Comparative pharmacology of guinea pig cardiac myocyte and cloned hERG (I(Kr)) channel.
BACKGROUND
This study used whole-cell, patch clamp techniques on isolated guinea pig ventricular myocytes and HEK293 cells expressing cloned human ether-a-go-go-related gene (hERG) to examine the action of drugs causing QT interval prolongation and torsades de pointes (TdP) in man. Similarities and important differences in drug actions on cardiac myocytes and cloned hERG I(Kr) channels were established. Qualitative actions of the drugs on cardiac myocytes corresponded with results obtained from Purkinje fibers and measurement of QT interval prolongation in animal and human telemetry studies.


RESULTS
Adult guinea pig ventricular myocytes were isolated by enzymatic digestion. Cells were continuously perfused with Tyrode's solution at 33-35 degrees C. Recordings were made using the whole-cell, patch clamp technique. Action potentials (APs) were elicited under current clamp. Voltage clamp was used to study the effect of drugs on I(Kr) (rapidly activating delayed rectifier potassium current), I(Na) (sodium current), and I(Ca) (L-type calcium current). Dofetilide increased the myocyte action potential duration (APD) in a concentration-dependent manner, with a pIC50 of 7.3. Dofetilide 1 microM elicited early afterdepolarizations (EADs) but had little affect on I(Ca) or I(Na). E-4031 increased APD in a concentration-dependent manner, with a pIC50 of 7.2. In contrast, 10 microM loratadine, desloratadine, and cetirizine had little effect on APD or I(Kr). Interestingly, cisapride displayed a biphasic effect on myocyte APD and inhibited I(Ca) at 1 microM. Even at this high concentration, cisapride did not elicit EADs. A number of AstraZeneca compounds were tested on cardiac myocytes, revealing a mixture of drug actions that were not observed in hERG currents in HEK293 cells. One compound, particularly AR-C0X, was a potent blocker of myocyte AP (pIC50 of 8.4). AR-C0X also elicited EADs in cardiac myocytes. The potencies of the same set of drugs on the cloned hERG channel also were assessed. The pIC50 values for dofetilide, E-4031, terfenadine, loratadine, desloratadine, and cetirizine were 6.8, 7.1, 7.3, 5.1, 5.2, and <4, respectively. Elevation of temperature from 22 to 35 degrees C significantly enhanced the current kinetics and amplitudes of hERG currents and resulted in approximately fivefold increase in E-4031 potency.


CONCLUSIONS
Our study demonstrates the advantages of cardiac myocytes over heterologously expressed hERG channels in predicting QT interval prolongation and TdP in man. The potencies of some drugs in cardiac myocytes were similar to hERG, but only myocytes were able to detect important changes in APD characteristics and display EADs predictive of arrhythmia development. We observed similar qualitative drug profiles in cardiac myocytes, dog Purkinje fibers, and animal and human telemetry studies. Therefore, isolated native cardiac myocytes are a better predictor of drug-induced QT prolongation and TdP than heterologously expressed hERG channels. Isolated cardiac myocytes, when used with high-throughput patch clamp instruments, may have an important role in screening potential cardiotoxic compounds in the early phase of drug discovery. This would significantly reduce the attrition rate of drugs entering preclinical and/or clinical development. The current kinetics and amplitudes of the cloned hERG channel were profoundly affected by temperature, significantly altering the potency of one drug (E-4031). This finding cautions against routine drug testing at room temperature compared to physiologic temperature when using the cloned hERG channel.
Davie
Christina
C
Lead Generation & Discovery BioScience, AstraZeneca, R&D Charnwood, Loughborough, Leicestershire, United Kingdom.
Pierre-Valentin
Jean
J
Pollard
Chris
C
Standen
Nick
N
Mitcheson
John
J
Alexander
Paul
P
Thong
Bob
B
D000818:Animals; D027682:Cation Transport Proteins / Q000187:drug effects* / Q000502:physiology; D020117:Cisapride / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D022062:Electrophysiologic Techniques, Cardiac; D051638:Ether-A-Go-Go Potassium Channels; D006168:Guinea Pigs; D066298:In Vitro Techniques; D017336:Loratadine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008297:Male; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000502:physiology; D010627:Phenethylamines / Q000494:pharmacology; D024642:Potassium Channels, Voltage-Gated / Q000187:drug effects* / Q000502:physiology; D013449:Sulfonamides / Q000494:pharmacology; D013997:Time Factors; D016171:Torsades de Pointes / Q000139:chemically induced* / Q000503:physiopathology
D027682:Cation Transport Proteins; D051638:Ether-A-Go-Go Potassium Channels; C092509:KCNH6 protein, human; D010627:Phenethylamines; D024642:Potassium Channels, Voltage-Gated; D013449:Sulfonamides; D017336:Loratadine; C121345:desloratadine; C063533:dofetilide; D020117:Cisapride