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ANIMAL MODELS AND THEIR VALUE IN

PREDICTING DRUG EFFICACY AND TOXICITY


SEPTEMBER 15 16, 2011
ABSTRACTS FOR POSTER SESSIONS
Scientific Organizing Committee
Maria Gabriela Belvisi, BSc, PhD
Imperial College London

Garret A. FitzGerald, MD
University 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, FBPharmacolS


Imperial College London

Ray Hill, PhD, DSc, (Hon), FMedSci


Imperial College London

Sandra J. Engle, PhD


Pfizer Inc.

Kerstin Hofmeyer, PhD


The New York Academy of Sciences
Simon Howell, PhD
King's College London

Poster Sessions 1 and 2 are Generously Sponsored By

TRAVEL FELLOWSHIP AWARDEES


Travel Fellowships have been awarded on a competitive basis to support the attendance and
participation as a poster presenter of the following junior investigators:
Elizabeth J. Cartwright, PhD
University of Manchester

Hannah C. Greenwood, BSc


Imperial College London

Ian F. Harrison, BSc (Hons), MRes


Imperial College London

Neil Hill, BMBS


Imperial College London

Paul Holloway
Imperial College London

Christina Nesarajah, BSc


Imperial College London

Emily S. Sena, PhD


University of Edinburgh

Anna Starr, PhD


Kings College London

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

Aisah A. Aubdool, BSc (Hons) Pharmacology, MRes


Kings College London

Jennifer V. Bodkin, BSc, MRes


Kings College London

Paul Holloway
Imperial College London

Emma S.J. Robinson, BSc(Hons), PhD


University of Bristol

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

POSTER PRESENTATION PRIZE


A certificate and prize of US$250.00 will each be awarded for the two best posters presented at the
conference. Posters will be judged on the basis of scientific merit by members of the Scientific
Organizing Committee. Poster prizes will be awarded during Joint Session VIII on the afternoon
of Friday, September 16, 2011.
Poster Presentation Prizes were made by possible by the generous support of the:

PRESENTING AUTHOR
POSTER SESSION 1
THURSDAY, SEPTEMBER 15, 2011
6:30PM 8:00PM
PS1 - 1

Anjali A. Amin

PS1 - 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 Greek

PS1 - 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

A LIST OF PRESENTERS FOR POSTER SESSION 2 IS AVAILABLE ON


PAGE 17 OF THIS BOOKLET.

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 double
between 2005 and 2030, driving the need for urgent and effective treatments in the management of
this condition. Treatments for type 2 diabetes remain suboptimal, with marked side-effects, and
treatment for the majority of patients with type 1 diabetes remains life-long subcutaneous insulin
therapy. The complex interplay of genetic and environmental factors which influence the nature of
diabetes make it difficult to use in vitro models to investigate the specific molecular mechanisms
responsible. In vivo models allow the investigation of these mechanisms under physiological and
path physiological conditions. Animal models of diabetes can be generated spontaneously, or
induced by chemicals such as streptozotocin, by dietary or surgical manipulations, or by a
combination of these techniques. The development of transgenic models of diabetes has allowed
further insight into the molecular mechanisms involving autoimmunity against -cells. The
development of new technologies such as transcriptomics, proteomics and metabolomics will aid the
identification 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 and
underlying aetiological mechanisms which cause diabetes. The use of rodent models is thus
essential 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
1

Cardiovascular Division, School of Medicine, Kings College London, United Kingdom


Centre of Integrative Biomedicine, Kings College London, United Kingdom
3
Wolfson CARD Centre, Kings College London, United Kingdom
2

Hypertension is associated with secondary conditions that include peripheral vascular and micro
vascular disease. Recent studies suggest that transient receptor potential ankyrin 1 (TRPA1) may
potentially regulate blood vessel tone, as activation of TRPA1 in the peripheral vasculature leads to
the release of the neuropeptide CGRP. It is well established that CGRP possesses potent
vasodilator and cardiovascular properties. We investigated the role of TRPA1 in cardiovascular
haemodynamics and peripheral blood flow in vivo using the Angiotensin IIinduced experimental
model of hypertension in transgenic mice. The use of subcutaneous osmotic mini-pumps to
chronically deliver Angiotensin-II potentially reduced animal stress by eliminating chronic repeated
daily manual infusions. Using the non-surgical tail-cuff method, 5 different blood pressure
parameters; systolic, diastolic and mean blood pressure, tail blood volume and flow were assessed
at baseline and several time-points following infusion of Angiotensin-II or saline (control) in each
restrained, conscious mouse. Heart rate, perfusion index and oxygen saturation were also monitored
non-invasively using the MouseSTAT pulse oximeter. Furthermore, peripheral blood flow was
recorded 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-Field
Laser Perfusion Imager. At Day 14, plasma and multiple tissues such as ears, brain, aorta, heart and
paws were collected for microarray and qRT-PCR experiments to map genes for hypertension. This
study design allowed each individual mouse to be serially used to assess multiple parameters over 2
weeks to study cardiovascular pathophysiology and hence, reduced the total number of animals
required for the study.

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


FIBROSIS
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, Geoff
Varty1, Rachel Yabkowitz1
1
2

Fibrosis and Wound Repair Therapeutic Strategy Unit, Sanofi U.S., Bridgewater, New Jersey
Molecular Innovative Therapeutics, Sanofi U.S., Bridgewater, New Jersey

Idiopathic pulmonary fibrosis (IPF) is a debilitating disease with limited treatment options. Prognosis
is poor, and patients have a median survival time of less than five years. The pathology of IPF is
likely driven by repeated damage to the airway epithelium that leads to dysregulated repair
mechanisms, fibroblastic foci in which myofibroblasts deposit extracellular matrix components such
as collagen and a progressive decline in lung function. Ideally, an animal model of IPF should allow
the effects of potential new therapies to be assessed within a reasonably short timeframe, and
should predict clinical efficacy. Bleomycin is often used to induce lung fibrosis in mice, and to profile
potential new therapies. However, the clinical predictability of this model has been questioned. Our
goal has been to establish a protocol and methods that will maximize the clinical predictability of this
model. We demonstrate that bleomycin causes deposition of collagen in lungs that can be
quantified by histopathology or assays of tissue homogenates, increased expression of the
myofibroblast marker alpha smooth muscle actin in lungs, increased serum titers of non-invasive
putative IPF biomarkers, impaired oxygen saturation of blood, and changes in lung mechanics that
model those associated with restrictive changes in IPF.
4. HUMANIZED NOG MOUSE: A NOVEL MODEL FOR STUDYING MYCOBACTERIUM
TUBERCULOSIS PATHOGENESIS AND HOST-IMMUNE RESPONSE AGAINST
INFECTION
Angelo Izzo1, Gerold Feuer2, 3 and Prabal Banerjee, PhD3
1

Department of Microbiology, Immunology & Pathology, Colorado State University, Colorado


Department of Microbiology and Immunology, SUNY Upstate Medical University, Syracuse, New
York
3
HuMurine Technologies, Inc., La Verne, California
2

Humanized mice are a recent breakthrough with the potential to circumvent the implicit obstacles of
conventional animal models. The technology used to develop and generate humanized mice has
improved significantly in recent years due to the development of the humanized NOD/SCID IL-2rgcnull
(NOG) mice that develop a complete lineage of human cells of the innate and adaptive immune
system including monocytes/macrophages, plasmacytoid and myeloid DC, NK cells and T and B
lymphocytes, post reconstitution with human hematopoietic stem cells. We have characterized and
standardized the parameters of humanization and the long-term multilineage hematopoiesis in NOG
mice thereby leading to the development of a humanized mouse model that supports a robust and
complete pattern of human hematopoiesis. These humanized mice provide a unique opportunity to
gain insight into pathogenesis of human-specific pathogens including intracellular pathogenic
bacterial 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 of
pulmonary granulomas when these mice are infected with a low dose aerosol infection of virulent
Mycobacterium tuberculosis H37Rv. Specifically, after infection, both human CD4+ and CD8+ T cells
expand in response to infection and produce IFN-, TNF- and IL-2, such that these cytokines are
significantly elevated by day 34 PI. In addition, engrafted mice produce lesions in the lungs that
resemble granulomas, suggesting that these mice may provide a suitable model to investigate the
interaction between the human host cells and the pathogen in an in vivo environment.

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


AND OBESITY
Katherine Banks, BSc, Kylie E. Beale, PhD, Stephen R. Bloom, MA, MD, DSc, FRCPath, FRCP and
Kevin 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 the
ventral tegmental area (VTA) signal to the nucleus accumbens and pre-frontal cortex to activate
reward pathways and associate food intake with pleasure. The VTA is heavily implicated in nonhomeostatic eating; the continued ingestion of food when already satiated. The ready availability of
convenient high-fat and high-sugar foods in the Western diet may trigger non-homeostatic eating
due to their high reward value and thus contribute to the current obesity pandemic. Hypothalamic
neuronal 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. In
vivo models can be used to investigate the neuronal circuitry involved in the pleasure-reward
system. Hormones such as leptin, released from adipose tissue, and ghrelin, released from the
stomach, signal to the VTA to influence food intake. Our current work focuses on how such
circulating signals modulate neural feeding impulses sent to dopaminergic VTA neurons, and how
they differentially regulate reward pathways depending on nutritional state. Altered indices of reward
in genetic and environmental models of obesity can also aid our understanding of the role of the
mesocorticolimbic system in food intake. The use of in vivo models to investigate reward and food
intake may identify new therapeutic targets for the treatment of obesity.
6. AN INTEGRATED METABOLOMICS STUDY OF GENTAMICIN-INDUCED
NEPHROTOXICITY
Richard Beger, PhD1, Laura Schnackenberg, PhD1, Jinchun Sun, PhD1, Sudeepa Bhattacharyya,
PhD1, Yosuke Ando, PhD1,2
1

National Center for Toxicological Research, US FDA, Jefferson, Arkansas


Daiichi Sankyo Co., Ltd., Tokyo, Japan

Gentamicin is an aminoglycoside antibiotic used in the treatment of bacterial infections. However, in


10-25% of patients, there is an increase in blood urea nitrogen and a reduction in glomerular
filtration rate. In this study, LC/MS- and NMR-based global metabolomics analyses of urine were
employed to discover metabolite biomarkers of kidney injury and recovery from toxic insult. Male
Sprague 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 on
day 0. Four animals from each group were sacrificed on days 1, 2, 3, 7, 10, 15, 18, 22, 29, 36, and
44. 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. Several
metabolites including amino acids and hydroxyproline were closely associated with the
histopathology during the injury and recovery periods. Glucosuria was noted prior to the increase in
BUN 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 sulfate
that may be indicators of renal adaptive response prior to gentamicin-induced injury. Biomarkers
related to the efficacy of gentamicin were also detected. Specifically, gut-microflora-related
compounds including hippurate, indole derivatives, and phenyl derivatives were decreased on days
2-10. An integrated LC/MS and NMR-based metabolomics approach identified potential biomarkers
of gentamicin-induced renal injury and efficacy.

7. APPLYING THE 3RS TO MURINE CARDIOVASCULAR PHENOTYPING


Jennifer V. Bodkin BSc, MRes, Aisah A. Aubdool, and Susan D. Brain
Kings College London, Cardiovascular Division, School of Medicine, London, United Kingdom
Transient Receptor Potential Ankyrin 1 (TRPA1) channels are membrane expressed, non-selective
cation channels, activated by noxious compounds. These include exogenous agonists such as
mustard oil, cinnamaldehyde and components of air pollution. In vivo, TRPA1 channels are
suggested to be activated by reactive oxygen species (ROS) and products of oxidative stress. A
growing number of publications have demonstrated TRPA1 agonists to mediate vasodilation
responses. We were the first to demonstrate the role of TRPA1 in this response by using TRPA1
KO mice. As peripheral blood vessel tone is an important component of peripheral resistance and
blood pressure control, we have gone on to conduct a novel cardiovascular characterization study
using TRPA1 wild-type (WT) and -KO mice. To do this we have adopted 3Rs strategies, refining our
techniques to maximize data output and reducing animal numbers. Our mice are profiled at baseline
by telemetry, showing conscious blood pressures, heart rate and activity levels. Their heart
morphology and function is also assessed non-invasively by echocardiography. They then undergo
experimental hypertension induction using subcutaneously implanted osmotic minipump infusion of
angiotensin II. This additional pathological stress is designed to exacerbate the effect of removing
TRPA1-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 then
collected and analyzed using a variety of methods, measuring remodeling and inflammatory
markers.
8. IMATINIB-RESISTANCE AND MICROCYTIC ERYTHROCYTOSIS IN A MOUSE MODEL
OF GASTROINTESTINAL STROMAL TUMOR (GIST)
Benedikt Bosbach1, Shayu Deshpande1, Ferdinand Rossi1, Cristina Antonescu2, Peter Besmer1
1

Developmental Biology Program, Sloan-Kettering Institute, New York, New York


Department 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 the
tyrosine kinase inhibitor imatinib tumor clones frequently grow out with second-site KIT mutations
that are thought to disrupt binding of the inhibitor. We had previously generated a mouse model of
GIST by introducing the KitV558 mutation found in a case of familial GIST into the Kit gene. Now, to
investigate the consequences of second-site KIT mutations on imatinib-susceptibility and GIST
development, we generated a mouse model introducing into the endogenous Kit locus
simultaneously the V558 and the kinase gatekeeper mutation T669I (human T670I) found in
imatinib-resistant GIST. Invariably, these KitV558;T669I/+ mice developed pronounced interstitial cell of
Cajal (ICC) hyperplasia in the stomach and colon, and cecal GIST. Treatment of single-mutant
KitV558/+ mice with imatinib, dasatinib, sunitinib or sorafenib significantly reduced GIST signal
transduction and cell proliferation. In contrast, treatment of double-mutant mice with imatinib or
dasatinib did not inhibit GIST growth. However, the resistance mediated by the gatekeeper mutation
could be overcome by treatment with sunitinib and sorafenib.
Interestingly, the KitV558;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 to
overcome gatekeeper-mediated imatinib-resistance in GIST and in the investigation of the
consequences of different levels of oncogenic KIT signaling in diverse KIT-dependent cell lineages.

9. MARKERS OF OXIDATIVE STRESS AND APOPTOSIS FOR PRECLINICAL


EVALUATION OF HEMOGLOBIN-BASED OXYGEN THERAPEUTICS IN
NONASCORBATE-PRODUCING SPECIES
Omer I. Butt, PhD, Paul W. Buehler, PhD, Felice DAgnillo, PhD
Division of Hematology, CBER/FDA, Bethesda, Maryland
Hemoglobin-induced oxidative stress and apoptosis may contribute to some of the unresolved
toxicities of hemoglobin-based therapeutics. Guinea pigs may be a useful species for examining
hemoglobin 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, we
examined sensitive and specific biomarkers of oxidative stress and apoptosis. Using a 50%
exchange transfusion model with polymerized bovine hemoglobin in guinea pigs, we analyzed
NRF2, a potent marker of oxidative stress; 4-hydroxynonenal (4-HNE)-modified protein adducts, an
index of lipid peroxidation; 8-hydroxy-2-deoxyguanosine (8-OHdG), a marker of oxidative DNA
damage; and cleaved caspase 3, a marker of apoptosis. Immunohistochemical and western blot
analyses revealed translocation of NRF2 from the cytoplasm to the nucleus. There was also
enhanced accumulation of 4-HNEmodified protein adducts, along with increased nuclear 8-OHdG
immunoreactivity in renal proximal tubules and glomeruli. Cleaved caspase 3 was detectable by
western blot in the kidneys but was not associated with significant increases in serum creatinine or
other common histopathological indices. Cleaved caspase 3 levels were also elevated in hearts and
lungs. Clinical trial results with some hemoglobin-based therapeutics have led to suggestions that
preclinical testing may not have been sufficiently predictive of safety in humans. The present findings
suggest that sensitive and specific markers of oxidative stress and apoptosis in a guinea pig animal
model may be useful in preclinical studies designed to evaluate the safety of these products.
10. HIGHLY-SENSITIVE IN VIVO PHOTONIC IMAGING OF APOPTOTIC CELLS BY
UTILIZATION 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 visualization
and analysis in live mice. This method is based on a caspase-3/7 activatable substrate (caged
luciferin, Z-DEVD-Aminoluciferin) and two luciferase-expressing transgenic models: 1) Taconic
ubiquitous luciferase mouse model (-actin-luc), which can be used for any mouse organ apoptosis
imaging, 2) Sanofis myelin basic protein-luciferase mouse model, which can be used for mouse
brain and intestine apoptosis imaging. The method has been used to detect cadmium-induced liver
apoptosis and radiation-induced intestinal apoptosis in live mice. Our data demonstrated that the
luminescent apoptosis probe was highly sensitive in vivo to detect apoptosis that was induced by
ionizing radiation and cadmium. The apoptosis imaging window is consistent with
immunohistochemical data in the literature. Our results indicated that this apoptosis imaging method
could be used to study apoptosis-related mouse disease mechanisms and to profile compounds in
vivo.

11. TREATMENT OF CARDIAC HYPERTROPHY WITH A NOVEL PHARMACOLOGICAL


INHIBITOR OF PLASMA MEMBRANE CALCIUM ATPASE 4 (PMCA4) MIMICS THE
PHENOTYPE 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 5
million people in the USA currently affected by this disease it is essential that we increase our
understanding of its mechanistic basis to develop effective treatment strategies. Pathological
hypertrophy, resulting from high blood pressure, myocardial infarction and aortic stenosis, is a prerequisite 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 pharmacological
inhibitor 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 was
identified, 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 and
in vivo AP2 significantly prevented the response to hypertrophic stimuli. Importantly AP2 was able to
effectively reverse established hypertrophy, which is a more realistic clinical scenario. Whole body
phenotyping 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 and
effective target for its treatment.
12. THE FUTURE OF ANIMALS IN RESEARCH PROJECT: AN INNOVATIVE APPROACH TO
BUILDING NEW RESEARCH STRATEGIES AT GLAXOSMITHKLINE
D. Kemp Covington, DVM1, Sarah Hawthorne, BEng (Hons) MIChemE2, Julie Huxley-Jones, PhD3,
Ghislaine Poirier, PhD, DVM3
1

GlaxoSmithKline, Research Triangle Park, North Carolina


GlaxoSmithKline, Brentford, United Kingdom
3
GlaxoSmithKline, Stevenage, United Kingdom
2

Animal research represents a small but vital role in the process of discovering medicines. Due to
limitations 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 to
delivering the best science while reducing reliance on animals, GlaxoSmithKline recently chartered a
global project to investigate the way we approach and conduct animal research, specifically in drug
efficacy models. The project, The Future of Animals in Research, was commissioned by GSKs
Head of Research and Development to challenge current processes and drive novel thinking in
company 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 objectives
included evaluating the companys application of the 3Rs (replacement, refinement, reduction),
improving internal and external communications regarding animal research, determining where
investments in innovative solutions make sense for the practice of best science, and partnering with
public stakeholders in the animal welfare community to examine how animals contribute to drug
discovery. The project has identified key opportunities to optimize efficacy-based animal research at
GSK, in a traditional 3Rs approach, and through enhanced consideration of animal model
relevance, robustness, and reproducibility. We will present an overview of how effective

communication and engagement, scientific peer review, and maximizing the value of data can
positively impact animal research.
13. ANTI- INFLAMMATORY AND CYTOTOXIC ACTIVITY OF THE PLANT CANNABIS
SATIVA (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
1

Department of Microbiology, Faculty of Pure and Applied Sciences, International University of


Africa, Khartoum, Sudan
2
Department of Biochemistry, Nutrition, Toxicology and Pharmacology Central of Veterinary
Research laboratory, Khartoum, Sudan
3
National Ribat University, Khartoum, Sudan
4
Department of Medicine, Pharmacology and Toxicology, University of Khartoum, Sudan
In this study, the plant Cannabis sativa seeds petroleum oil extract was investigated for antiinflammatory activity and explore the toxicity on albino rats. All extracts showed no significant
cytotoxic activity on the Vero cell line. The inflammation was firstly obtained by using carrageenan
suspension 0.1 ml of 10% saline injected at the sub plantar region of the left limb for inducing a
local acute oedema. A decrease in oedema size was reported after 24 hours for the rats pretreated
with 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.), compared
to Indomethacin (standard anti-inflammatory drug), which reported a decrease in oedema size
diameter to 0.55mm, which indicated an increase inhibition percentages were reported for the
different pretreated groups 0.00, 87.03, 79.56 and 87.91 including the comparative Indomethacin
treated 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 maximum
concentration 1 ml/day of C. sativa oil extract in comparison to the standard drug. Hence, such
results recommend the prospectice focus for the preventive medical use of the extract.
14. ELUCIDATION OF THE ROLE OF ENDOGENOUS NO AND ENDOTHELIAL NO
SYNTHASE IN REGULATING PLATELET FUNCTION IN VIVO
Christopher Moore, PhD, Michael Emerson, PhD
Platelet Biology Group, National Heart and Lung Institute, Imperial College London, London, United
Kingdom
Nitric oxide (NO) regulates vessel tone and platelet function in vitro. The role of endogenous NO and
the eNOS (endothelial nitric oxide synthase) isoform in regulating platelets in vivo remains unclear
since conflicting reports have been published concerning the thrombotic phenotype of eNOS-/- mice
and models for assessing platelet function in vivo in the mouse have not been available. We
developed and employed a mouse model for measuring platelet aggregation in vivo as the
accumulation of radiolabelled platelets in the pulmonary vasculature via external scintillation probes.
Collagen- and thrombin-induced in vivo platelet aggregation were enhanced by NOS inhibitors. In
contrast, 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 by
Western blotting. The duration of platelet aggregation in eNOS-/- mice was potentiated following
moderate thrombotic stimulation. NOS inhibition enhanced platelet aggregation in wild-type mice but
had no effect on aggregation responses in eNOS-/- mice. In addition, iNOS and nNOS inhibitors were
ineffective in both WT and eNOS-/- mice. Endogenous NO therefore negatively regulates platelets in
vivo through a direct action on platelets and not a secondary vascular effect. The source of bioactive
NO in vivo is exclusively eNOS, with a negligible contribution by other NOS isoforms. Platelets are
regulated 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 potential
therapeutic target for platelet-driven disorders.
10

15. MOUSE AND HUMANIZED MOUSE MODELS FOR EVALUATING THROMBOTIC


PLATELET 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, United
Kingdom
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 therefore
developed methods for measuring platelet aggregation in situ in the mouse. Platelets were isolated
from donor mice following cardiac puncture or human volunteers following venepuncture,
radiolabelled with 111Indium Oxine and infused into anaesthetized C57Bl/6 or platelet depleted NODscid recipients respectively. Circulating radiolabelled platelets were monitored via external 1cm
diameter scintillation probes over the pulmonary region. The platelet agonists ADP, thrombin and
collagen induced platelet aggregation responses, detected as rapid and transient increases in
radioactive counts as platelets aggregated and became trapped in the pulmonary region. Counts
then returned to baseline in a variable time frame. The peak responses and area under curve
measurements indicated dose-dependency. There were no detectable changes in counts upon
injection 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 in
lungs from mice exposed to platelet agonists. Administration of aspirin significantly reduced platelet
aggregation in response to collagen. We therefore present a novel method for evaluating human and
mouse platelet aggregation responses in vivo which may find applications in the identification of
novel anti-thrombotic targets as well as in drug evaluation and safety profiling.
16. SHROB RAT MODEL OF METABOLIC SYNDROME REVEALS UNEXPECTED BENEFITS
AND OFF-TARGET EFFECTS FOR MULTIPLE AGENTS
Paul Ernsberger, PhD and Richard J. Koletsky
Department 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 human
metabolic syndrome. We hypothesized that antidiabetic and antihypertensive agents may have a
spectrum of therapeutic activity on components of metabolic syndrome. Antidiabetic agents: All
agents tested normalized glucose tolerance. PPAR-gamma agents also corrected
hypertriglyceridemia, and ameliorated steatohepatitis and hypertension while increasing
subcutaneous fat. Glyburide did not affect body fat but worsened left ventricular hypertrophy.
Sitagliptin normalized elevated glucagon levels and reduced visceral fat without affecting overall
adiposity. Antihypertensive agents: All agents tested normalized blood pressure. Hydralazine and
alpha-methyldopa worsened glucose tolerance and insulin resistance, whereas both were improved
by another sympathoinhibitor, moxonidine. Captopril worsened glucose tolerance but lowered
triglycerides, LDL cholesterol and proteinuria. Allylmercaptocaptoril, with an allyl moiety from garlic,
improved glucose tolerance. Relative to captopril, it induced greater reductions in blood pressure
and proteinuria. Both agents reduced fatty acid turnover in isolated abdominal adipocytes. Another
garlic compound, diallyl sulfide, was effective in lowering blood glucose during an oxidative stress
challenge. Thus, preclinical drug trials in the SHROB model parallel those in humans and suggest
unexpected benefits of new agents and possible risks of older agents. The SHROB model may
identify useful agents for treating metabolic syndrome.

11

17. EVALUATION OF THE PRECLINICAL EFFICACY AND SAFETY IN NONHUMAN


PRIMATE OF A RECOMBINANT VACCINE AGAINST HEPATITIS E
Mabel Tinoco, DVM1, Isabel Gracia, MSc1, Braulio Hernndez, Phys. Ant.2, Alejandra Ibaez, Biol2,
Jorge Revilla, Dr,3, Aarn Molina, MSc3, Laura Cobos, PhD
1

National Autonomous Universitiy of Mexico, Mexico City, Mexico


Proyecto Camina A.C. Mexico
3
Probiomed S.A. de C.V. Mexico
2

Infection with hepatitis E virus (EHV) is one of the most common liver infections in developing
countries. It is transmitted through the gastrointestinal tract by ingestion of contaminated water with
the virus. Like hepatitis A, EHV is a short-live entity that can cause liver failure in rare cases. Major
epidemics have been observed in countries in Asia and South America where sanitary measures are
not implemented properly. The development of a recombinant DNA vaccine, produced in Mexico, will
provide a therapeutic option for this disease, increasing in numbers, and in our country is
underestimated. The advantages of this new vaccine are the development of a broad spectrum of
protective antibodies, and achieving an early and complete immunization as the viral structure is
contained in this recombinant formulation. Based on the above, we decided to perform preclinical
studies to evaluate the safety and efficacy of a new vaccine modified in Mexico against hepatitis E
virus in healthy rhesus monkeys (Macaca mulatta) treated with different doses, performing
anthropometric 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 is
immense empirical evidence calling this practice into question. An oft-overlooked consideration of
using animals to predict human response is the fact that animals and humans are examples of
complex systems that have different evolutionary histories. Complex systems exhibit the properties
of emergence, nonlinearity, robustness, and modularity. Perhaps the most important characteristics
of complex systems as they pertain to efficacy and toxicity are: 1. complex systems are very
dependent 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. the
whole is greater than the sum of the parts. The reason evolution, as manifest by changes in allele
frequency over time as well as new genes and gene functions, must be considered can perhaps best
be illustrated by the fact of intra-species differences. Personalized medicine is based on the fact that
even individual humans may not respond similarly to the same drug. Inter-species differences should
be even more profound and indeed this has been revealed to be the case. To put all this in the
context of using animals in efficacy and toxicity evaluation, very small differences in the genetic
makeup 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 to
perturbations such as drugs.

12

19. THE USE OF ANIMAL MODELS TO STUDY ENERGY HOMEOSTASIS AND THERAPIES
FOR 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 America
being second only to tobacco-related deaths. This has driven great research interest in the
physiological systems that regulate body weight and identifying potential targets for the treatment of
obesity. Animal models are invaluable to indentify the signaling molecules that regulate food intake
and energy expenditure. However, a reduction in food intake following administration of a novel
agent does not necessarily reflect the activation of a physiological satiety pathway. It can also result
from non-specific toxic or behavioural effects. It is therefore crucial to establish robust protocols to
establish the specificity of an anorectic effect before human studies can commence. Rodent models
are widely used to study energy homeostasis. However, as rats and mice are unable to
communicate feelings of discomfort or to vomit, it is critical to use other techniques to determine
whether anorectic agents have non-specific effects. We have studied the effects of specific
micronutrients on food intake in rodent models, and used behavioural analysis, conditioned taste
aversion 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 of
anorectic agent on food intake are likely to reflect a specific or a non-specific effect. Such studies
should 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 Universitt 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 be
evoked 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 the
occurrence 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 occur
and dose-dependently increase in occurrence and intensity after the treatment with fluoxetine, 5HTP, and tranylcypromine as well as their combinations. Here, we investigated which signs of the 5HT-syndrome are mediated by the 5-HT1A-receptor. We administered a full 5-HT1A-receptor agonist
and a partial 5-HT1A-receptor agonist at different doses to evaluate the effect of 5-HT1A-receptor
activation on the occurrence of 15 behavioral and physiological responses including body
temperature. Both agonists produced all five core responses. The full 5-HT1A-agonist induced one
additional 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 presynaptic
5-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 tail
response is not a general parameter for describing the 5-HT-syndrome in mice.

13

21. MAGNETIC RESONANCE IMAGING AS A TOOL FOR LONGITUDINAL MONITORING IN


THE LACTACYSTIN MODEL OF PARKINSONS DISEASE AND STUDYING THE
NEUROPROTECTIVE EFFECTS OF VALPROATE
Ian F. Harrison, BSc (Hons) MRes, David T. Dexter, BSc (Hons), PhD
Imperial College London, London, United Kingdom
Parkinsons disease (PD) is the second most common neurodegenerative disease, with cardinal
clinical symptoms of rigidity, tremor and bradykinesia, resulting from degeneration of the
dopaminergic nigrostriatal pathway. Dopaminergic replacement strategies are the primary point of
therapy for PD; however such drugs do not stop the progressing neurodegeneration and are only
able to provide temporary symptomatic relief. Hence novel neuroprotective agents are sought.
Recent evidence has highlighted a pathological imbalance of the epigenetic acetylation apparatus in
favor of histone deacetylation, in neurodegenerative conditions such as PD. This imbalance has
been shown to induce apoptosis and neurodegeneration following excessive chromatin
condensation. Therefore it is theorized that this imbalance can be corrected with histone
deacetylase inhibitors (HDACIs) and the neurodegeneration avoided. The HDACI Valproate (VPA) is
the most commonly prescribed anti-epileptic drug. Recent in vitro evidence has suggested that VPA
is neuroprotective towards dopaminergic neurons. Similarly, in vivo, VPA treatment in the Rotenone
rat 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, by
combining behavioral analysis, and immunohistochemistry of the nigrostriatal pathway, with the
relatively novel technique of preclinical structural magnetic resonance imaging (MRI).
Lactacystinlesioned animals are treated daily with either VPA or saline, for 28 days, starting 7 days
after lesioning. By using volumetric and T2 signal intensity analysis the use of MRI provides us with
longitudinal information of the neuropathological progression in the disease model alongside
behavioral information.
22. DEVELOPMENT OF A LONG-TERM RODENT MODEL OF CRITICAL ILLNESS
Neil Hill1, Kevin Murphy1, Stephen Brett2, Duncan Wilson3, Gary Frost1, Waljit Dhillo1, Mohammad
Ghatei1, Steve Bloom1, Mervyn Singer4
1

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


Centre for Peri-operative Medicine and Critical Care Research, Imperial College Healthcare NHS
Trust, London, United Kingdom
3
Academic Department of Military Medicine, Royal Centre for Defence Medicine, Birmingham,
United Kingdom
4
Wolfson Institute for Biomedical Research, University College London, London, United Kingdom
2

Patients with critical illness develop a catabolic state of negative energy balance resulting in rapid
loss of lean body mass, which can take months to recover from. Reducing the period of rehabilitation
and the time to return to normal function in this population is challenging. Evidence suggests the
orexigenic 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 specific
muscle groups but does not reflect whole-body allostatic adaptations to critical illness. Trauma
models focus on specific injury patterns e.g. spinal cord injury, haemorrhage; and are not designed
to cause cachexia. Burns models do cause cachexia but the principals of 3R means that they are
infrequently used. Cancer cachexia is an effective model of muscle wasting but does not induce the
same metabolic response as critical illness, thus limiting its applicability. Sepsis models are wellestablished and include injection of live bacteria into the bloodstream, pneumonia models and
administration of lipopolysaccharide. Intra-peritoneal sepsis is considered the gold standard in
sepsis research. We have developed a long-term rodent model of critical illness cachexia using
injection of intra-peritoneal faecal slurry in which acute sepsis is followed by recovery of food intake
14

and body mass occur. This will enable the novel investigation of the effects of ghrelin on recovery of
body mass, food intake, muscle strength, and protein turnover after critical illness.
23. THE MELANOCORTIN RECEPTOR SYSTEM AS AN ANTI-INFLAMMATORY
THERAPEUTIC TARGET FOR STROKE
Paul Holloway1, Stephen Getting2, Felicity N.E. Gavins1
1

Wolfson Neuroscience Laboratories, Imperial College London, London, United Kingdom


School of Life Sciences, University of Westminster, London, United Kingdom

A disproportionate inflammatory response has been shown to play a major role in the pathogenesis
of a wide variety of disorders and is increasingly being recognised as a significant factor in the
pathophysiology of stroke. Therefore targeting the inflammatory response in stroke may dramatically
increase the time window for therapeutic intervention, improving functional outcome in this
debilitating disease. The melanocortin receptor system activates potent neuro-protective and antiinflammatory circuits and is rapidly becoming acknowledged as an exciting pharmacological target
for 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 the
melanocortin 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 of
leukocytes in the cerebral microcirculation. BCCAo (5 minutes ischemia, 40 minutes reperfusion)
induced a considerable increase in leukocyte rolling and adhesion. Treatment with the pan receptor
agonist, -MSH, dramatically reduced ischemia reperfusion induced leukocyte rolling (69%) and
adhesion (81%). Combination treatment with -MSH and SHU9119 (MC3/MC4 antagonist) failed to
abrogate the anti-inflammatory effects of -MSH, suggesting anti-inflammatory circuits independent
of MC3 and MC4. These preliminary results suggest an important role for MC1 in mediating the
protective anti-inflammatory effects of the melanocortins following BCCAo.
24. PDE INHIBITORS: A TREATMENT FOR MOTHERS AND BABIES AT RISK OF
PRETERM 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 death
and handicap. Although some treatments reduce the risk of preterm labour (PTL), none have been
shown to improve neonatal outcomes. Cyclic adenosine monophosphate (cAMP) has been shown to
down-regulate the oxytocin receptor and is also known to be an immunomodulator, inhibiting both
NFkB-driven transcription and mitogen activated kinase (MAPK) activation, suggesting that cAMP
agonists 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, and
PDE3 inhibitors have been found to have neuroprotective effects in cultured neurons. We
administered 10 g of LPS into the right horn of the uterus, at laparotomy, performed on E16 of
gestation in CD1 outbred mice. 2 hours prior to this we administered 2mg/kg of Milrinone (PDE3
inhibitor) or control via intraperitoneal injection. The Milrinone group of mice showed no delay in
delivery 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 of
PDE3 and 4 inhibition is a better combination for neuroprotection and PTL prevention than either
alone.
15

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
2

Disposition Safety and Animal Research, Sanofi US, Bridgewater, New Jersey
Fibrosis and Wound Repair, Sanofi US, Bridgewater New Jersey

The development of discovery and preclinical animal models of postoperative abdominal adhesions
are a critical component of the drug discovery process. Abdominal adhesions are bands of fibrous
tissue that form abnormal attachments between abdominal structures and are considered to be a
major source of post-operative morbidity and mortality. Abdominal adhesions can lead to chronic
pelvic pain, bowel obstruction and infertility, resulting in significant health care costs. The rabbit is a
commonly used pharmacology model for studying surgical adhesions in the abdomen, and
antiadhesive treatments. One commonly used surgical model is the sidewall and cecal abrasion
procedure. This model is used for profiling barrier and non-barrier antiadhesive agents, studying
adhesions in the formation and reformation mode, and investigating adhesion formation with
minimally invasive surgical approaches (example: laparoscopy). This animal model produces
consistent 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 antiadhesive
treatments.

16

PRESENTING AUTHOR
POSTER SESSION 2
FRIDAY, SEPTEMBER 16, 2011
1:15PM 2:45PM
PS2 - 1

Deborah Iglesias-Bregna

PS2 - 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 Scheer

PS2 - 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

17

1. TRANSCRANIAL MAGNETIC STIMULATION IS A USEFUL METHOD TO ASSESS


MOTOR FUNCTION IN THE DA-RAT MODEL OF EAE
Deborah Iglesias-Bregna1, MS, Zhongqi Ji1, MS, Margaret Petty1, PhD, Li Liu2, PhD, Donghui
Zhang2, PhD, Kathleen McMonagle-Strucko1, MBA, and Susan Hanak1, PhD
1

Immuno-inflammation Therapeutic Strategy Unit, Sanofi-aventis, Bridgewater, New Jersey


Department of Statistics, Sanofi-aventis, Bridgewater, New Jersey

Transcranial magnetic stimulation (TMS) is a technique that uses head-mounted wire coils that send
strong but short magnetic pulses directly into specific brain regions, thus safely and painlessly
inducing eddy currents which cause brain cells to fire. TMS is currently being evaluated in humans
and in several animal models for the study of various diseases, including depression, epilepsy,
chronic pain, Parkinsons dystonia, spinal cord injury and Multiple Sclerosis (MS). In MS patients,
TMS-motor evoked potentials (tcMMEPs) have longer conduction times and significantly reduced
amplitudes. Teriflunomide, an oral immunomodulatory compound currently in Phase III studies for
the treatment of MS, has been reported to delay onset and progression of experimental autoimmune
encephalomyelitis (EAE) in the Dark Agouti (DA) rat model of MS and electrophysiologically, has
demonstrated preservation of the somatosensory pathway. In this study, functional assessment of
the descending motor spinal tracts was determined using tcMMEPs in the presence of therapeutic
treatment 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 to
sham animals and to EAE-teriflunomide-treated animals. This methodology provides a functional
assessment of the nervous system that translates well between the human condition and animal
models.
2. EVALUATION OF ASPIRIN-TRIGGERED 15-EPI-LIPOXIN A4 AS A BIOMARKER OF
COX2 EXPRESSION IN VIVO
Nicholas S. Kirkby, PhD1,2, Martina H. Lundberg, BSc1, William Edmands3, PhD, Timothy D. Warner,
PhD2, Jane A. Mitchell, PhD1
1

National Heart & Lung Institute, Imperial College London, London, United Kingdom
William Harvey Research Institute, Barts & the London School of Medicine, London, United
Kingdom
3
Department of Surgery & Cancer, Imperial College London, London, United Kingdom
2

The inducible cyclo-oxygenase isoform, COX2, has been implicated in the pathogenesis of several
diseases but the benefits of COX2 inhibition are limited by cardiovascular toxicity. COX2 ordinarily
converts arachidonic acid to prostaglandin H2, but when acetylated by aspirin, conversion is diverted
to 15-R-HETE. This undergoes further metabolism by leucocyte 15-lipoxygenases to form 15-epilipoxin A4. We investigated whether the aspirin-triggered formation of 15-epi-lipoxin A4 might provide
a novel biomarker for COX2 expression in vivo. Methods: WT, COX1-/- and COX2-/- mice were
treated 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 was
determined by ELISA.
Vascular COX2 expression was determined by en face aortic
immunohistochemistry. COX activity was determined as TXB2 formation in A23187-stimulated blood,
and PGE2 formation in lung homogenates. Results: Aortic COX2 immunoreactivity was weak in
untreated mice and significantly increased after LPS administration. TXB2 formation in whole blood
and PGE2 production by lung homogenates were strongly inhibited by aspirin administration. Plasma
levels of 15-epi-lipoxin A4, however, were not altered by any combination of LPS and aspirin, in any
genotype 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 aspirin
administration in vivo, despite confirmed vascular COX2 expression and inhibition of prostanoid
synthesis by aspirin. As such, these data suggest that aspirin-triggered 15-epi-lipoxin A4 cannot be
used as a biomarker of COX2 expression in vivo.

18

3. INNOVATIVE TOOLS TO EVALUATE GENETIC TARGETS AND THE EFFICACY OF


NOVEL CANCER THERAPEUTICS
Yan Xu1, Marina Tugusheva1, Jost Seibler1, Holger Kissel1, Jos Jonkers2, David Grass1 and Olesia
Buiakova1
1

Taconic Farms Inc., Hudson, New York


Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands

More predictive small animal models for compound and genetic target assessment are needed to
improve development of more efficacious drugs. For assessment of novel cancer therapeutics we
have developed an in vivo and ex vivo bioluminescent imaging platform to evaluate compound
efficacy. In these mouse models orthotopic tumor grafts are being used that are more relevant with
respect to host-tumor interactions, display metastatic potential and allow the evaluation of responses
to novel therapeutics. These models closely mimic the progression of human disease and allow
quantitative analysis and high-throughput in vivo assessment of potential anti-tumor activity. In
addition we will present an oncology platform using mouse models for breast cancer. The Brca1/p53
breast cancer model resembles hormone receptor- and ERBB2-negative (triple-negative)
mammary carcinomas and shows responses to therapies similar to humans, including the
emergence of drug resistance. We will present an orthotopic allograft system using primary tumors
from 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 resembling
human disease. Finally, we will present a transgenic RNAi system that can be used for studying the
loss-of-function of target genes. We will present how this system can be used to develop novel
disease models and how potentially this model can be combined with disease models to study the
genetic loss of a drug target in an established disease state.
4. IMMUNOMODULATION IN PRE TERM LABOUR: AN ANSWER TO IN UTERO
NEUROLOGICAL 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 most
problems occurring in babies born before 32 weeks. In this group, preterm labour (PTL) is most
commonly caused by infection/inflammation, prompting the search for agents that can modulate the
maternal inflammatory response, thereby reducing rates of PTL. We use a proven mouse model of
inflammatory PTL based on the administration of lipopolysaccharide (LPS), a major cell-wall
component of gram-negative bacteria, which activates the innate immune system via the toll-like
receptor type 4. In this study, we used Rolipram, a phospodiesterase (PDE) 4 inhibitor to modulate
the 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 of
gestation in CD1 outbred mice. 2 hours prior to this we administered 2mg/kg of Rolipram or DMSO
vehicle diluted in PBS (control) via intraperitoneal injection. The Rolipram group of mice showed a
significant 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 prolong
pregnancy, but that it does not appear to improve fetal outcomes. Further studies will define the
mechanisms by which rolipram delays parturition and define whether this approach might be
clinically useful.

19

5. PDE INHIBITORS: A TREATMENT FOR MOTHERS AND BABIES AT RISK OF


PRETERM 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 death
and handicap. Although some treatments reduce the risk of preterm labour (PTL), none have been
shown to improve neonatal outcomes. Cyclic adenosine monophosphate (cAMP) has been shown to
down-regulate the oxytocin receptor and is also known to be an immunomodulator, inhibiting both
NFkB-driven transcription and mitogen activated kinase (MAPK) activation, suggesting that cAMP
agonists 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, and PDE3 inhibitors have been found to
have neuroprotective effects in cultured neurons. We administered 10g of LPS into the right horn of
the uterus, at laparotomy, performed on E16 of gestation in CD1 outbred mice. 2 hours prior to this
we administered 2mg/kg of Milrinone (PDE3 inhibitor) or control via intraperitoneal injection. The
Milrinone group of mice showed no delay in delivery 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 of PDE3 and 4 inhibition is a better combination for
neuroprotection and PTL prevention than either alone.
6. THE CIEA NOG MOUSE: A SUPER IMMUNODEFICIENT MOUSE FOR HUMANIZATION
AND CANCER RESEARCH
Megan MacBride1, Holger Kissel1, Hirsohi Suemizu2 and Mamoru Ito2
1

Taconic Farms Inc., Hudson, New York


Central Institute for Experimental Animals, Kawasaki, Kanagawa, Japan

Humanized mice, in which human cells and tissues are engrafted and partially function, have
become important tools for the study of human disease and direct testing of therapeutic agents. The
establishment of humanized mice is largely attributed to the development of severe
immunodeficient 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 the
generation of humanized mice, i.e. mice with a human immune system, due to extremely high
engraftment rates and differentiation of human hematopoietic stem cells exceeding those in other
immunodeficient mice, e.g. NOD scid mice. The advantages of the super immunodeficient CIEA
NOG mouse also allow the use of these mice in various fields of cancer research including the
identification of cancer stem cells and the factors responsible for metastasis. Numerous
hematological and solid tumor cell lines have been engrafted successfully in the CIEA NOG mouse
using considerably fewer human cells than in other models. In some cases, as few as 10 cells have
resulted in successful engraftment. Tumor models include adult T-cell leukemia/lymphoma (ATL),
multiple myeloma, Hodgkins disease as well as metastasis models for pancreatic, colon and
melanoma cell lines. Data on the engraftment of the human immune system in these mice and
grafting rates of various cancer cell lines will be presented.

20

7. THE USE OF IN VIVO MODELS TO INVESTIGATE THE ROLE OF GASTROINTESTINAL


NUTRIENT-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 regulation
of food intake and metabolism. The gut uses hormonal and neural signaling mechanisms to
communicate the acute nutritional state to the central nervous system. Determining the nutrients
capable of inducing satiation and prolonging satiety, and elucidating the mechanisms by which they
mediate their effects on appetite may help identify novel treatments for obesity. Receptor systems
that 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, in
viv omodels 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 specific
macronutrients. Rodent models are particularly relevant as their gut-brain signaling pathways are
similar to those of humans. In rodents, nutrients can be administered via different routes, including
directly 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 using
vagotomised models and the importance of particular receptors in sensing nutrients can be
determined by the use of knock-out mouse models. These in vivo model systems have the potential
to aid the discovery of new drug targets for the treatment of obesity.
8. HUMANIZED MICE AND IN VIVO BIOLUMINESCENCE IMAGING: TOOLS FOR TESTING
ANTIVIRAL 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 of
chicken pox and shingles. Recently, SCID/beige mice implanted subcutaneously with full-thickness
human fetal skin and a recombinant virus, VZV-BAC-Luc that constitutively expresses firefly
luciferase, 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 are
treated with antiviral compounds. VZV replication is measured repeatedly in the same mouse by in
vivo bioluminescence imaging using a Caliper/Xenogen IVIS-200 instrument to detect photons
emitted 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 mice
that receive vehicle. Tests of acyclovir and valacyclovir, approved VZV therapies, found that they
were less effective than several experimental antiviral compounds, even at high doses given by oral
gavage twice per day at 120 mg/kg/day. The L conformation bromovinyl uracil derivatives, L-BHDU
and 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 gavage
at 30 mg/kg in 0.4% carboxymethylcellulose. Other compounds are currently being tested in this
model with equally encouraging results. We are the first to offer a standardized system for preclinical in vivo evaluation of compounds with potential anti-VZV activity.

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9. GENETIC AND PHARMACOLOGICAL MANIPULATION OF DDAH1 IN ENDOTOXIC


SHOCK: A NOVEL THERAPEUTIC TARGET
Manasi Nandi, PhD1, Zhen Wang2 and James Leiper2
1

Pharmacology and Therapeutics Group, Institute of Pharmaceutical Science, Kings College


London, London, United Kingdom.
2
MRC 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 elicits
an overwhelming inflammatory response to a microbial infection. Initial hypotension can quickly escalate
to 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 inflammatory
response. In the cardiovascular system, this excess NO contributes to circulatory collapse. Asymmetric
dimethylarginine (ADMA) is an endogenously produced competitive inhibitor of NOS. ADMA is
metabolized by the enzyme dimethylarginine dimethylaminohydrolase (DDAH) of which there are 2
isoforms, with distinct tissue distributions (DDAH1 and 2). We hypothesized that reducing DDAH1
activity, would elevate endogenous ADMA and hence inhibit NOS activity in a tissue-specific
manner, thereby providing a novel pathway to treat hypotension in septic shock. The cardiovascular
effects of reduced DDAH activity were investigated using a heterozygous DDAH1-knockout mouse
(DDAH1+/- ) and a novel DDAH1 inhibitor, NG-(2-Methoxyethyl)-L-arginine, in rodent models of
endotoxic shock [using lipopolysaccharide (LPS)]. LPS was administered intravenously and all
animals presented with circulatory shock. Intervention with a DDAH inhibitor at 3mg/kg attenuated
the observed hypotension with a concomitant improvement in the extent of acidosis developed
compared to vehicle treated animals. DDAH1+/- mice similarly displayed attenuation in the rate of
developed hypotension and furthermore demonstrated enhanced responses to adrenergic
vasopressor challenge. Both pharmacological and genetic reduction of DDAH1 activity is protective
against the vascular changes observed during endotoxic shock.
10. ANALYSIS OF SYNAPTIC REARRANGEMENTS FOLLOWING LASER-MEDIATED
MICROLESIONS IN THE ADULT BRAIN
Alison Canty, PhD, Lieven Huang, PhD, Johanna Jackson, PhD, Christina Nesarajah, BSc, and
Vincenzo De Paola, PhD
Neuroplasticity and Disease Group, MRC Clinical Science Centre, Imperial College, London, United
Kingdom
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. We
studied axonal degeneration and associated synaptic responses caused by focal lesions in the
cerebral cortex using in vivo two-photon imaging through a cranial window. Excitatory axons were
visualised using transgenic mice expressing green fluorescent protein (GFP) in a subset of neurons
and were cut using a pulsed high-energy femtosecond laser (800 nm). Damaged axons were
tracked to assess the fragmentation kinetics of the disconnected part and the synaptic
reorganisation of the surviving part. The surviving part of terminauxbouton (TB)-rich axons showed
an increase in synaptic turnover due to TB losses one day post-lesion (n=13 axons, 9 mice; p<0.01).
However, four days post-lesion both TB gains (p<0.05) and losses (p<0.001) contributed to
heightened synaptic turnover (n=13 axons, 9 mice). These data indicate a compensatory synaptic
response of damaged neurons to focal damage. We are now in the process of testing the hypothesis
that this is a general phenomenon affecting other neuronal types. Insight into the mechanisms that
regulate axon responses to injury in vivo may lead to new therapies to treat the substantial number
of neurological conditions characterized by axonal injury.

22

11. EVALUATION OF NON-APOPTOTIC CELL DEATHS IN DEVELOPING MOUSE


EMBRYOS 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 death
processes. The deregulation of these processes can result in embryonic lethality and sometimes in
diseases such as cancer. Traditionally, cell death has been classified as occurring through
programmed apoptosis or non-programmed necrosis. However, recent investigations suggest the
existence of programmed non-apoptotic cell death morphologies, such as autophagy, necroptosis,
mitotic catastrophe, and others. These discoveries suggest that cell death events in mouse embryos
previously classified as apoptotic may need to be re-evaluated. Here, we explore whether there are
developmental cell death events distinct from apoptosis in the developing mouse embryo. We
developed 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 such
as lysosomes. Lysosomes, which are organelles that degrade cellular proteins, are crucial to cell
death 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 distinct
occurrences of cell death in the dorsal root ganglion of developing mouse embryos on gestation
days 10 and 11. To confirm that Lysotracker red does detect dying cells, we induced death in
embryos 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, a
well characterized biomarker for apoptosis were done to confirm that this mode of cell death is
indeed 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 of
many client proteins. Hsp90 is a promising anticancer drug target, as cancerous cells are more
susceptible to Hsp90 inhibition than normal cells. A group of Hsp90 inhibitors was synthesized at our
department. Compound binding to Hsp90 was measured by isothermal titration calorimetry and the
thermal shift assay. The most potent compounds bound to Hsp90 with the dissociation constant of
about 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 Lipinski
rules of 5, the acute toxicity was predicted to be moderate, and the bioavailability per os of most
compounds was predicted to be more than 30%. Moreover, all compounds were predicted not to be
extensively 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 to
be 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 be
useful 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
1

Cardiovascular Disease-Atherosclerosis, Merck Research Laboratories, Rahway, New Jersey


In Vivo Pharmacology-Pharmacoanalytics, Merck Research Laboratories, Rahway, New Jersey
3
Modeling and Simulation, Merck Research Laboratories, Rahway, New Jersey
4
Laboratory Animal Resources, Merck Research Laboratories, Rahway, New Jersey
2

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 differentiate
lipid and protein kinetics. Although rodents and non-human primates are widely used as preclinical
models to study the pathophysiological basis of dyslipidemia, including pharmacological efficacy,
there is a shortage of methods for determining the contribution of the respective pathways. We
demonstrate the ability to dissect lipid flux using stable isotope tracers in rodents and non-human
primates, e.g. How can I determine the contribution of newly made lipid to the circulating pool? If I
change fatty acid synthesis will I impact lipoprotein production? The methods that we have
developed/implemented are well suited for studying free-living animals (i.e. tracers are given via a
single 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 in
humans which ensures translatability. The initial phases of our platform development considered
fundamental questions, e.g. what type and how much tracer do I dose? When should I collect
samples? What is the "best" way to measure endpoints? The later phases of our platform
development considered physiologically oriented questions, e.g. how does metabolic flux change
with a dietary and/or pharmacological intervention? Our data suggest that the methods are suitable
for routine investigations in support of drug discovery programs aimed at modulating dyslipidemia;
specifically, we focused on "high-throughput" analytical approaches (which predominantly rely on
mass spectrometry to quantify the abundance and/or the isotopic labeling of various metabolites). In
total, our approach is primarily directed towards decision-making as related to drug efficacy. The
utilization 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 the
gap(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 Snchez, M. en C.1
1

Unidad de Experimentacin Animal, UNAM, Mexico


Nekutli S.A. de C.V., Mexico

Dietary fiber is an essential part of diets, contributing to the proper functioning of the gut; the
recommended fiber intake for adults according to different countries, is in the range of 25 to 35
grams per day. Fructans are specific dietary fibers which provide energy and have demonstrated
prebiotic activity. Fructans are polymers of fructose where one or more fructosylfructose linkage
constitutes a majority of the linkages. Metlin and Metlos are purified organic fructans extracted
from Blue Agave. The aim of the study was to assess the acute toxicity and genotoxicity of two
dietary fibers, Metlin and Metlos. For genotoxicity and acute toxicity study, 6 week-old male and
female ICR (Hsd:ICR) mice were used. The objective was to identify the dose causing adverse
effects 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 products
are 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
1

School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom


Global Safety Pharmacology, Pfizer Global Research and Development, Sandwich, United Kingdom
3
Neuropsychopharmacology Unit, Division of Experimental Medicine, Imperial College, London,
United Kingdom
2

Negative biases in cognition and emotional processing are increasingly accepted as having a central
role in the causation of depression, and to be an important target for antidepressant drugs. It has
been hypothesised that negatively valenced emotional processing and its effects on memory may
underlie the development and persistence of mood disorders and that antidepressants work by
remediating these biases. We have developed a novel assay for rats to facilitate basic research into
how affective state influences the memory for positive experiences. The assay uses a bowl-digging
task where rats form two independent memories for a substrate-reward association under control or
treatment conditions. Memory bias is then assayed using a subsequent choice test. Induction of
negative 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 for
rewarding experiences. These data suggest that memories formed during negative affective states
have reduced value, which biases responding away from the associated cue. Antidepressants
induced a bias towards the associated cue suggesting acute antidepressant treatment enhances the
value of the memory. We hypothesise that memories formed during negative affective states have
reduced positive affective valence, which leads to the anhedonia and negative mood observed in
affective disorders, an effect which can be reversed by antidepressant treatment. These data also
support the predictive validity of this new rodent assay.
16. ROOSTERS AS A MODEL FOR AGING: DECREASED SENSITIVITY OF TESTICULAR
LEYDIG CELLS TO HYPOPHYSIAL GONADOTROPHINS
Avi Rosenstrauch, PhD
Achva 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. In
contrast, exposing gonadotrophins from low fertility adeno-hypophysis show (a) high fertility Leydig
showed an increase of 210% in testosterone release after 30 min incubation. (b) low fertility Leydig
cells reach this peak of testosterone only after 120 min of incubation. Then, aging Leydig cells show
decreased sensitivity of response to hypophysial gonadotrophins.

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17. AMELIORATION OF SIDE EFFECTS OF CHEMOTHERAPEUTICS IN ONCOLOGY


MODELS IMPROVES ANIMAL HEALTH AND SURVIVAL TIME
Sara Tobias Savage, DVM, David Lee-Parritz, DVM, DACLAM
Genzyme Corporation, a Sanofi Company, Framingham, Massachusetts
Neuropathy, pain, nausea, cachexia, dehydration and depression are commonly recognized
complications 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 animals
on therapeutic efficacy studies may be significantly decreased as a result. Oncology studies often
rely upon survival time to determine efficacy of novel treatments. Duration of survival time from the
primary disease cannot be determined accurately if animal models die of dehydration, malnutrition or
other secondary complications of treatment. Additionally, IACUC compliance depends upon
adherence to previously determined measures of animal welfare. Side effects of cancer treatments
may complicate interpretation of clinical signs or moribund status due to the primary disease
process, cause premature death or euthanasia of animals and require the use of larger experimental
cohorts to achieve statistical significance. Methods used to support these animals are not routinely
reported on in the scientific literature. Consistent application of nursing and supportive care
techniques currently used in veterinary and human clinical oncology may significantly improve
consistency and animal welfare in experimental cancer treatment models. These measures include
supplemental nutrition, fluid therapy, administration of analgesics, antiemetics, antidepressants, and
simple husbandry or housing modifications. Investigators are justifiably concerned with the potential
for confounding factors to adversely impact studies. Inclusion of proper experimental controls,
standardization of supportive techniques across all treatment groups and full characterization of
disease 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 both
improve animal welfare and permit more accurate modeling of the human disease for which novel
treatments are being investigated.
18. GENERATION OF HUMANIZED MOUSE MODELS AND THEIR USE IN DRUG-DRUG
INTERACTION, PHARMACOKINETIC AND SAFETY TESTING OF COMPOUNDS
Nico Scheer, PhD1, Martin R. Wilson1, Maki Hasegawa2, Yury Kapelyukh3, Harunobu Tahara2 and C.
Roland Wolf3
1

TaconicArtemis GmbH, Kln, Germany


Kyowa Hakko Kirin Co. Ltd., Sunto-gun Shizuoka, Japan
3
CXR Biosciences Ltd., Dundee, United Kingdom
2

More than 80% of drugs on the market are metabolized by Cytochrome P450 (CYP) 3A4, CYP2D6
or CYP2C9. The extrapolation of preclinical in vivo results to humans is often difficult due to species
differences in regulation of expression and substrate specificity of CYPs. For example, significant
differences in the ligand-receptor specificity, which regulates CYP3A4 expression, exist between
mouse and man. To overcome these difficulties, we have generated a unique portfolio of humanized
and knockout mouse models for key proteins involved in drug metabolism and disposition to
investigate the pharmacokinetic (PK) and drug-drug interaction (DDI) profile of both known and novel
compounds. In particular, a new multiple humanized model has been generated, which combines
the humanization of the two Xenoreceptors that regulate CYP3A4 expression, namely Pregnane X
Receptor (PXR) and Constitutive Androstane Receptor (CAR), along with replacement of the mouse
CYP3a cluster with a human region carrying both CYP3A4 and 3A7. This humanized model was
found 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 and
CYP2C9, will be presented. We expect that these models will be useful tools to better predict the PK
and 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.

26

19. LOW STUDY QUALITY AND PUBLICATION BIAS OF EXPERIMENTAL STUDIES


OFNEUROLOGICAL DISEASES RESULT IN OVERSTATEMENTS OF REPORTED
EFFICACY
Emily S. Sena, PhD, Hanna M. Vesterinen, BSc (Hons), Kieren J. Egan, BSc (Hons), and Malcolm
R. Macleod, PhD, FRCP
Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, United Kingdom
Discordance between animal and human studies has been attributed in part to compromised internal
and external validity of animal experiments. Systematic review and meta-analysis have proved
useful tools in deriving quantitative estimates of the impact of validity on reported study outcome. We
have systematically collected data from over 2500 studies describing 4700 experiments modeling six
neurological diseases reporting outcome from over 60,000 animals. Assessment of publication bias
in 499 publications in focal ischaemia suggests that one in six experiments remain unpublished, with
efficacy inflated by at least 30%. Few studies reported measures to avoid bias and those which did
reported smaller effect sizes. These findings are not unique to experimental stroke. Only 16% of 428
publications describing transgenic mouse models of Alzheimers disease reported random allocation
to group, 22% reported blinded assessment of outcome and none reported a sample size
calculation. In 1117 publications describing experimental autoimmune encephalitis used as a model
of multiple sclerosis efficacy was substantially overstated in those which did not report measures to
avoid bias (random allocation to group: 41.6% [36.746.5] versus 20.6% [95%CI 11.429.7] in
randomized studies; blinded assessment of outcome: 41.0% [36.2-45.8] versus 29.8% [19.839.8] in
blinded studies. These observations are unlikely to be unique to neurological disorders; Good
Laboratory Practice in disease modeling and evidence-based clinical trial design may improve
translation from bench to bedside.
20. REFINED MEASUREMENT OF CARDIOVASCULAR HEMODYNAMICS IN RODENT
SEPTIC SHOCK
Anna Starr, PhD and Manasi Nandi, PhD
Pharmacology and Therapeutics Group, Institute of Pharmaceutical Science, Kings College London,
London, United Kingdom
Sepsis is a leading cause of death in intensive care patients worldwide, occurring when the body elicits
an overwhelming inflammatory response to a microbial infection. Initial hypotension can quickly escalate
to serious circulatory collapse (septic shock) with ensuing multiple organ failure and death. One of the
key mediators produced as part of the inflammatory response is nitric oxide (NO), which in response to
an infection, is produced in excessive quantities, contributing to the observed circulatory collapse.
Tetrahydrobiopterin (BH4) is an essential cofactor for NO synthase (NOS). Limiting BH4 biosynthesis
may regulate excessive NO production, representing a therapeutic target for septic shock. To date, the
translation of findings in animal sepsis models into therapeutic strategies has been difficult. Intensive
care patients represent a highly heterogeneous population, which makes preclinical laboratory
modelling complex. Moreover, the methods used to induce septic shock vary significantly from lab to
lab and the monitoring of cardiovascular function has been highly confounded, through the use of
techniques such as tethered blood pressure recording. We have compared two models of sepsis,
employing refined methods for monitoring cardiovascular function. Conscious radiotelemetry and high
resolution non-invasive ultrasound measurements of cardiac contractility were used and temporal
changes in BH4 were measured. The use of these refined techniques has resulted in a significant
improvement in animal welfare compared to traditionally used methods of cardiovascular monitoring.
Furthermore, a clear reduction in confounding factors has resulted in high quality, reproducible data,
with each animal acting as its own control.

27

21. USE OF ANIMAL MODELS IN THE QUEST TO DISCOVER A NOVEL, HUMAN, CHRONIC
WOUND
HEALING AGENT
Paritosh Suman, MD, Harikrishnan Ramachandran, PhD, Sossy Sahakian, BS, Shanta Modak PhD,
Mark Hardy, MD
Department of Surgery, Columbia University Medical Center, New York, New York
Chronic non-healing venous stasis and decubitus ulcers are a significant cause of morbidity and
mortality in elderly patients, especially those with diabetes mellitus. Surgical debridement and
removal of constant pressure remain the mainstays of treatment. We hypothesized that a topical
formulation of natural proangiogenic protein Thymosin4 (T4) and Silvathymosin, a synergistic
combination of T4 and antimicrobial SilverSulfadiazine (Silvadene) could promote chronic wound
healing. In view of the non-availability of a true preclinical animal wound healing model for chronic
pressure and venous stasis ulcers, we used a surgically created 3x3 cm rat skin wound model to test
the healing properties of Thymosin4 and Silvathymosin. Compared to placebo, the T4 and
Silvathymosin led to the most rapid wound healing. Evaluation of wound histopathology, including
immunohistochemical assessment of angiogenesis and cellular proliferation, confirmed the
effectiveness of the tested agents in the healing of the wounds. Thymosin4 was previously
demonstrated to result in more rapid wound healing in human decubitus and venous stasis ulcers.
Although, human pressure ulcers did not show a statistically significant difference in healing with the
application of T4, preliminary clinical trials demonstrated more rapid wound healing when T4 was
used in venous stasis ulcers. The non-availability of a true animal model for chronic decubiti or
ischemic venous stasis pressure ulcers proves to be a major hindrance in optimizing and validating
the preliminary clinical findings. We conclude that better animal models are needed to reliably
predict wound healing in chronically ischemic wounds resulting from constant pressure in diabetic
and non-diabetic animals.
22. PRECLINICAL EVALUATION OF THE SAFETY AND EFFICACY OF A
BIOCOMPARABLE IN B CELL NON-HODGKIN LYMPHOMA
Mabel Tinoco, DVM1, Isabel Gracia, MSc1, Marisol Rivera, DVM1, Emilio Medina, PhD2, Jorge
Revilla, Dr2, Norma Garca, BTI2
1

National Autonomous University of Mexico. Mexico


Probiomed S.A. de C.V. Mexico

Treatment for non-Hodgkin lymphoma during the 1980s was a boom due to the creation of
monoclonal antibodies (MAB's). These recombinant proteins are currently modeled with a higher
proportion of human protein in its structure, obtaining chimeric and humanized antibodies. Looking
for a treatment against this neoplasia and manufactured in our country for the benefit of the
population, we carried out preclinical studies to evaluate the toxicological effects as well as efficacy
according to FDA guidelines. To determine these effects, we conducted the acute and subchronic
toxicity, the genotoxicity, pharmacodinamics and pharmacokinetics in a comparative way between
the innovator and our biosimilar (Kikuzubam). Both products showed similar activity, being nontoxic and with good effectiveness against B lymphocytes, with appropriate blood levels during 7
days, without statistical differences.

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23. DEVELOPMENT OF A PORCINE MODEL FOR EXPOSURE TO ENDOCRINEDISRUPTING CHEMICALS


Eric M. Walters, PhD1,2, T.J. Evans, DVM, PhD1, Y. Agca, PhD1, and V.K. Ganjam, DVM, PhD3
1

Department of Pathobiology, College of Veterinary Medicine University of Missouri, Columbia


Missouri
2
National Swine Resource and Research Center, Columbia, Missouri
3
Department of Biomedical Sciences, College of Veterinary Medicine University of Missouri,
Columbia, Missouri
Similarities in size and physiology, as well as genomic complexity, between humans and swine
make the pig a novel animal model for studying the potential effects of putative endocrine-disrupting
compounds (EDCs) and xenoestrogneic compounds. One of the advantages of the porcine model is
that samples can be routinely collected during the dosing period prior to euthanization of the animal.
Relatively little is known about the effects of EDCs on reproductive function in non-rodent mammals.
It was hypothesized that subacute oral exposure to vinclozolin (VCZ) would adversely affect
reproductive parameters in peripubertal and post-pubertal boars. Peripubertal and post-pubertal
boars were treated with various doses (0, 0.25, 5, or 100 mg/kg) of VCZ for 14 days. The two
highest dosages of VCZ were both associated with morphologic changes in the reproductive tract.
VCZ-associated histologic changes were more severe in swine than those reported in rodents.
Additionally, there are financial and logistical concerns associated with the use of porcine models in
comparative research. A recent development in testicular tissue xenografting using immunodeficient
murine recipients may address some of those concerns. The results of a preliminary study
demonstrate that altered development of porcine-to-murine testicular xenografts can be used as a
novel bioassay. Furthermore, prepubertal gilts can also be used to develop a non-invasive model to
evaluate the effects of xenoestrogens. Xenoestrogenic treated gilts had larger vulvas and heavier
reproductive tracts than controls. The results of these studies demonstrate the usefulness and
sensitivity of porcine models for studying the adverse effects of endocrine-disrupting chemicals.
24. ADME STUDIES IN GENETICALLY MODIFIED RATS LACKING KEY DRUG
TRANSPORTERS
Edward Weinstein, PhD
Sigma Aldrich, St. Louis, Missouri
Gene knockout models for disease have been limited mostly to mice, which may not accurately
reflect the physiology in humans. Advances in genetic engineering have expanded the number of
species that can be manipulated, enabling the creation of targeted knockouts in rats, rabbits,
zebrafish and swine. We demonstrate a genetic engineering technology, called the Zinc Finger
Nuclease, which has been used to develop translational animal models. We show data that through
utilization of this technology we have created the knockout of Mdr1a, Mrp1, Mrp2, and Bcrp genes in
rats. Data from our initial characterization studies demonstrate these single gene transporter
knockouts serve as useful models for absorption, distribution, metabolism, and excretion (ADME)
studies and have potential implications for more efficient drug development studies.

29