title: Optimisation and validation of a medium-throughput electrophysiology-based hNav1.5 assay using IonWorks.
abstract: BACKGROUND
  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 (IonWorks HT). A pharmacological comparison was also made between IonWorks HT and canine cardiac Purkinje Fibre action potential upstroke data.
  
  
  RESULTS
  Activation curve parameters for hNav1.5 in IonWorks HT were not statistically different (p>0.05) from those generated using conventional electrophysiology. IonWorks 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 IonWorks 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 IonWorks 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 IonWorks HT or IonWorks Quattro, although the latter yielded more consistent data.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Safety Assessment UK, AstraZeneca R&D Alderley Park, Macclesfield, SK10 4TG, UK. alex.harmer@astrazeneca.com
authors.forename: A R
authors.initials: AR
authors.lastname: Harmer
authors.forename: N
authors.initials: N
authors.lastname: Abi-Gerges
authors.forename: A
authors.initials: A
authors.lastname: Easter
authors.forename: A
authors.initials: A
authors.lastname: Woods
authors.forename: C L
authors.initials: CL
authors.lastname: Lawrence
authors.forename: B G
authors.initials: BG
authors.lastname: Small
authors.forename: J-P
authors.initials: JP
authors.lastname: Valentin
authors.forename: C E
authors.initials: CE
authors.lastname: Pollard
mesh_terms: D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004594:Electrophysiology; D005260:Female; D006801:Humans; D020128:Inhibitory Concentration 50; D008297:Male; D008564:Membrane Potentials; D009124:Muscle Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D062554:NAV1.5 Voltage-Gated Sodium Channel; D011237:Predictive Value of Tests; D011690:Purkinje Fibers / Q000187:drug effects; D015203:Reproducibility of Results; D026941:Sodium Channel Blockers / Q000494:pharmacology; D015222:Sodium Channels / Q000378:metabolism*
chemical_list: D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; C568320:SCN5A protein, human; D026941:Sodium Channel Blockers; D015222:Sodium Channels