Variability in the measurement of hERG potassium channel inhibition: effects of temperature and stimulus pattern.
BACKGROUND
In vitro evaluation of drug effects on hERG K(+) channels is a valuable tool for identifying potential proarrhythmic side effects in drug safety testing. Patch-clamp recording of hERG K(+) current in mammalian cells can accurately evaluate drug effects, but the methodology has not been standardized, and results vary widely. Our objective was to evaluate two potential sources of variability: the temperature at which recordings are performed and the voltage pulse protocol used to activate hERG K(+) channels expressed in HEK293 cells.


METHODS
A panel of 15 drugs that spanned a broad range of potency for hERG inhibition and pharmacological class was evaluated at both room and near-physiological temperatures using several patch-clamp voltage protocols. Concentration-response analysis was performed with three stimulus protocols: 0.5- and 2-s step pulses, or a step-ramp pattern.


RESULTS
Block by 2 of the 15 drugs tested, d,l-sotalol (antiarrhythmic) and erythromycin (antibiotic), was markedly temperature sensitive. hERG inhibition measured using a 2-s step-pulse protocol underestimated erythromycin potency compared with results obtained with a step-ramp protocol. Using conservative acceptance criteria and the step-ramp protocol, the IC(50) values for hERG block differed by less than twofold for 15 drugs.


CONCLUSIONS
Data obtained at near-physiological temperatures using a step-ramp pattern are highly repeatable and provide a conservative safety evaluation of hERG inhibition.
Kirsch
Glenn E
GE
ChanTest, Inc., 14656 Neo Parkway Cleveland, OH 44128, USA. gkirsch@chantest.com
Trepakova
Elena S
ES
Brimecombe
Jessica C
JC
Sidach
Serguei S
SS
Erickson
Hope D
HD
Kochan
Mary C
MC
Shyjka
Lisa M
LM
Lacerda
Antonio E
AE
Brown
Arthur M
AM
D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D000072237:ERG1 Potassium Channel; D004558:Electric Stimulation; D051638:Ether-A-Go-Go Potassium Channels; D006801:Humans; D020128:Inhibitory Concentration 50; D018408:Patch-Clamp Techniques; D004364:Pharmaceutical Preparations / Q000008:administration & dosage*; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D024642:Potassium Channels, Voltage-Gated / Q000037:antagonists & inhibitors* / Q000187:drug effects*; D015203:Reproducibility of Results; D013696:Temperature; D014162:Transfection
D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D004364:Pharmaceutical Preparations; D026902:Potassium Channel Blockers; D024642:Potassium Channels, Voltage-Gated