drog concentration cell number and hERG current

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zhangxu04's picture
drog concentration cell number and hERG current

ok, I will record the hERG to calculate IC50 for  drug candidate.
I have some questions:
1. how many cells I should record for each drug ?  10 20 0r more ?
2. what  concentration of IC50 can be regaded as safe for a drug candidate? what is the standard?
3 how many point of drug concentrations shouls I used and how to decide the starting concentration point ? 
4 I read some paper. I found usually a cell was used to different drug concentration. I think this should  need a long time, maybe more than one hour. so the rundown of the hERG current could be obvious. so I want to know if I can used one cell for just one drug concentration.
5 If I used only one cell for different concentration, how long of the interval to washing out the previous cincentration drog is enough? I worry much the interactive effect caused by rudimental drug at different concentration.
thank you very much .

Bluejay's picture

I haven't seen any required numbers for these steps.  The current best practice still seems to be evolving.  You might want to check www.Chantest.com or www.Cerep.fr to see how they do hERG assays.  Also, you can review postnigs on the Scientist solutions website and the ICH (international committee on harmonization) guidelines.  Here's my take on these regulations and perhaps other experts have more precise answers.
1)  6 good readings at one concentration or 3 good readings per concentration for a dose response.  Depends on teh scatter or variability of your data.
2)  In general, an IC50 for herg of less than 100nM is a problem, 100-1000 is moderate and 1uM to 10uM is slight.  It depends on the drug level in the blood.  My understanding is that you are OK at 30 times the test drug concentration, i.e. if your drug is at 10nM in the blood, then herg IC50 of above 300nM would be tolerable.
3)  Most people use 4-6 concentrations with traditional patch clamp, bracketing the IC50 concentration.
4)  It is best to use one cell per concentration.  You could try washing the cell and see if it returns to a non-drug status.  If so, then the cell could be re-used.
5)  See #4 above.