parent compound or its metabolite(s) act on non-hERG cardiacchannels to produce this QT prolongation. NBI-1 has a diversifiedmetabolic pathway and testing its major metabolite did not revealany activity on hERG channel. NBI-1 was then tested in HEK cellsexpressing human IKs or IK1 potassium channels. Results indicatedthat whereas NBI-1 was inactive against IK1 channels, it produced adose dependent inhibition (up to 43.1%) of IKs current suggestingthat NBI-1 may be an IKs blocker which may explain its QTprolongation in vivo.

doi:10.1016/j.vascn.2009.04.130

Simultaneous cardiovascular and pharmacokinetic data collectionin freely moving guinea pigs

Brian M. Roche⁎, Thomas Vinci, S. Peter Hong, Jeremy Smith,Brandon Wood, Jerry Johnson, Steve Graves, Craig HasslerBattelle Memorial Institute, Columbus, OH, United States

The pharmaceutical industry is placing more emphasis oneliminating compounds with deleterious side effects earlier in thedevelopment process. Combining methodologies to maximize thedesired endpoints (i.e., BP, ECG, PK, etc.) from a single study wouldbe an efficient paradigm for early decision making. Additionally,utilizing a smaller animal with cardiac ion channels similar tohumans would provide a more relevant and test article efficientmodel. Automation allows for dosing and blood collection to occurwhen appropriate without human presence while minimallyaffecting cardiovascular endpoints. Combining the use of theCulex/Empis automated device to collect blood and/or to doseguinea pigs with a cardiovascular telemetry study is a model thatenhances the amount of data (knowledge) about a potential newdrug from a single study.

We evaluated the use of guinea pigs, instrumented with atelemetry device capable of collecting ECG, blood pressure and bodytemperature, in conjunction with the Culex/Empis automated deviceto establish the PK/PD relationship of several known compounds: QTprolonging agent, terfenadine; hypotensive agent, nitroprusside;hypertensive agent, phenylephrine; chronotropic agent, zatebridine;and negative QT control enalapril.

doi:10.1016/j.vascn.2009.04.131

Effect of pacing rate and beta blockade on cardiac refractoryperiods and his-bundle conduction

Jinbao HuangCorDynamics, Inc., Chicago, IL, United States

Stimulation of the sympathetic nervous system releases norepi-nephrine, activating beta receptors and triggering increases in heartrate (HR) and atrioventricular (AV) conduction velocity. Metopro-lol is a selective beta receptor blocker, which causes a decrease insinus HR and slows AV conduction. The purpose of this study wasto assess the effects of pacing and beta blockade on cardiacelectrophysiologic parameters in the anesthetized dog. Two bipolarplunge electrodes were used to determine atrial and ventricularrefractory periods (AERP and VERP), as well as to record atrialelectrograms. A multipolar electrophysiology catheter positioneddistal to the bicuspid valve was also used to record His bundleelectrograms to determine atria to His (AH) and His to ventricle(HV) interval measurements. The AV nodal refractory period(AVNERP) was also calculated. In nontreated dogs (n=11),

AVNERP and AH intervals were prolonged at 180 bpm comparedto 120 bpm (AVNERP: 201±17 vs. 192±18 msec and AH: 128±6vs. 119±5 ms, respectively). Atrial ERP, VERP and HV intervalswere similar when hearts were paced at 120 and 180 bpm.Treatment with 0.1, 0.3 and 1.0 mg/kg metoprolol dose-depen-dently decreased sinus HR (10 to 20% from baseline) and increasedAVNERP (10 to 25% from baseline) with 180 or 120 bpm pacing.Metoprolol also prolonged AH conduction time. In conclusion,increasing pacing rate prolongs AVNERP and AH conduction time.In contrast, beta blockade decreases HR, but also increases AHinterval, suggesting slowing of supraventricular conduction.

doi:10.1016/j.vascn.2009.04.132

Inductionof Torsades dePointes by FPL64176,DPI-201106, dofetilide,and chromanol 293B in isolated guinea pig and rabbit hearts

Hsien C. Cheng⁎, Josephine IncardonaSanofi-Aventis, Bridgewater, NJ, United States

Introduction: For predicting torsades de pointes (TdP) liability ofa compound, most of previous studies have used surrogate markerssuch as hERG inhibition or QT prolongation which may or may notlead to TdP. In this study, we have used isolated hearts for testinginduction of TdP.

Method: Spontaneously beating isolated guinea pig and rabbithearts were perfused according to the Langendorff method inhypokalemic (2.1 mM) solution. Lead II ECG and incidence of TdPwere monitored for 1 h.

Results: FPL 64176, a calcium channel activator, and DPI-201106,a sodium channel modulator, produced TdP in isolated guinea pigand rabbit hearts in a concentration dependent manner; guinea pighearts were more sensitive than rabbit hearts. In contrast,dofetilide, an IKr inhibitor, gave a low incidence of TdP in guineapig hearts (1/24), and no TdP at all in rabbit hearts. Chromanol293B, an IKs inhibitor, elicited TdP concentration dependently inguinea pig but not in rabbit hearts.

Conclusion: Thus IKs inhibition, rather than IKr inhibition, appearsto be critical in inducing TdP in isolated guinea pig hearts. Rabbitheart did not produce TdP by IKs inhibitor presumably due to a lowlevel of IKs channels in the heart. TdP produced in this study areconsistent with the notion that their production may be aconsequence of reduced repolarization reserve, thereby causingventricular abnormalities (Roden, J internal Med., 2006; Jost et al.,A.N.E., 2007). Thus, these isolated guinea pig and rabbit hearts maybe useful for predicting TdP liability of compounds in drugdevelopment.

doi:10.1016/j.vascn.2009.04.133

Possible role of the ultra-rapid delayed rectifier potassium current(IKur) in action potential repolarization in rabbit heart

Carol Wilson⁎, Samantha Robinson, Sara Graham,Nick McMahon, Bronagh HeathGlaxoSmithKline, The Frythe, Welwyn, Hertfordshire, United Kingdom

The ultra-rapid delayed rectifier K+ current (IKur) plays animportant role in early cardiac action potential repolarization in anumber of species. Although the rabbit heart has proven useful for

Abstracts / Journal of Pharmacological and Toxicological Methods 60 (2009) 210–258 241

studying cardiac repolarization, the role of IKur in rabbit heart isless well known, as is the potential contribution of this ion channelto drug-induced changes in repolarization. The aim of this studywas to investigate the effects of 4-aminopyridine (4-AP) and 2-isopropyl-5-methylcyclohexyl diphenyphosphine oxide (DPO-1)on action potential duration (APD) in isolated rabbit atria andventricular papillary muscles. Intracellular action potentials wererecorded at 36–37 °C and APD was measured at 20, 50 and 90%repolarization (APD20, 50 and 90). Exposure to 4-AP at 50 µM for30min caused a significant prolongation in APD20 of 29.0±3.3% and39.4±9.4% in left atria and right ventricular papillary musclesrespectively (stimulated at 1 Hz). In contrast, late repolarizationwasless affected; increases in APD90 were 8.6±2.8% and 18.7±5.0% foratria and papillary muscle respectively. The prolongation wasreverse frequency-dependent (0.2–1 Hz). Similarly, application ofDPO-1 at 0.3 µM for 30 min significantly prolonged APD20 by 21.9±8.8% and 45.8±23.0% in atria and papillary muscle, respectively.APD90was less affected, with increases of 10.8±2.2 and 25.2±13.2%in atria and papillary muscle respectively. In conclusion, it is likelythat IKur may play a functional role in repolarization in both rabbitatria and ventricle.

doi:10.1016/j.vascn.2009.04.134

Electrophysiological assessment of the effect of NBI-1 on theaction potential kinetics of cardiac human Purkinje fibers in thepresence or absence of dofetilide and b-adrenoceptor stimulation

Aida Sacaana,⁎, Andrea Ghettib, Guy Pageb, Antonio Guiab,Shirley Lioa, Haig Bozigiana

aNeurocrine Biosciences, San Diego, CA, United StatesbAviva Biosciences, San Diego, CA, United States

The purpose of this study was to investigate the effects of NBI-1 onthe function of human Purkinje fibers. Prior experiments hadestablished that NBI-1 has measurable inhibitory effects on recombi-nant IKs channels (see accompanying abstract). Several groups havepreviously shown that under normal circumstances IKs blockade hasonly minor effects on cardiac action potential. However, in thepresence of b-adrenergic stimulation and/or reduced repolarizationcapacity, the inhibition of IKs current can precipitate prolongation ofthe action potential. In light of these data, both NBI-1 and Chromanol293B (Chrom, a known IKs blocker) were tested in the present studyalone or, in the presence of Isoproterenol (b-adrenergic activator) anddofetilide (IKr blocker).

NBI-1 produced a dose dependent increase in action potentialduration (APD) and amplitude (APA) without altering the restingmembrane potential (RMP). By contrast, Chrom had no effect onAPD, APA or RMP when tested alone. When either NBI-1 or Chromwere tested in the presence of isoproterenol and dofetilide, a clearprolongation of the APD was seen by both compounds. Again, NBI-1but not Chrom produced an increase in APA.

The results of the present study support the notion that NBI-1 hasphysiologically relevant activity on human IKs conductance. Thecomparison with the prototypical IKs channel blocker suggests thatNBI-1 may be a more potent and efficacious blocker of IKs. In addition,the effect of NBI-1 on APA suggests a potential action on Na+ and/orCa2+ conductance.

doi:10.1016/j.vascn.2009.04.135

Analysis of dynamic restitution of ECG parameters in the rabbitlangendorff heart exposed to drugs that lengthen or abbreviate QT

Y. Panyasing⁎, A. Kijtawornrat, C. del Rio, L. Snedden,T. Mongkolmaneepol, J. Schmidt, R. HamlinQTest Labs, LLC/The Ohio State University, Columbus, OH, United States

Dynamic restitution has been used to characterize ECG parameters,but not to cardiac electrograms recorded from Langendorff prepara-tions. This study was designed to determine if expressions of dynamicrestitution detect effects of drugs that either prolong or abbreviate QTin rabbit Langendorff preparations. Dynamic restitution was charac-terized as hysteresis of beat-to-beat QT-TQ, an expression known topredict arrhythmogenic liability in vivo. Rabbit hearts perfusedaccording to the methods of Langendorff were exposed to vehicle orto escalating concentrations of dofetilide (that prolongs QT) andpinacidil (that shortens QT). RR, QT, QTc (F, B, V), TQ and TQ/QT weremeasured from bipolar transventricular electrograms during baselineand exposure to drugs. Plots of each parameter versus concentration, orversus time for vehicle, were made. Opposite changes were producedby dofetilide and pinacidil; RR and QT lengthened significantly fordofetilide and shortened significantly for pinacidil. QTbtb (the width ofthe envelope surrounding the cloud of points generated by a plot of QTversus RR and an RR of 1000 ms) increased for dofetilide and decreasefor pinacidil. The ratio, QT/TQ, lengthens for dofetilide and shortens forpinacidil. Thus effects on expressions dynamic restitution in the in vitroLangendorff preparation mimic effects observed in vivo.

doi:10.1016/j.vascn.2009.04.136

Blockade of hERG K+ channel by the Histamine H1 receptorantagonist, diphenhydramine

Ki-Suk KimKorea Institute of Toxicology, Yuseong-gu, Seo-gu, Daejoen,Republic of Korea

Treatment with second generation histamine H1 receptor antago-nists has been associated with lengthening of the Q–T interval andproarrhythmia. Similarly, lengthening of the Q–T interval has beenreported in patients after overdosing with diphenhydramine (DPH), afirst generation agent. The inhibition of the potassium current IKr andQT prolongation has been known to be associated with drug-inducedarrhythmias. In the present study, we investigated the molecularmechanisms of voltage-dependent inhibition of the human ether-a-go-go-related gene (hERG) by DPH in HEK-293 cells expressingdelayed rectifier K+ channel. DPH inhibited the hERG current in aconcentration-dependent manner with a half-maximal inhibitoryconcentration (IC50) value of 22.2 µM. In voltage-dependent inhibi-tion of the hERG current, the activation curve was shifted in anegative voltage from −25.4 mV in controls to −36.1 mV afterapplication of 100 µM DPH. The block of the hERG by DPH was foundto be time-dependent-, and occurred rapidly. The blockades of thewild-type hERG channel by DPH were comparable to those of boththe S6 residue hERG mutants (F656A and Y652A) and the pore regionmutants (T623A and S624A). In a conclusion, these results indicatethat DPH preferentially inhibits the hERG potassium channel, but theblockade of hERG channel by DPH may not involve or involve a weakinteraction with the S6 residue and the pore region. However theinhibition may be induced through other pathways such as bindingthrough other regions or effects by drug binding receptors.

doi:10.1016/j.vascn.2009.04.137

Abstracts / Journal of Pharmacological and Toxicological Methods 60 (2009) 210–258242