Professional Documents
Culture Documents
Garret A. FitzGerald, MD
University of Pennsylvania School of Medicine
Paul Holloway
Imperial College London
The aforementioned travel fellowships were made possible by the generous support of the:
Paul Holloway
Imperial College London
The aforementioned travel fellowships were made 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
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.
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
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.
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.
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
11
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
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
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
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
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
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
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
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
21
22
23
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
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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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.
25
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.
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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
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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