Optimisation and validation of a medium-throughput electrophysiology-based hNav1.5 assay using IonWorkstrade mark.

Optimisation and validation of a medium-throughput electrophysiology-based hNav1.5 assay using IonWorkstrade mark. Research Abstract Details 

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Optimisation and validation of a medium-throughput electrophysiology-based hNav1.5 assay using IonWorkstrade mark. Abstract Text:

A R Harmer,N Abi-Gerges,A Easter,A Woods,C L Lawrence,B G Small,J-P Valentin,C E Pollard,

INTRODUCTION: The safety implications of blocking the human cardiac Na(+) channel (hNav1.5) make it prudent to test for this activity early in the drug discovery process and design-out any potential liability. This needs a method with adequate throughput and a demonstrable predictive value to effects in native cardiac tissues. Here we describe the validation of a method that combines the ability to screen tens of compounds a day, with direct assessment of channel function. METHODS: The electrophysiological and pharmacological properties of hNav1.5 were compared using two methods: conventional, low-throughput electrophysiology and planar-array-based, medium-throughput electrophysiology (IonWorkstrade mark HT). A pharmacological comparison was also made between IonWorkstrade mark HT and canine cardiac Purkinje Fibre action potential upstroke data. RESULTS: Activation curve parameters for hNav1.5 in IonWorkstrade mark HT were not statistically different (p>0.05) from those generated using conventional electrophysiology. IonWorkstrade mark HT V(1/2)=-22+/-0.8 mV, slope=6.9+/-0.2 (n=11); conventional electrophysiology V(1/2)=-20+/-1.6 mV, slope=6.4+/-0.3 (n=11). Potency values for a range of hNav1.5 blockers determined using IonWorkstrade mark HT correlated closely with those obtained using conventional electrophysiology (R=0.967, p<0.001). The assay was able to distinguish between highly use-dependent blockers (e.g. tetracaine) and blockers that do not display strong use-dependence (e.g. quinidine). Comparison of the degree of hNav1.5 inhibition and decrease in canine Purkinje fibre action potential upstroke velocity (V(max)) showed that the IonWorkstrade mark HT assay would have predicted the outcome in Purkinje fibres in the majority of cases, with false negative and positive rates estimated at 8 and 7%, respectively. Finally, hNav1.5 pharmacology was similar when determined using either IonWorkstrade mark HT or IonWorkstrade mark Quattro, although the latter yielded more consistent data. DISCUSSION: The assay described combines a functional assessment of hNav1.5 with medium-throughput. Furthermore the assay was able to reveal information on the use-dependency of compound block, as well as predicting Na(+) channel effects in more integrated systems such as the cardiac Purkinje fibre action potential. This makes it possible to determine quantitative potency data, and mechanistic information about use-dependence, in a timeframe short enough to influence medicinal chemistry.

Optimisation and validation of a medium-throughput electrophysiology-based hNav1.5 assay using IonWorkstrade mark. Publishing Authors By Initials

AR Harmer,N Abi-Gerges,A Easter,A Woods,CL Lawrence,BG Small,JP Valentin,CE Pollard,

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Optimisation and validation of a medium-throughput electrophysiology-based hNav1.5 assay using IonWorkstrade mark. Journal Published:

PUBLICATION TYPE: Journal Article

Journal: Journal of pharmacological and toxicological metho

VOLUME: 57

Page Numbers: 30-41

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ISSN: 1056-8719

DAY: 26

MONTH: 09

YEAR: 2007

Optimisation and validation of a medium-throughput electrophysiology-based hNav1.5 assay using IonWorkstrade mark. Information

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LANGUAGE: eng

NlmUniqueID: 9206091

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AFFILIATION: Safety Assessment UK, AstraZeneca R&D Alderley Park, Macclesfield, SK10 4TG, UK.

Country: United States

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MEDLINETA: J Pharmacol Toxicol Methods

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