Hello community,
We are currently studying different drugs and how they interact with each other, but I am struggling with data interpretation. I've looked through many questions here and I see many others experience the same problems, so I would like to know your experience on this topic.
What we are doing is to calculate CI values using Cell Titer Glo and analysing with CompuSyn the (normalized) values obtained. However, the results vary significantly (from extremely synergistic to mildly antagonistic) depending on the data we introduce.
The main issues we have are:
1. Should we repeat the dose-response curves with every experiments, or can we use previous ones? For the drug combination we use lower doses of the drugs alone, as we don't want effects over 90% that would be irrelevant for the combination later on. However, the drug alone has only a 60% max effect, which gives us bad curves (with r= 0.6-0.8). We are not sure whether we should still use these curves for the analysis or use other curves calculated before with higher doses (and r values of >0.95).
2. What range of Fa should we consider? As many people point out, extremely low or high values always affect the calculations and should be excluded. Is there a optimal range/criteria to follow, or is it just lab-based? We experience more problems with low Fa, with higher errors and variability from experiment to experiment.
3. Is there a better alternative to CompuSyn? We are overall happy with the software, but it has some limitations and we see that the parameters change considerably when adding/removing a single value, which makes me think the results are not so strong.
4. Is it better to use more points while keeping the drug ratio constant (e.g.: 8 points with a ratio 1:1) or less points with different ratios (e.g.: 4 points with a ratio 1:1; 4 points with a ratio 1:2)?
Thanks a lot for your time and your advice!
Heathcliff Dorado
Personally, to do CI calculations, I just use excel. It forces you to understand what your data means, and how the calculation works, which is a good thing, and also lets you set up the data however you like, which is also a good thing.
As for your specific questions: 1) How reliable/reproducable are your IC50 values? If they're pretty consistent, it doesn't matter much. If you get large fluctuations based on the exact cell density/settling/etc., it's probably a good idea to do all the experiments simultaneously. And for CI, all that matters for the combination dose is that you use a constant ratio of inhibitors: basing that around IC50 is common, but not required, so if your IC50 shifts, it won't make the combination data less valid.
2) In the original paper, Chou and Talalay plotted the CI for every value of Fa from 0.5 to 0.95. That's not a bad way to do it - it gives you a plot that gives you a feel for where your inhibitors should or should not synergize nicely. As an alternative, you can simply calculate the CI for the specific concentrations you used and Fa's you measured - this can be a bit harder to plot, but also lets you report direct data, while the full plot is really a bit more of a model of the data.
3) Again, I just use excel, but: my guess is that Compusyn is linearizing IC50 plots, the same way the original paper recommended. This means that removing one or two points, especially at either end of your curve, can have a huge result. This is simply because in a typical dose curve, you want to draw the no-inhibition and max-inhibition plateaus cleanly - when you linearize, those extended points tend not to be on the line. If you do it manually, you can get a feel for which points should be included and which are outside the linear range.
4) For the traditional CI calculation, your drug ratio has to be constant (e.g. 1:1). So if you do 4 points at 1:1, and 4 at 1:2, you're effectively doing two different experiments. This isn't bad, per se, but it does mean you're giving half as much data for any given experiment as 8 points at 1:1. So this is really more a question of what you want: 8 points at one ratio gives you a better picture of CI at that ratio. 4 points at two ratios lets you know if the drugs synergize/antagonize somewhat better at one ratio or another.
If it helps, a reviewer for Katt et al. (Mol Pharm, 2015) asked that we put a step-by-step sample calculation for CI with two drugs into the supplemental information of that paper, so you could look there for such a guide. It is, arguably, a bit easier to follow than the original Chou and Talalay paper, but doesn't cover the contingencies Chou and Talalay described either.
Thank you for your answers! They have been very useful to better understand the CI!!
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