produce any abnormal symptoms/behavior like convulsion, vocaliza-tion, diarrhea/defecation; change in posture, excessive urination andrespiration.However, NP-355produced significant increase (P<0.05) inspontaneous activity as compared to saline group. Inhomecage removaland handling, NP-355 did not significantly alter the lacrimation,salivation and body tone. NP-355 did not show significant increase inexcitability/handling reactivity. In open field activity, NP-355 did notsignificantly alter surface righting, aerial righting, tail pinch, pinnareflexes, visual placing, palpebral closure and auditory response butincreases the arousal. There was significant increase (P<0.05) in totallocomotor activity (AUC 0-180 Min) observed between mice treatedwith NP-355 as compared to saline. No abnormality in motorcoordination was observed in mice treated with NP-355.

Conclusions: Its potent antiepileptic activity and good CNS SafetyPharmacology profile make it a promising candidate for furtherevaluation.

doi:10.1016/j.vascn.2010.11.073

Poster Number: 70Board Number: 70

Validation of the FOB method following a single oraladministration in ratAurlie Boucard, Guillaume Froget, Alain Simmonard

CIT, Evreux Cedex, France

The purpose of the safety pharmacology core battery is toinvestigate the effects of a test substance on vital functions, amongthem the Central Nervous System (CNS). The Functional ObservationBattery (FOB) is a behavioral screening battery generally used todetect CNS dysfunctions by the means of detailed clinical observa-tions and determination of reactivity to different stimuli. However,considering the density and the heterogeneity of the results, theirinterpretation at the level of the CNS high functions remainsubjective in most cases. The aim of the study was to define andvalidate an index calculated from the profiles of informationobtained, which would better describe the effects of substances onhigh level CNS broad domains: neurologic, autonomic and behavioral.The effects of two sedative agents (chlorpromazine and clonidine) onCNS activity were first evaluated by means of systematic observationsof symptoms induced by a single oral administration to consciousSprague-Dawley rats. Then, FOB parameters were combined inseverity scores to provide an index of the toxicity of the test itemson the three CNS broad domains. This describes the domain(s)impaired and quantifies the level of this impairment. Moreover, bycomparing substances and dose ranges, it was demonstrated thatseverity scores were sensitive enough to discriminate moderateto severe effects. Altogether, these results showed that combiningFOB data in severity scores was an appropriate interpretation toolallowing a more accurate measurement of the neurotoxicity potentialof a candidate substance.

doi:10.1016/j.vascn.2010.11.074

Poster Number: 71Board Number: 71

Use of Zebrafish in safety pharmacologyWill S. Redferna, Matthew J. Winterb, Gareth Waldronc, Paul Butlerc,Mark Holbrookc, Rob Wallisd, Jean-Pierre Valentina

aAstraZeneca R&D, Macclesfield, Cheshire, United KingdombAstraZeneca Safety, Brixham Environmental Laboratory, Devon,United KingdomcPfizer Global R&D, Sandwich, Kent, United KingdomdPfizer, Ltd., Groton, CT, United States

Convenient screens to identify potential safety hazards duringearly drug discovery phases would be highly valuable, and could helpto reduce associated attrition rates. Such front-loaded assays mayalso have a beneficial 3R's impact: as well as weeding-out ‘weak’candidate drugs prior to in vivo testing in mammalian species, inprinciple those compounds that did progress would be expected tohave reduced toxicity in the mammalian species. Recently, a variety oflarval Zebrafish assays have been proposed for safety pharmacologyapplications. At least three pharmaceutical companies have publishedvalidation data on the following assays in larval Zebrafish: QT liability,cardiac contractility, gastrointestinal motility, seizure liability, andvisual function, and other assays exist (Redfern et al., 2008). Zebrafishlarvae can be maintained in multiwell plates, and the assays fall into 2types: free-swimming and immobilised larvae. These assays need tobe rigorously assessed, not just in terms of sensitivity and specificity,but also for face validity, compound bioavailability, assay reliability,throughput, compound requirements, and cost. This presentationwill address these issues, aligning each larval Zebrafish assay with itsnearest equivalent mammalian cell-based/tissue-based assay.

References

Redfern, W. S., Waldron, G., Winter, M. J., Butler, P., Holbrook, M.,Wallis, R., & Valentin, J. -P. (2008). Zebrafish assays as early safetypharmacology screens: Paradigm shift or red herring? JPTM, 58,110−117.

doi:10.1016/j.vascn.2010.11.075

Poster Number: 72Board Number: 72

Cardiotoxicity testing in Zebrafish: Relevance of bioanalysisAdrian Hilla, Martin Traebertb, Berengere Dumotierb

aEvotec AG, Abingdon, Oxfordshire, United KingdombNovartis Institutes for BioMedical Research NIBR, Cambridge,MA, United States

In a study with Novartis, 20 blinded drugs were assessed in aZebrafish in vivo model by researchers at Evotec (UK) Ltd. for pro-arrhythmogenic potential. Larvae were exposed to a five-point doserange for 3 h and then rates of atrial (A) and ventricular (V) contractionwere determined; thereby revealing any tachycardia, bradycardia andchanges in the A:V ratio. Extra doses were also used to further evaluatesome drugs. Bioanalysis was used to determine that amount of drugs inthe larvae. Based on dog or human data,14 compoundswere expected tocause QT-prolongation. In Zebrafish, 9 of these were correctly identifiedas they caused A/V decoupling: e.g. the LOEC for a potent hERG blocker,Astemizole, versus that for Amiodarone, was determined as 8.8 and182 ng/larva, respectively. The LOEC for these compounds in dog(plasma) is 0.01 and 1.8 μg/ml, respectively. Of the 5 compounds whichfailed to induce the A/V decoupling, 1 caused bradycardia (Quinidine),but as uptake was relatively low (7.8 ng/ larvae), the A/V effect may beachievable if a higher dose was used as bradycardia typically precedesthe A/V arrhythymia in a dose response (N.b. LOEC in dog is 6.5 μg/ml). 4further compounds caused no adverse effect but this may be related to

Abstractse22

poor uptake as this only reached 0.02, 0.5, 9.1 and 108 ng/ larva,respectively, and those <10 ng were unclassified pending furtherinvestigation. Finally 5 compounds expected to cause bradycardia and1 negative control were correctly identified. Therefore the overallpredictivity for this assay excluding bioanalysis was 75%, and if thosecompounds failing to achieve a body burden of >10 ng/larva areremoved from the data set pending further investigation, this wouldincrease to 88%. The Zebrafish is therefore a promising model to test forcardiac dysfunction.

doi:10.1016/j.vascn.2010.11.076

Poster Number: 73Board Number: 73

ECG in methoxamine-sensitized rabbits for screeningproarrhythmic risk— Comparison with ECG data in dogsPhilippe Guillaume, Sabrina Serpillon, Cecile Chauvin,Christophe Legrand, Stephane Herve, Sandra Picard

Porsolt & Partners Pharmacology, Boulogne-Billancourt, France

Whereas QT interval prolongation assessment is mainly carried outin conscious large animals, the anesthetized methoxamine-sensitizedrabbit model (Carlsson) has been proposed for evaluating torsadogenicrisk. However, its sensitivity is highly dependent on the experimentalconditions used. The present worked has compared rabbit and dogmodels for responsiveness to the effects of known torsadogenicsubstances. ABP and ECG were monitored in anesthetized male orfemale rabbits infused with methoxamine (520.2 μg/ml starting 10 minbefore treatment) or in conscious telemeteredmale dogs. Clofiliumwasmore proarrhythmic in female thanmale rabbits at 100 nmol/kg/min for45 min (40% vs 20% with ventricular tachycardia, VT). At 200 nmol/kg/min for 20 min, it induced VT and torsades de pointes, TdeP, in 71% and29% females, with increased QTc by +40.8±6.6% (Fridericia) or +46.1±7.6% (Carlsson). Sotalol induced VT or ventricular fibrillation (VF) in75% rabbits and QTc prolongation in both species (rabbits: +9.3±6.4%,Fridericia, and +11.0±6.6%, Carlsson ; dogs: +16% max vs -1% incontrols p<0.001, Fridericia). Terfenadine induced VF in 33% rabbitswith QTc prolongation (+23.2±4.9%, Fridericia, and +23.7±5.0%,Carlsson) but no significant effects on QTc in dogs. These findingsdemonstrate that, when used under appropriate experimental condi-tions, the rabbit TdeP model is a powerful tool for screeningproarrhythmic risk and QT prolongation as it can reveal effectssometimes difficult to detect in conscious dogs after single oral dosing.

doi:10.1016/j.vascn.2010.11.077

Poster Number: 74Board Number: 74

Compound-X-induced TdP in the anesthetized guinea-pig:Unraveling the mechanism of actionBrigitte G.P. Loenders, Jan Verrelst, Hua Rong Lu, Eddy Vlaminckx,Jutta Rohrbacher, Ard Teisman, David J. Gallacher

Johnson & Johnson Pharmaceutical Research & Development,Beerse, Belgium

The anesthetized guinea-pig is a sensitive and relevant model thatcan easily be used as a first-in-line in vivo screen for the earlydetection of compounds that affect cardiac electrophysiology. Indeed,compounds that alter the conduction of several cardiac ion-channels

can be detected in the guinea-pig at clinically relevant exposures.Therefore this model is part of our integrative approach to determinethe cardiovascular risk of drug candidates. Female Dunkin-Hartleyguinea-pigs were anesthetized with sodium pentobarbital. Heart rate,blood pressure and the PQ, QRS, QT and QTcB intervals weremeasured after intravenous administration of compound or vehicle.A detailed screening of ECG morphology was performed. We recentlytested compound-X in this anesthetized guinea-pig model andobserved TdPs in 2 out of the 7 compound-dosed animals. Aninteraction with IKr and IKs channels and an increase in intracellularcalcium are reported to be prerequisites for TdP induction in theguinea-pig. The observed increase in blood pressure concurs with anincrease in Ca2+. However, compound-X did not notably prolong theQTc interval nor decreased heart rate, both of which are associatedwith IKr blockade by dofetilide in this model. Moreover, the intrinsicIKs blocking potential of the compound was low. This implies theinvolvement of mechanisms other than IKr and IKs blockade in thedevelopment of these TdPs. We further evaluated the electrophysio-logical effects of compound-X in other preclinical safety models toexplore its mechanism of action. The implications for drug safetyassessment are discussed.

doi:10.1016/j.vascn.2010.11.078

Poster Number: 75Board Number: 75

Accurate and repeatable measurement of effects of test-articles onvenous toneRobert L. Hamlina,b, C.L. del Rioa,b, A. Kijtawornrata,b, Y. Panyasinga,b,L. Sneddenb, D. Probstb, Y. Ueyamaa,b, D. Hamlina, W.W. Muira,b

aThe Ohio State University, Columbus, OH, United StatesbQTest Labs, Columbus, OH, United States

If affected by drugs, changes in venous capacitance (VC) and meancirculatory filing pressure (MCFP)may lead to postural hypotension (PH),an adverse drug effect. We have developed a facile, humane, minimally-invasive, accurate, and repeatable method of measuring VC and MCFP indogs. Under aseptic conditions, an automatic fibrillator-defibrillator (St.Jude, PROMOTETM RF) is inserted into the right ventricle and units (DSI)to telemeter arterial (AP) as well venous (vP) pressures are implanted.Following recovery, AP/vP are continuously recorded under anesthesia,while the dogs are put briefly into ventricular fibrillation (VF) for ~30 s,and then defibrillated; because duration of VF is brief and animals areunconscious, defibrillation occurs painlessly andwithout incident. Duringcardiac arrest, AP decreases and vP increases (approaching each other).The AP decay is determined by systemic vascular resistance (svr), bloodviscosity (h), and elastic recoil of the aorta (e). Meanwhile, theequilibration pressure to which vP rises is determined by the ratioof blood volume (BV) to the VC; however, if neither BV nor h change,then the AP fall is determined by arterial properties (svr and e), and thepressure to which vP approaches is the MCFP (determined by tone ofvenous smooth muscle). For instance, in α-chloralose anesthetizednormal dogs, MCFP decreased following onset of isoflurane anesthesia(from 9.6±1.0 to 6.7±0.6 mmHg), suggesting venorelaxation. Further-more, we have shown that this methodology identifies both venor-elaxation produced by sildenafil and nitroglycerine, as well asvenocontraction produced by norepinepherine. This methodology canquantify the effects of test articles on these important parameters.

doi:10.1016/j.vascn.2010.11.079

Abstracts e23