The impressive list of drugs, already on the market or still under development that have been reported to adversely prolong repolarisation, makes it imperative to investigate any new chemical entity for this potential side effect before its first use in man.
1Haverkamp W, Breithardt G, Camm AJ, Janse MJ, Rosen MR, Antzelevitch C, Escande D, Franz M, Malik M, Moss A and Shah R. (2000) Eur Heart J 21 (15); 1216-31.
|Instrument||Ionworks™ HT System (Molecular Devices)|
|Cell Line||CHO-hERG cells|
|Perforating Agent||Amphotericin B|
|Test Article Concentration||0.008, 0.04, 0.2, 1, 5, 25 μM (different concentrations available)|
|Final DMSO Concentration||0.25%|
|Number of Replicates||4 replicates per concentration|
|Quality Controls||0.25% DMSO (negative control)
Quinidine (positive control)
Seal resistance must be > 50 MOhms
Pre-compound current must be ≥ 0.1 nA)
|Test Article Requirements||100 µL of 10 mM solution|
|Data Delivery||IC50 determination|
For the validation, a literature search was performed to identify a selection of compounds which were known to inhibit the hERG current with a range of potencies.
A single hERG-expressing cell is positioned by negative pressure over a pore in the bottom of each well of a specially designed patch plate containing 384 wells. The aperture separates two isolated fluid filled upper and lower chambers. The positioned cells form stable seals over the apertures impeding electrical flow between the two chambers. A cell membrane pore-forming agent (Amphotericin B) is introduced into the lower chamber creating an electrical pathway through the portion of the cell membrane exposed via the small aperture in each well. An electronics head containing 48 electrodes is positioned in the upper chamber clamping the cell membrane potential and subsequently recording ionic currents from up to 48 cells in parallel. Current is monitored before and after test compound addition.
Why is it important to investigate hERG inhibition?
The human ether-a-go-go related gene (hERG) encodes the inward rectifying voltage gated potassium channel in the heart (IKr) which is involved in cardiac repolarization. Inhibition of the hERG current causes QT interval prolongation resulting in potentially fatal ventricular tachyarrhythmia called Torsade de Pointes. A number of drugs have been withdrawn from late stage clinical trials due to these cardiotoxic effects, therefore it is important to identify inhibitors early in drug discovery1.
Please provide an overview of Cyprotex hERG Safety assay.
The hERG inhibition assay uses a high throughput single cell planar patch clamp approach. Chinese hamster ovary cells transfected with the hERG gene (CHO-hERG) are dispensed into the PatchPlate. Amphotericin is used as a perforating agent to gain electrical access to the cells. The hERG tail current is measured prior to the addition of the test compound by perforated patch clamping. Following addition of the test compound (typically 0.008, 0.04, 0.2, 1, 5, and 25 µM, n= 4 cells per concentration, final DMSO concentration = 0.25%), a second recording of the hERG current is performed.
Post-compound hERG currents are expressed as a percentage of pre-compound hERG currents (% control current) and plotted against concentration for each compound. Where concentration dependent inhibition is observed the Hill equation is used to fit a sigmoidal line to the data and an IC50 (concentration at which 50% inhibition is observed) is determined.
What controls are included in the hERG assay?
Only cells with a seal resistance greater than 50MOhm and a pre-compound current of at least 0.1nA are used to evaluate hERG blockade. Quinidine, a known hERG inhibitor, is used as a positive control for the experiment. DMSO is included as a negative control.
How does in vitro hERG inhibition relate to in vivo cardiotoxicity?
The hERG channel inhibition assay is a highly sensitive measurement which will identify compounds exhibiting cardiotoxicity related to hERG inhibition in vivo. It is important to note, however that not all compounds which inhibit hERG activity in vitro will proceed to cause cardiotoxicity in vivo. The relevance of the in vitro data will be dependent on other factors such as the plasma concentrations reached in vivo.
1 Haverkamp W et al. (2000) Eur Heart J 21(15); 1216-31
2 Kiss L et al. (2003) Assay Drug Dev Technol 1; 127-135
3 Schroeder K et al. (2003) J Biomol Screen 8(1); 50-64