELIVERY?

Laura Howe, BSc (Hons), Johann Malawana, MBBS, Bronwen Herbert, PhD, Mark Johnson, MBBS,PhD, MRCP, MRCOG

Institute of Reproductive and Developmental Biology, Faculty of Medicine, Imperial College London,United Kingdom

Premature delivery is the most important problem in obstetrics causing over 70% of neonatal deathand handicap. Although some treatments reduce the risk of preterm labour (PTL), none have beenshown to improve neonatal outcomes. Cyclic adenosine monophosphate (cAMP) has been shown todown-regulate the oxytocin receptor and is also known to be an immunomodulator, inhibiting bothNFkB-driven transcription and mitogen activated kinase (MAPK) activation, suggesting that cAMPagonists may be ideal agents for the prevention of PTL. Some data support the role ofPhosphodiesterase 4 (PDE) inhibitors in the management of PTL, but the role of PDE3 has not beenassessed. PDE3 is abundant in embryonic neuroepithelium, and PDE3 inhibitors have been found tohave neuroprotective effects in cultured neurons. We administered 10µg of LPS into the right horn ofthe uterus, at laparotomy, performed on E16 of gestation in CD1 outbred mice. 2 hours prior to thiswe administered 2mg/kg of Milrinone (PDE3 inhibitor) or control via intraperitoneal injection. TheMilrinone group of mice showed no delay in delivery time compared with the control group, but animprovement in pup survival (=0.02). These data suggest that PDE3 inhibition may have aneuroprotective effect in our model of PTL. Further studies will establish the mechanisms involvedand define whether a combined approach of PDE3 and 4 inhibition is a better combination forneuroprotection and PTL prevention than either alone.

6. THE CIEA NOG MOUSE®: A SUPER IMMUNODEFICIENT MOUSE FOR HUMANIZATIONAND CANCER RESEARCH

Megan MacBride1, Holger Kissel1, Hirsohi Suemizu2 and Mamoru Ito2

1Taconic Farms Inc., Hudson, New York2Central Institute for Experimental Animals, Kawasaki, Kanagawa, Japan

“Humanized mice“, in which human cells and tissues are engrafted and partially function, havebecome important tools for the study of human disease and direct testing of therapeutic agents. Theestablishment of “humanized mice” is largely attributed to the development of severeimmunodeficient mice. Recently, a new immunodeficient mouse was developed, the CIEA NOG ® mouse (NOG), by introducing the Il2rg- null gene into NOD scid mice. These mice contributed to thegeneration of “humanized mice”, i.e. mice with a human immune system, due to extremely highengraftment rates and differentiation of human hematopoietic stem cells exceeding those in otherimmunodeficient mice, e.g. NOD scid mice. The advantages of the super immunodeficient CIEANOG® mouse also allow the use of these mice in various fields of cancer research including theidentification of cancer stem cells and the factors responsible for metastasis. Numeroushematological and solid tumor cell lines have been engrafted successfully in the CIEA NOG ® mouseusing considerably fewer human cells than in other models. In some cases, as few as 10 cells haveresulted in successful engraftment. Tumor models include adult T-cell leukemia/lymphoma (ATL),multiple myeloma, Hodgkin’s disease as well as metastasis models for pancreatic, colon andmelanoma cell lines. Data on the engraftment of the human immune system in these mice andgrafting rates of various cancer cell lines will be presented.


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7. THE USE OF IN VIVO MODELS TO INVESTIGATE THE ROLE OF GASTROINTESTINALNUTRIENT-SENSING SYSTEMS IN APPETITE REGULATION

Anne McGavigan, MSc, Hannah C. Greenwood, BSc, Stephen R. Bloom, MA, MD, DSc, FRCPath,FRCP, Kevin G. Murphy, PhD

Section of Investigative Medicine, Imperial College London, London, United Kingdom

Recent research has highlighted the importance of gastrointestinal nutrient-sensing in the regulationof food intake and metabolism. The gut uses hormonal and neural signaling mechanisms tocommunicate the acute nutritional state to the central nervous system. Determining the nutrientscapable of inducing satiation and prolonging satiety, and elucidating the mechanisms by which theymediate their effects on appetite may help identify novel treatments for obesity. Receptor systemsthat respond to particular nutrients have been characterized and localized to the gut, and in vitro studies have identified specific nutrients that stimulate anorectic gut hormone release. However, inviv o models are crucial to determine the utility of such nutrient-sensing systems as drug targets.Functional physiological responses to the ingestion of nutrients can only be studied using in vivo models. It is relatively simple to manipulate diet in rodent models to determine the effect of specificmacronutrients. Rodent models are particularly relevant as their gut-brain signaling pathways aresimilar to those of humans. In rodents, nutrients can be administered via different routes, includingdirectly into regions of the gastrointestinal tract, and the effects on gut hormone release and food

intake determined. The role of vagal nerve signaling in any anorectic effects can be identified usingvagotomised models and the importance of particular receptors in sensing nutrients can bedetermined by the use of knock-out mouse models. These in vivo model systems have the potentialto aid the discovery of new drug targets for the treatment of obesity.

8. HUMANIZED MICE AND IN VIVO BIOLUMINESCENCE IMAGING: TOOLS FOR TESTINGANTIVIRAL DRUGS FOR VARICELLA ZOSTER VIRUS

Jennifer Moffat, PhD, Chandrav De, Nancy Fiore, and Dongmei Liu, MS

SUNY Upstate Medical University, Syracuse, New York

SCID mice implanted with human tissues are used to study varicella-zoster virus (VZV)

pathogenesis because it is restricted to humans. VZV causes skin lesions during episodes ofchicken pox and shingles. Recently, SCID/ beige mice implanted subcutaneously with full-thicknesshuman fetal skin and a recombinant virus, VZV-BAC-Luc that constitutively expresses fireflyluciferase, were developed as a model system for evaluating the efficacy of antiviral drugs.Engrafted human skin tissues are inoculated with virus by direct scarification, and then the mice aretreated with antiviral compounds. VZV replication is measured repeatedly in the same mouse by invivo bioluminescence imaging using a Caliper/Xenogen IVIS-200 instrument to detect photonsemitted by luciferase activity in VZV-infected cells, which are proportional to VZV plaques in culture.Antiviral efficacy is determined by comparing the VZV growth rates in treated mice compared to micethat receive vehicle. Tests of acyclovir and valacyclovir, approved VZV therapies, found that theywere less effective than several experimental antiviral compounds, even at high doses given by oralgavage twice per day at 120 mg/kg/day. The L conformation bromovinyl uracil derivatives, L-BHDUand valyl-L-BHDU, were both effective at low doses. L-BHDU was given once per day by

subcutaneous injection at 8 mg/kg in DMSO. Valyl-L-BHDU was give once per day by oral gavageat 30 mg/kg in 0.4% carboxymethylcellulose. Other compounds are currently being tested in thismodel with equally encouraging results. We are the first to offer a standardized system for pre-clinical in vivo evaluation of compounds with potential anti-VZV activity.


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9. GENETIC AND PHARMACOLOGICAL MANIPULATION OF DDAH1 IN ENDOTOXICSHOCK: A NOVEL THERAPEUTIC TARGET

Manasi Nandi, PhD1, Zhen Wang2 and James Leiper2

1Pharmacology and Therapeutics Group, Institute of Pharmaceutical Science, King’s CollegeLondon, London, United Kingdom.2MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, London, United

Kingdom

Sepsis is a leading cause of death in intensive care patients worldwide, occurring when the body elicitsan overwhelming inflammatory response to a microbial infection. Initial hypotension can quickly escalateto serious circulatory collapse (septic shock) with ensuing multiple organ failure and death. Nitric oxide(NO) is produced in excess from inducible nitric oxide synthase (iNOS) as part of the inflammatoryresponse. In the cardiovascular system, this excess NO contributes to circulatory collapse. Asymmetricdimethylarginine (ADMA) is an endogenously produced competitive inhibitor of NOS. ADMA ismetabolized by the enzyme dimethylarginine dimethylaminohydrolase (DDAH) of which there are 2isoforms, with distinct tissue distributions (DDAH1 and 2). We hypothesized that reducing DDAH1activity, would elevate endogenous ADMA and hence inhibit NOS activity in a tissue-specificmanner, thereby providing a novel pathway to treat hypotension in septic shock. The cardiovasculareffects of reduced DDAH activity were investigated using a heterozygous DDAH1-knockout mouse

(DDAH1+/- ) and a novel DDAH1 inhibitor, N G-(2-Methoxyethyl)-L-arginine, in rodent models ofendotoxic shock [using lipopolysaccharide (LPS)]. LPS was administered intravenously and allanimals presented with circulatory shock. Intervention with a DDAH inhibitor at 3mg/kg attenuatedthe observed hypotension with a concomitant improvement in the extent of acidosis developedcompared to vehicle treated animals. DDAH1+/- mice similarly displayed attenuation in the rate ofdeveloped hypotension and furthermore demonstrated enhanced responses to adrenergicvasopressor challenge. Both pharmacological and genetic reduction of DDAH1 activity is protectiveagainst the vascular changes observed during endotoxic shock.

10. ANALYSIS OF SYNAPTIC REARRANGEMENTS FOLLOWING LASER-MEDIATEDMICROLESIONS IN THE ADULT BRAIN

Alison Canty, PhD, Lieven Huang, PhD, Johanna Jackson, PhD, Christina Nesarajah, BSc, andVincenzo De Paola, PhD

Neuroplasticity and Disease Group, MRC Clinical Science Centre, Imperial College, London, UnitedKingdom

Axonal damage is a hallmark of many neurological disorders including neurodegeneration and injury.However, the factors that trigger axonal degeneration and repair are not fully understood. Westudied axonal degeneration and associated synaptic responses caused by focal lesions in thecerebral cortex using in vivo two-photon imaging through a cranial window. Excitatory axons werevisualised using transgenic mice expressing green fluorescent protein (GFP) in a subset of neuronsand were cut using a pulsed high-energy femtosecond laser (800 nm). Damaged axons weretracked to assess the fragmentation kinetics of the disconnected part and the synapticreorganisation of the surviving part. The surviving part of terminauxbouton (TB)-rich axons showedan increase in synaptic turnover due to TB losses one day post-lesion (n=13 axons, 9 mice; p


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11. EVALUATION OF NON-APOPTOTIC CELL DEATHS IN DEVELOPING MOUSEEMBRYOS VIA LYSOTRACKER RED

Egberanmwen Ode, BS, and Brent R. Stockwell

Howard Hughes Medical Institute, Department of Biological Sciences, Department of Chemistry,Columbia University, New York, New York

The proper development of mammals depends on a series of well-orchestrated cell deathprocesses. The deregulation of these processes can result in embryonic lethality and sometimes indiseases such as cancer. Traditionally, cell death has been classified as occurring throughprogrammed apoptosis or non-programmed necrosis. However, recent investigations suggest theexistence of programmed non-apoptotic cell death morphologies, such as autophagy, necroptosis,mitotic catastrophe, and others. These discoveries suggest that cell death events in mouse embryospreviously classified as apoptotic may need to be re-evaluated. Here, we explore whether there aredevelopmental cell death events distinct from apoptosis in the developing mouse embryo. Wedeveloped an assay using the dye Lysotracker Red to study cell death in whole mounted embryos.Lysotracker Red is a weakly basic amine that selectively accumulates in cellular compartments suchas lysosomes. Lysosomes, which are organelles that degrade cellular proteins, are crucial to celldeath processes. Though they have been implicated in all three of the morphologically distinct cell

death pathways, their involvement remains unclear, motivating this study. We found distinctoccurrences of cell death in the dorsal root ganglion of developing mouse embryos on gestationdays 10 and 11. To confirm that Lysotracker red does detect dying cells, we induced death inembryos via different methods, such as intra-peritoneal injection of lethal compounds, and in vitro incubation of embryos in lethal compound solutions. Further analyses via caspase 3 antibodies, awell characterized biomarker for apoptosis were done to confirm that this mode of cell death isindeed non-apoptotic.

12. DEVELOPMENT OF HSP90 INHIBITORS AS ANTICANCER COMPOUNDS

Vilma Petrikaite, PhD, Egidijus Kazlauskas, Jurgita Matuliene, PhD, Daumantas Matulis, PhD

Vilnius University Institute of Biotechnology, Vilnius, Lithuania

Heat-shock protein 90 (Hsp90) is responsible for ATP-depended folding, stability and functioning ofmany “client” proteins. Hsp90 is a promising anticancer drug target, as cancerous cells are moresusceptible to Hsp90 inhibition than normal cells. A group of Hsp90 inhibitors was synthesized at ourdepartment. Compound binding to Hsp90 was measured by isothermal titration calorimetry and thethermal shift assay. The most potent compounds bound to Hsp90 with the dissociation constant ofabout 1 nM. The compounds were efficient inhibitors of HeLa and osteosarcoma cell line growth.Main ADMET properties have been estimated in silico . The tested compounds followed the Lipinskirules of 5, the acute toxicity was predicted to be moderate, and the bioavailability per os of mostcompounds was predicted to be more than 30%. Moreover, all compounds were predicted not to beextensively metabolized in the body. Toxicity was determined in mice by method of fixed doses.Preliminary data of toxicological experiments in mice showed that these compounds are supposed tobe moderately toxic (MDL is about 400 mg/kg). Obtained in vivo results were very similar to the

predicted parameters. Tumour xenograft experiments are being planned. The compounds may beuseful for further development and clinical applications.


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13. LIPID TRAFFICKING: MOVING FROM MOUSE TO MONKEY TO MAN

Stephen Previs1, Douglas Johns1, Ablatt Mahsut1, Kithsiri Herath1, Gowri Bhat1, Paul Miller1,Haihong Zhou1, Jose Castro-Perez1, Vinit Shah1, Dave McLaren2, Sheng-Ping Wang1, Karim Azer3,Alison Kulick4, Keiana Dunn4, Christopher Johnson4, Thomas Roddy2 and Brian Hubbard1

1Cardiovascular Disease-Atherosclerosis, Merck Research Laboratories, Rahway, New Jersey2In Vivo Pharmacology-Pharmacoanalytics, Merck Research Laboratories, Rahway, New Jersey3

Modeling and Simulation, Merck Research Laboratories, Rahway, New Jersey

4 Laboratory Animal Resources, Merck Research Laboratories, Rahway, New Jersey

Lipid homeostasis is controlled by (i) dietary intake, (ii) de novo synthesis, (iii) tissue efflux and (iv)elimination. In addition, when considering lipoprotein metabolism it is often necessary to differentiatelipid and protein kinetics. Although rodents and non-human primates are widely used as preclinicalmodels to study the pathophysiological basis of dyslipidemia, including pharmacological efficacy,there is a shortage of methods for determining the contribution of the respective pathways. Wedemonstrate the ability to dissect lipid flux using stable isotope tracers in rodents and non-humanprimates, e.g. How can I determine the contribution of newly made lipid to the circulating pool? If Ichange fatty acid synthesis will I impact lipoprotein production? The methods that we havedeveloped/implemented are well suited for studying free-living animals (i.e. tracers are given via asingle oral and/or intravenous bolus) therein making it possible to perform studies in numerous

locations with a minimum of technical expertise. In addition, the methods are suitable for use inhumans which ensures translatability. The initial phases of our platform development consideredfundamental questions, e.g. what type and how much tracer do I dose? When should I collectsamples? What is the "best" way to measure endpoints? The later phases of our platformdevelopment considered physiologically oriented questions, e.g. how does metabolic flux changewith a dietary and/or pharmacological intervention? Our data suggest that the methods are suitablefor routine investigations in support of drug discovery programs aimed at modulating dyslipidemia;specifically, we focused on "high-throughput" analytical approaches (which predominantly rely onmass spectrometry to quantify the abundance and/or the isotopic labeling of various metabolites). Intotal, our approach is primarily directed towards decision-making as related to drug efficacy. Theutilization of tracer kinetics in preclinical models can help predict target engagement and validation;since the methods utilize stable isotopes and require blood sampling it is possible to bridge thegap(s) to clinical investigations.

14. ASSESSMENT OF ACUTE TOXICITY IN RODENTS OF AGAVE INULIN

Marisol Rivera, MVZ1, Ma. Isabel Gracia, M en C.1, Manuel Cruz, Ing.2, Mabel Tinoco, MVZ1,Francisco Sánchez, M. en C.1

1Unidad de Experimentación Animal, UNAM, Mexico2 Nekutli S.A. de C.V., Mexico

Dietary fiber is an essential part of diets, contributing to the proper functioning of the gut; therecommended fiber intake for adults according to different countries, is in the range of 25 to 35grams per day. Fructans are specific dietary fibers which provide energy and have demonstratedprebiotic activity. Fructans are polymers of fructose where one or more fructosylfructose linkageconstitutes a majority of the linkages. Metlin ® and Metlos ® are purified organic fructans extractedfrom Blue Agave. The aim of the study was to assess the acute toxicity and genotoxicity of twodietary fibers, Metlin ® and Metlos ® . For genotoxicity and acute toxicity study, 6 week-old male andfemale ICR (Hsd:ICR) mice were used. The objective was to identify the dose causing adverseeffects and to determine the average oral lethal dose in mice ICR (Hsd:ICR) and rats Wistar(Hsd:Wi). The results shown that no death at any doses, so it can be concluded that these productsare not toxic, at the administrated doses.


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15. A NEW ANIMAL MODEL TO STUDY DEPRESSION AND ANTIDEPRESSANT EFFICACY

S.A. Stuart1, P. Butler, PhD2, D.J. Nutt, PhD 3, Emma S.J. Robinson, PhD1

1School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom2Global Safety Pharmacology, Pfizer Global Research and Development, Sandwich, United Kingdom3Neuropsychopharmacology Unit, Division of Experimental Medicine, Imperial College, London,United Kingdom

Negative biases in cognition and emotional processing are increasingly accepted as having a centralrole in the causation of depression, and to be an important target for antidepressant drugs. It hasbeen hypothesised that negatively valenced emotional processing and its effects on memory mayunderlie the development and persistence of mood disorders and that antidepressants work byremediating these biases. We have developed a novel assay for rats to facilitate basic research intohow affective state influences the memory for positive experiences. The assay uses a bowl-diggingtask where rats form two independent memories for a substrate-reward association under control ortreatment conditions. Memory bias is then assayed using a subsequent choice test. Induction ofnegative affective state in rats induced a negative memory bias for reward-associated experiences.In contrast, treatment with antidepressants had the opposite effect, inducing a positive recall bias forrewarding experiences. These data suggest that memories formed during negative affective stateshave reduced value, which biases responding away from the associated cue. Antidepressants

induced a bias towards the associated cue suggesting acute antidepressant treatment enhances thevalue of the memory. We hypothesise that memories formed during negative affective states havereduced positive affective valence, which leads to the anhedonia and negative mood observed inaffective disorders, an effect which can be reversed by antidepressant treatment. These data alsosupport the predictive validity of this new rodent assay.

16. ROOSTERS AS A MODEL FOR AGING: DECREASED SENSITIVITY OF TESTICULARLEYDIG CELLS TO HYPOPHYSIAL GONADOTROPHINS

Avi Rosenstrauch, PhDAchva Academic College, M.P. Shikmim, Israel

Leydig cells respond to hypophysial gonadotrophins stimulation. I examined this response in aging

roosters by cross incubation of Leydig cells from high fertility (32 weeks) and low fertility (70 weeks),exposed to gonadotrophins media from adeno-hypophysis of high and low fertile roosters,preincubated with GnRH. Exposing gonadotrophins from high fertility adeno-hypophysis show (a)high fertility Leydig cells showed 360% increment testosterone level release after 30 min incubation.(b) low fertility Leydig cells reached this peak of testosterone only after 60 min of incubation. Incontrast, exposing gonadotrophins from low fertility adeno-hypophysis show (a) high fertility Leydigshowed an increase of 210% in testosterone release after 30 min incubation. (b) low fertility Leydigcells reach this peak of testosterone only after 120 min of incubation. Then, aging Leydig cells showdecreased sensitivity of response to hypophysial gonadotrophins.


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17. AMELIORATION OF SIDE EFFECTS OF CHEMOTHERAPEUTICS IN ONCOLOGYMODELS IMPROVES ANIMAL HEALTH AND SURVIVAL TIME

Sara Tobias Savage, DVM, David Lee-Parritz, DVM, DACLAMGenzyme Corporation, a Sanofi Company, Framingham, Massachusetts

Neuropathy, pain, nausea, cachexia, dehydration and depression are commonly recognizedcomplications of cancer treatment in humans. Animal models of cancer may experience similar side

effects from novel or standard treatments intended to treat cancer, and the survival rate of animalson therapeutic efficacy studies may be significantly decreased as a result. Oncology studies oftenrely upon survival time to determine efficacy of novel treatments. Duration of survival time from theprimary disease cannot be determined accurately if animal models die of dehydration, malnutrition orother secondary complications of treatment. Additionally, IACUC compliance depends uponadherence to previously determined measures of animal welfare. Side effects of cancer treatmentsmay complicate interpretation of clinical signs or moribund status due to the primary diseaseprocess, cause premature death or euthanasia of animals and require the use of larger experimentalcohorts to achieve statistical significance. Methods used to support these animals are not routinelyreported on in the scientific literature. Consistent application of nursing and supportive caretechniques currently used in veterinary and human clinical oncology may significantly improveconsistency and animal welfare in experimental cancer treatment models. These measures includesupplemental nutrition, fluid therapy, administration of analgesics, antiemetics, antidepressants, andsimple husbandry or housing modifications. Investigators are justifiably concerned with the potentialfor confounding factors to adversely impact studies. Inclusion of proper experimental controls,standardization of supportive techniques across all treatment groups and full characterization ofdisease models will permit evaluation of proposed new supportive practices on established models,and may allow animals to remain on study that would be otherwise excluded. These methods bothimprove animal welfare and permit more accurate modeling of the human disease for which noveltreatments are being investigated.

18. GENERATION OF HUMANIZED MOUSE MODELS AND THEIR USE IN DRUG-DRUGINTERACTION, PHARMACOKINETIC AND SAFETY TESTING OF COMPOUNDS

Nico Scheer, PhD1, Martin R. Wilson1, Maki Hasegawa2, Yury Kapelyukh3, Harunobu Tahara2 and C.Roland Wolf3

1TaconicArtemis GmbH, Köln, Germany2Kyowa Hakko Kirin Co. Ltd., Sunto-gun Shizuoka, Japan3CXR Biosciences Ltd., Dundee, United Kingdom

More than 80% of drugs on the market are metabolized by Cytochrome P450 (CYP) 3A4, CYP2D6or CYP2C9. The extrapolation of preclinical in vivo results to humans is often difficult due to speciesdifferences in regulation of expression and substrate specificity of CYPs. For example, significantdifferences in the ligand-receptor specificity, which regulates CYP3A4 expression, exist betweenmouse and man. To overcome these difficulties, we have generated a unique portfolio of humanizedand knockout mouse models for key proteins involved in drug metabolism and disposition toinvestigate the pharmacokinetic (PK) and drug-drug interaction (DDI) profile of both known and novelcompounds. In particular, a new multiple humanized model has been generated, which combinesthe humanization of the two Xenoreceptors that regulate CYP3A4 expression, namely Pregnane XReceptor (PXR) and Constitutive Androstane Receptor (CAR), along with replacement of the mouseCYP3a cluster with a human region carrying both CYP3A4 and 3A7. This humanized model wasfound to rank different PXR activators according to their potency of inducing CYP3A4 in humans.Furthermore, the model was able to quantitatively predict PXR/CYP3A4-mediated DDI in the clinic.These data, as well as characterization studies on novel humanized mouse lines for CYP2D6 andCYP2C9, will be presented. We expect that these models will be useful tools to better predict the PKand DDI of target compounds in humans, therefore reducing: (i) total animal numbers used; (ii)potential risk of new compounds and (iii) time to market of new drugs.


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19. LOW STUDY QUALITY AND PUBLICATION BIAS OF EXPERIMENTAL STUDIESOFNEUROLOGICAL DISEASES RESULT IN OVERSTATEMENTS OF REPORTEDEFFICACY

Emily S. Sena, PhD, Hanna M. Vesteri

Documents

Animal Models Abstract Booklet 20110915