Solubility of drugs, EC50 determination, etc? What parameters or specifications should I consider, while working with signalling mechanisms involving AEDs?
There are a lot of things to consider in your study to ensure that it is scientifically relevant. First you need to know what the EC50 is. This may necessitate the use of a dose response experiment. You do not want to saturate a receptor as it may not be physiologically relevant. The EC50 may also give some insight to how lethal your compound may be (another consideration). you also need to know what you have to dissolve it in. This is because the vehicle may have an effect on its own. Thus you will have to make a control for it (e.g. using DMSO to dissolve some drugs). however you would need to give us some more information regarding your study, if you want more help on how to design appropriate controls etc....
I don't know what really you want to do. If you give some more explanation about your work I may have more suggestion. in general, you must be careful about the solution PH, some times acidic solutions have a suppressant effect on seizure, in vitro. an other issue you must be careful is how long your drug can stay effective after making solution some drugs can not stay effective in solution for a long time. then you have to prepare the drug solutions freshly . the vehicle is important since the vehicle may have an effect on epileptiform activities. The dose of drug is important also, some drug may have biphasic effect on seizure dose dependently. you must have a dose response cure for any drug you use.
Hi Josef and Ehsan,
My question is to check the uptake activity of receptor using radio labeled uptake assay.
I am working with neuronal cell line and have data on neurotransmitter uptake through cell lines implicating role of WT and Mutant receptors.
now i want to check whether addition of these drugs would help in rescue. Si ideally I would be dissolving the drugs in the uptake buffer, incubate the cells in culture with this drug - buffer at a pH 7.2 for 5 mins with/without the neurotransmitter.
Control of the vehicle alone is needed. One of the drugs is water soluble but the other is ethanol soluble. I don't know how can I possibly dissolve that in ethanol and use it in a buffer and what will happen to my cells!.. So basically a final conc of drug in the buffer which is not killing my cells have to be determined so dosage based on a constant amount of neurotransmitter but a range of drug and determine the rate of uptake. Best fit conc. can be used further.
So how to go about a dose-response curve the fit equations for its kinetics is a question too.
Hi Kalpita
I am sorry I can not give a suggestion to help you in your investigation, because I have never had an experience working with radio labeled uptake assay in cell culture. Generally, I use brain slices and/ or whole hippocampus to study epileptiform activities. If you are interested please look at saboory et al 2007
So one of the issues is a difference between ethanol and buffer soluble drugs. Often EtOH soluble drugs will fall out of solution when placed into the buffer or medium in the cell culture dish. While you haven't mentioned experimental design, I will assume you are doing these experiments without serum in the medium. If you are, then you need to take into account that the albumin may bind your drug of interest, thereby lowering the true free concentration.
Either way, just remember if you are comparing the impact of these two drugs, make sure EtOH is added to both groups to make the comparison valid.
Good luck with your experiments.
Hi Eric,
I would be working on few drugs where the comparison is essentially among the WT and mutant groups rather than among the drugs. So, one of the drugs is EtOH soluble and others are water soluble. My concern was exactly what would happen to the one's in EtOH when put in buffer of pH-7.2!
No My dishes will not have serum. It will be washed and replaced with HEPES based buffer to maintain cells and drugs will be added along with radio labeled amino acid to assess its uptake through scintillation.
I want to check with and without drugs, what is uptake rate and hence compare each group of Control Vs mutant constructs expressing the protein.
Hi Sarang,
Fluorescence labelling for amino acids is difficult, I mean in radiolabeled ones you can manupilate one hydrogen or carbon and use it for detection. Tags like biotin is available for fluorescence which can be used in IF to check uptake but since uptake is a fast reaction, this is the best way although its radioactive. And since its Tritium or C14, its not that harmful also. Counts are stable. In fluorescence, an ELISA based approach is possible but little tricky I would say. Since uptake is more of a kinetic phenomena, radiolabeled one is more standard and classical.
What is the pKa of the EtOH soluble drug? Often times the pH at which you make up a drug is highly important for solubility and manipulating that aspect will help keep a drug in solution for the stock solution and then dilution is generally less of an impact. The big issue is will the drug be in solution when you put it into the medium as a EtOH stock solution.
No effect may very well be merely due to the drug not being in solution. So be careful.
Also, when washing cells be aware that many g-protein coupled processes may be initiated after washing, e.g. ARA release, PLA2 activated, PLC activated etc. So, being gentle is important and give the cells some time to settle down after the washing step.
I agree with you that radiotracer is the way to go on this experiment.
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