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ADME PK

hERG safety

  • 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  • A number of drugs have been withdrawn from late stage clinical trials due to these cardiotoxic effects, therefore it is important to identify inhibitors early in drug discovery.
  • The Cyprotex hERG Safety service is a cell-based assay which employs the Ionworks™ HT System (Molecular Devices) as an automated patch clamp electrophysiology measurement.
  • The Ionworks™ HT system delivers high quality, accurate and sensitive data which is comparable with the traditional single cell patch clamp method.
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.

Protocol

hERG safety protocol

Instrument Ionworks™ HT System (Molecular Devices)
Analysis Method Electrophysiology
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

Data

Data from Cyprotex's hERG safety assay

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.

 
hERG patch clamp diagram
Figure 1
hERG safety experimental approach.

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.

hERG safety data
Figure 2
Comparison of hERG safety data with published traditional patch clamp data.

The graphs illustrates that the hERG safety assay using the Ionworks™ HT system generates data comparable with traditional single cell patch clamp measurements.
hERG IC50 data
Figure 3
hERG safety IC50 data generated for a set of compounds over 3 separate days.

The data illustrate that good consistency is achieved over a number of different days for compounds with a range of different potencies. The method used in the Cyprotex hERG Safety assay has also been extensively validated by other groups: Kiss et al., 2003; Schroeder et al., 2003.

Q&A

Questions and answers on hERG safety

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.

hERG inhibition
Figure 4
Inhibition of hERG by the positive control compound, quinidine.

IC50 (± standard error) = 1080 ± 98.0 nM

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.

References

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

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