dogs, having at least 95% of segments within the specified range ofQTc interval, were for each heart rate interval (b75;75–100, 100–150,150–200, N200): Van de Water (3,5,4,3,3), Bazett (3,0,0,0,0), Spence(4,5,3,0,0) and Linear regression (4,5,5,5,3).Conclusion: Linear regres-sion method is considered superior to Spence , van de Water andBassett for drug-induced tachycardia.
doi:10.1016/j.vascn.2008.05.066
Validation of the Dynaflow® temperature control system for thehERG test
Fabian Haeusermanna, Susanne Braumb, Alexander Breidenbacha,Liudmila Polonchuka (F. Hoffmann-La Roche AG, Basel, Switzerland)(Cellectricon AB, Göteborg, Sweden)
Nowadays testing new drug candidates for their potency to affectcardiac repolarisation in the hERG assay is an indispensable step in thein vitro cardiac safety assessment required by regulatory agenciesworldwide. Among the experimental parameters that have to becontrolled in the hERG test temperature and drug exposure areparticularly important. Dynaflow® technology has been successfullyused for the drug application in the electrophysiological experimentsin various labs over the last decade. Recently, the Dynaflow®Temperature Control System has been developed in order to performpatch clamp experiments under controlled temperature with veryprecise and fast solution exchange. The objective of the study was toevaluate a newly developed Dynaflow® system in the hERG test atphysiological temperature (+37 °C). A set of reference compounds wasused to validate the novel technology using a CHO cell line stablytransfected with hERG. The Dynaflow® Temperature Control Systemgreatly facilitated the handling of drug testing in patch clampexperiments. The system provided higher success rate in sealformation and increased stability of recordings. A good correlationwas found between IC50 values for the reference drugs obtained withthe Dynaflow® System and those generated in the conventional patchclamp perfusion system at physiological temperature. Dynaflow®Temperature Control System technology enables to perform highquality tests at physiological temperature (+37 °C). In addition, itincreases the throughput as well as minimizes the amount ofcompounds used.
doi:10.1016/j.vascn.2008.05.067
Effect of pentamidine on QT interval in the conscious guinea-pigand hERG trafficking
Samantha Robinson, Stella Worton, Nikol Simecek, Lorraine Camp-bell, Nick McMahon, Yi Cui, Bela Patel, Bronagh Heath (SafetyPharmacology, GlaxoSmithKline, The Frythe, Welwyn, Hertfordshire,AL6 9AR, UK)
Pentamidine is known to cause QT prolongation and cardiacarrhythmias in the clinic. The purpose of this work was to investigatethe effect of a single dose of pentamidine (a known hERG traffickinginhibitor) on QT interval in conscious guinea-pigs and to confirmhERG trafficking inhibition using HEK-293 cells stably transfectedwith hERG-1 cDNA. The effects of acute (10min) and chronic (16–20 h)exposure to pentamidine (1–300 mM) on hERG current and currentdensity were studied using whole-cell patch-clamp configuration.Western blots were conducted to support electrophysiological find-ings. Conscious guinea-pigs (n=4), implanted with DSI telemetryunits, received a single dose of vehicle i.m. followed by a single dose ofpentamidine i.m at 18.5 mg/kg. ECG parameters were monitored for
2 h prior to and for 14 days after each dose. Signals were captured byPONEMAH OpenART, recorded at 15 s every 15 min. Chronicpentamidine exposure caused a marked reduction in hERG currentdensity (70.9% at 30 µM; Pb0.05) and hERG protein expression, yetin vivo, pentamidine was not associated with QTcf prolongation,moreover a sustained reduction in QTcf of 8 msec was noted betweenday 4 and 14.Based on PK modelling data from the rat, exposure topentamidine was estimated to be within the range showing a hERGtrafficking effect in vitro. To conclude, inhibition of hERG traffickingdid not translate to a QTc prolongation after a single dose in vivo,instead a reduction of QTc was apparent. The biological relevance ofthis has yet to be defined.
doi:10.1016/j.vascn.2008.05.068
Effects of arsenic trioxide, an inhibitor for hERG channeltrafficking, on the electrocardiogram of the guinea pig
Mototsugu Sakakibara, Atsushi Sasaki, Yoshikazu Tsumura, KeitaroHashimoto, Yasushi Yamashita (Mitsubishi Chemical Safety InstituteLtd., Kamisu, Japan)
Previous in vitro studies have demonstrated that some drugsinhibit the trafficking of hERG channel to the cell membrane, whichcauses reduction of the channel density on the cell surface. Thereforethe trafficking defect as well as direct blocking of hERG channel resultsin prolongation of the cardiac repolarization, which may beresponsible for generation of arrhythmias. This study aimed atexamining effects of an inhibitor of hERG channel trafficking, arsenictrioxide, on induction of arrhythmias, and comparing the effects withthose of a direct hERG blocker, astemizole. In the in vitro study, hERG/HEK293 cells were exposed to arsenic trioxide or astemizole and thehERG current was measured by the whole-cell clamp method. Thecells were also cultured in a medium containing arsenic trioxide orastemizole for analysis of hERG protein changes by Western blotting.In the in vivo study, male guinea pigs were administered once dailyarsenic trioxide by i.v. or astemizole by p.o.. The electrocardiogramwas recorded under a restrained condition. Although arsenic trioxideinhibited the hERG channel trafficking, the hERG current was notreduced directly. In contrast, astemizole directly reduced the hERGcurrent without inhibiting the hERG channel trafficking. Arsenictrioxide induced arrhythmias on the 9th day whereas astemizole didat the first day of experimentation. These results suggest that repeatedadministration is required to induce arrhythmias by drugs that inhibithERG channel trafficking.
doi:10.1016/j.vascn.2008.05.069
High throughput screening of a panel of ion channel expressingcell lines
Barbara A. Wible, Yuri A. Kuryshev, Glenn E. Kirsch, Arthur M. Brown(ChanTest Corp., 14656 Neo Parkway, Cleveland, OH 44128, USA)
Purpose: To develop and validate a catalog of cell lines over-expressing ion channel cDNAs for high throughput functional screen-ing in drug discovery and development. Methods: Human cDNAsencoding 80 of the ion channels most relevant to drug discovery,development, and safety are being cloned and stably transfected intoHEK293 or CHO cells. Cotransfection with GFP allows the selection oftransfected cells by fluorescent-activated cell sorting. Screening ofindividual subclones is carried out on two automated electrophysiol-ogy systems: PatchXpress and IonWorks Quattro. Both instrumentsprovide a dramatic increase in throughput compared to manual patch
162 Journal of Pharmacological and Toxicological Methods 58 (2008) 147–178
