Validation of a voltage-sensitive dye (di-4-ANEPPS)-based method for assessing drug-induced delayed repolarisation in beagle dog left ventricular midmyocardial myocytes.
BACKGROUND
Evaluation of drug candidates in in-vitro assays of action potential duration (APD) is one component of preclinical safety assessment. Current assays are limited by technically-demanding, time-consuming electrophysiological methods. This study aimed to assess whether a voltage-sensitive dye-based assay could be used instead.


METHODS
Optical APs were recorded using di-4-ANEPPS in electrically field stimulated beagle left ventricular midmyocardial myocytes (LVMMs). Pharmacological properties of di-4-ANEPPS on the main cardiac ion channels that shape the ventricular AP were investigated using IonWorks and conventional electrophysiology. Effects of 9 reference drugs (dofetilide, E4031, D-sotalol, ATXII, cisapride, terfenadine, alfuzosin, diltiazem and pinacidil) with known APD-modulating effects were assessed on optically measured APD at 1 Hz.


RESULTS
Under optimum conditions, 0.1 microM di-4-ANEPPS could be used to monitor APs paced at 1 Hz during nine, 5 s exposures without altering APD. di-4-ANEPPS had no effect on either hI(ERG), hI(Na), hI(Ks) and hI(to) currents in transfected CHO cells (up to 10 microM) or I(Ca,L) current in LVMMs (at 16 microM). di-4-ANEPPS had no effect on APs recorded with microelectrodes at 1 or 0.5 Hz over a period of 30 min di-4-ANEPPS displayed the sensitivity to record changes in optically measured APD in response to altered pacing frequencies and sequential vehicle additions did not affect the optically measured APD. APD data obtained with 9 reference drugs were as expected except (i) D-sotalol-induced increases in duration were smaller than those caused by other I(Kr) blockers and (ii) increases in APD were not detected using low concentrations of terfenadine.


CONCLUSIONS
Early in drug discovery, the di-4-ANEPPS-based method can reliably be used to assess drug effects on APD as part of a cardiac risk assessment strategy.
Hardy
Matthew E L
ME
Safety Pharmacology Department, Safety Assessment UK, AstraZeneca R&D, Alderley Park, Macclesfield, Cheshire SK104TG, UK.
Pollard
Chris E
CE
Small
Ben G
BG
Bridgland-Taylor
Matthew
M
Woods
Ann J
AJ
Valentin
Jean-Pierre
JP
Abi-Gerges
Najah
N
D000200:Action Potentials / Q000187:drug effects*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004195:Disease Models, Animal; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery; D064420:Drug-Related Side Effects and Adverse Reactions; D004558:Electric Stimulation; D004594:Electrophysiology; D005260:Female; D005456:Fluorescent Dyes / Q000494:pharmacology; D006352:Heart Ventricles / Q000166:cytology; D066298:In Vitro Techniques; D007473:Ion Channels / Q000502:physiology; D009200:Myocardial Contraction / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000502:physiology; D011726:Pyridinium Compounds / Q000494:pharmacology
D005456:Fluorescent Dyes; D007473:Ion Channels; D011726:Pyridinium Compounds; C050019:1-(3-sulfonatopropyl)-4-(beta)(2-(di-n-butylamino)-6-naphthylvinyl)pyridinium betaine