Calorimetric mapping of complex interplay between rifampicin, isoniazid, pyrazinamide and ethambutol: Identifying Thermodynamic signatures of interaction between small molecules in an Alliance

The alliance of four anti-tubercular drugs, namely rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB), is considered incompatible in tablet formulation. Several mechanistic investigations have been proposed to interpret this complex interplay in the literature. We employed isothermal titration calorimetry (ITC) to map the self-, hetero-, and catalytically modulated interactions among rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA), and ethambutol (EMB). Each drug displayed distinct thermodynamic signatures during self-association (KD ≈ 5.16 × 10 -7), primarily governed by enthalpy–entropy compensation. The self-association strength followed the order of PZA > INH > EMB > RIF, with PZA showing the strongest, entropy-driven aggregation via π–π stacking and RIF exhibiting the weakest. In binary systems, hetero-association patterns revealed strong, spontaneous complexation between INH–EMB and RIF–PZA, followed by RIF–INH, RIF–EMB ≈, INH–PZA, and weak PZA–EMB interactions. These differences stemmed from variations in hydrogen bonding, desolvation, and hydrophobic reorganization. Ternary mixtures demonstrated catalytic modulation of RIF–INH interaction, where PZA enhanced (positive catalyst) and EMB reduced (negative catalyst) their association. In the quaternary system, these opposing effects balanced to yield a weakly bound, entropy-dominated multicomponent network, representing a thermodynamic compromise among all four drugs. Overall, ITC provided a direct, quantitative insight into molecular compatibility and reactivity under isothermal conditions, thereby circumventing the confounding effects of decomposition typically observed in DSC or TGA. The calorimetric mapping approach, as explained in the manuscript, establishes a powerful strategy for identifying favourable and adverse small-molecule interactions in complex drug combinations.