ICH S7B and hERG assay

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lazy
lazy's picture
ICH S7B and hERG assay

I have questions regarding ICH S7B guideline, especially regarding in vitro cardiotoxicity (hERG assay). The item 3.1.1 of guideline mentions about the positive controls and reference compounds.
1: Positive control: is it possible to interprete that positive control should be used always in any recording of hERG assay for FDA? For example, a result must be: control / postive control/ washout /test compound / complete block of hERG current. Or, can I interprete that control / positive control / complete block of hERG current in independent cells?
2: Super-maximally effective postitive compounds cannot be used as the positive control?
3: The definition of reference compounds is not clear, and should such compounds be used always?
I am currently working in BioTech and I like to understand this difficult guideline. 
Thanks. 
 
 
 

Fraser Moss
Fraser Moss's picture
Terfenadine, Astemizole,

Terfenadine, Astemizole, Cisapride and Quinidne are common positive control compounds.
 
You run positive AND negative (DMSO) control drug applications in cells prepared under the same conditions used to test the drug of interest to show that the cell line and assay is behaving normally.
 
See www.moleculardevices.com/pages/instruments/ionworks_herg.html for an example of running a hERG screen using positive controls.
 
You use the means, Standard deviations of both th eposotive and negative controls to calulate the "Z factor"

Where σp = Standard deviation positive control; σn = Standard deviation negative control; µp = Mean positive control; µn = Mean negative control
 
Th Z factor value can be interpreted as a measure of how good your assay is.

Z-factor
Interpretation

1.0
Ideal. Z-factors can never exceed 1.

0.5 - 1.0
An excellent assay. Note that if σp = σn, 0.5 is equivalent to a separation of 12 standard deviations between μp and μn.

0-0.5
A marginal assay.

< 0
There is too much overlap between the positive and negative controls for the assay to be useful.

See Zhang JH, Chung TD, Oldenburg KR, "A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays." J Biomol Screen. 1999;4(2):67–73 for hte reference
 

lazy
lazy's picture
Thanks, Frasermoss.

Thanks, Frasermoss.
In terms of regulation (not research),
Shoul all positive control/negative contorls and test drug be applied to one cell?
Should postive control be sub-maximally effective concentration? Is super-maximally effective concentration not reasonable for verifying the cell functionality?
Thanks.
 

Fraser Moss
Fraser Moss's picture
You cannot apply all

You cannot apply all compounds to a single cell because you wont get complete washout of the hERG blockers.
If you apply IC50 and IC80 doses of the positive control compound it will show that your assay is accurately calibrated.
Super-maximal doses are less informative because you wont know whether or not the 100% block occurred at an artificially low dose or not.