Hi Alexandra,

I've done tests like this testing for synergy between antiviral molecules and their inhibition of virus replication.

A simple test is to add small amounts of each drug together and quantify their inhibition. Start with well less than the IC50 - if you see any synergy, you'll want to start with less. So, say take 10% of the IC50 of each drug. Test 10%IC50 of each drug individually and get quantification of those individual inhibitions, and then put these two concentrations together and quantify the resulting combinatory inhibition. If you get more inhibition than 20% (really 19% if the effects multiply as one would expect if there's no synergy or antagonism), then you have reason to believe they're synergistic. If you get less than 19% inhibition, then they may be competing with one another.

That is really useful but preliminary, incomplete data.

In order to fully test for synergism between any two drugs, you need to test a concentration matrix, with a number of concentrations of each drug. Prepare a dilution curve of each drug (n dilutions), with the IC50 in the middle of your dilution curve. Then put these dilutions sets for each substance perpendicular to each other in a test plate and test the full number of concentration combinations (n^2) for enzyme inhibition.

Then you'll have data in a matrix that shows the total effect of these two substances at each concentration pair. You can then analyze these data to look for synergy. Take a look at my paper link here for ideas of how to set up these experiments and do the analysis:

Article Kinetic Differences and Synergistic Antiviral Effects Betwee...

Good luck!

Thank you for your answer! So, to make it simple.... in the first step it is good if I test substance A alone at concentration of 10 mM (less that the IC50) and substance B alone at 40 mM (which is less than its IC50) and then the ocmbination substance A 10 mM:substance B 40 mM=1:1 (volume/volume)? And then i can compare the percentages of inhibition?

Isobologram is the best method and gold standard to study drug interaction. Its simple and the most accepted one. If you need help you can mail me [email protected].

Loewe S. The problem of synergism and antagonism of combined drugs. Arzneimittelforschung. 1953;3:285–290.

Parasitology. 2016 Sep;143(11):1421-32. doi: 10.1017/S0031182016000937

Hi again Alexandra,

I think you are on the right track. What I would do, just to start, is put in IC25 dose of substance A all by itself in volume X, IC25 dose of substance B all by itself also in volume X, and do your reaction. Each of these by itself should inhibit 25% of your reaction by definition, but re-test in your assay. At the same time, put the substance A IC25 dose together with the substance B IC25 dose both in the same final volume X, and do your reaction. If your substances simply have their normal effects in the combination (no synergy or competition), you should get around 50% inhibition (closer to 44% most likely) from the combination. If the substances synergize, you will get significantly more than 50% inhibition. If the substances are competitive with one another, you'll get less than 50% inhibition.

Once you do that, then you move on to the matrix with combinations of substances A and B at many different doses. (See Figure 6 in my paper I linked above for how the resulting data can be plotted). From the results of that, you can interpolate the isoboles that Swaroop is mentioning above (the red lines in Fig 6 in my paper - see the paragraphs in my paper link above that follow Figure 1 for how to do it using the provided equation). The isoboles are one way of looking at the data, but mathematically speaking not the most useful - they tell you whether or not your drugs are strictly additive in nature, but not if they truly synergize.

Real synergy, defined as 2 drugs that enhance each other's action, goes beyond Loewe Independence (2 drugs that simply have similar effects adding to each other) presented in the paper Swaroop mentions first. Figure 1 of my paper explains the difference. To decide whether you have true drug synergy (rather than their effects simply multiply out) you can re-create the plots I present in Figure 7 of the paper, and see if your datapoints from your matrix experiment follow the predicted inhibitory line or not.

I'm happy to answer any further questions you have if you. I know mathematicians working with a group that studies synergy between antibiotics who are devising a much improved strategy for analyzing synergy, and I expect this field will go a long way past the Loewe model and such in the coming few years.

Good luck!