Figure 1. Schematic representation of suppressive drug interactions: Single concentrations and gradients.

Here we show suppressive drug interactions in two-drug and three-drug combinations, in both single concentrations per drug and gradients of drugs. The two most widely used systems for classifying drug interactions are Loewe additivity¹⁰⁸ and Bliss independence.²⁸ Loewe additivity relies on entire gradients of each drug, while Bliss independence categorizes drug interactions based on single-point concentrations per drug. Two-drug combinations: (a, b) Loewe and Bliss differ in how they define additivity. Loewe defines additive drugs as those whose inhibition is constant along lines of equal effective dosage in a two-drug concentration graph. In this case, isoboles (lines of constant inhibition) can be plotted based on data for two drugs across a range of dosages. Following previous papers,^{29, 35, 44} in (a), the non-monotonic concave isobole (red) defines synergy, the linear isobole (orange) defines additivity, the convex isobole (green) defines antagonism, and the isobole with even greater convexity (blue) defines suppression. (Note that this is a simplified depiction, since isoboles indicating suppression are often non-monotonic.³⁵) Bliss defines additivity as the result when the relative effect of a drug at a certain concentration is independent of the presence of the other drug. If Drug X and Drug Y are additive, then w_XY = w_Xw_Y, where w is relative fitness. Suppression occurs when the combined effect of two drugs is less than the effect of one or both (b) of the drugs. In (b), any growth response caused by Drug X + Drug Y that is above the dotted line indicates that Drug X and Drug Y are suppressive. Three-drug combinations: (c, d) Recently these systems have been extended to classify higher-order drug interactions. Interactions are considered suppressive if the combined effect of the three drugs is less than the effect of any of the pairwise combinations or the single drugs.⁵³ Loewe’s extension to three drugs is shown in (c), and is characterized by an increase in growth (fitness) when all three drugs are present in high concentration. Growth response (fitness) increases as the combination becomes more suppressive (where red is synergistic, orange is additive, green is antagonistic, and blue is suppressive), as shown by the increasing convexity on the surfaces. In (d), Bliss’s extension to three drugs is shown. Any growth rate caused by Drug X + Drug Y + Drug Z that is above either dotted line (one for the highest single drug growth response (black) and one for the highest pairwise drug growth response (green)) indicates reciprocal suppression.