Drug Development
technique, positive pressure is applied to the glass pipette to keep the tip of the pipette clean. Subsequently, mild suction is applied when the tip of the pipette touches the cell. As a result, the membrane of the cell enters the pipette and a tight seal forms between the cell membrane and the inner surface of the pipette. Finally, more suction is applied to disrupt the membrane and an electric circuit is established between the electrode in the micropipette and the cytoplasm. In this way, the potential difference between a bath electrode and the electrode in the pipette directly reflects the membrane potential. By measuring cells that over- express the hERG ion channel (such as HEK-293 cells stably transfected with hERG-1 cDNA), it is possible to accurately assess the effect that a drug candidate has on the current IKr that is conducted through the hERG ion channel.
An estimated 25-40% of all drug candidates show some level of hERG-related inhibition, which results in a high level of attrition due to QT-pro- longation in the preclinical phase8. The early detec- tion of hERG cardiac toxicity and use of the data for compound development with better safety pro- files has therefore been proposed by the pharma- ceutical industry as a useful strategy9. Toxicity screening in the lead optimisation phase requires assays and techniques that have a relative high throughput and need a very small amount of test
Figure 2
The principle of the automated cytocentring
technique mimics conventional (manual) patch clamping. After catching the cell the principle of both techniques is equal.
Positive pressure is applied to the pipette to keep it clean. Suction is applied to form a tight seal between the membrane of the cell and the surface of the glass pipette. More suction is applied to disrupt the membrane and an electrical circuit is established
sample4. Yet, the manual patch clamp technique is low throughput and requires a high amount of compound, which makes this standard technique less suitable during the lead optimisation phase of drug development.
A new option: automated patch clamping
There are several automated patch clamp plat- forms on the market9-12 that are designed to address the drawbacks of manual methods. All have their own characteristics, throughput and data quality.
Automated systems suspend cells that are inject- ed into a recording chamber containing a planar substrate with one or more small apertures. In this chamber, the cells are captured by negative pres- sure and directed on to the patch aperture. The negative pressure is then increased to help form a tight seal between the cell membrane and the chamber10. The formation of a tight seal is prereq- uisite for high-quality patch clamp recordings (good voltage control of the cell as well as a stable measurement). An electrical circuit is established after disrupting the membrane either by membrane rupture or the application of pore forming chemi- cals. By using a specific voltage protocol and cells with an overexpression of the hERG ion channel, researchers can determine a drug candidate’s
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Drug Discovery World Spring 2013
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