Current investigation presented an experimental and theoretical drug-drug interaction study between nitazoxanide (NTZ) and azithromycin (AZT) in aqueous solution. Understudy drugs alone, in combination, and with other drugs are widely prescribed for the early treatment of COVID-19. Interaction was primarily studied by using UV/Vis, fluorescence, ATR-FTIR and CD spectroscopy. While molecular docking studies was performed to established the drugs interaction computationally. Two drugs when allowed to interact, the bright yellow color was observed giving hyper chromic band at 420 nm. The rate of absorbance was linearly increased with increasing drug concentrations and in a time-dependent manner. Stability of the interaction complex (i.e. NTZ: AZT) was measured at variable temperature (25–80 °C), pH (5.0–10.0) and ionic strength (0.05-2.0 M NaCl), and not only proved stable but also retain antimicrobial potential with reduced cellular toxicity. Mole ratio and Job`s method of continuous variation were used to established the binding stoichiometry between NTZ and AZT and found to be 2:1. While, the calculated binding constant (kb = 8400 M− 1) and Gibb’s free energy (ΔG° = -22.4 KJ/mol) were also suggested the energetically favorable interaction. FTIR spectra of NTZ: AZT complex in comparison with two drugs alone revealed significant interactions which was also complemented from molecular docking studies. The interaction was also successfully demonstrated in presence of carrier protein HSA and by spiking the two drugs in real samples of fresh human plasma and urine. Cumulatively, present study provided new protocol for drug’s interaction, functional efficacy and bioavailability in combination therapy.

Herein, we present an experimental and theoretical drug-drug interaction study between nitazoxanide (NTZ) and azithromycin (AZT) in an aqueous solution. Interaction was studied by using UV/Vis, fluorescence, attenuated total reflectance-fourier transform infrared (ATR-FTIR), and circular dichroism (CD) spectroscopy, while molecular docking studies were performed to establish the interaction computationally. A bright yellow color was observed when the two drugs interacted, giving a hyperchromic band at 420 nm. The rate of absorbance was linearly increased by increasing drug concentrations and in a time-dependent manner. Stability of the interaction complex (i.e., NTZ: AZT) was measured at variable temperatures (25-80 C), pH (5.0-10.0) and ionic strength (0.05-2.0 M NaCl), and not only proved stable but also retained antimicrobial potential with reduced cellular toxicity. Mole ratio and Job's method of continuous variations were used to establish the binding stoichiometry and found to be 2:1. The calculated binding constant (k b = 8,400 M À1 ) and Gibb's free energy (ΔG = À22.4 KJ/mol) also suggested an energetically favorable interaction. FTIR spectra of NTZ: AZT complex in comparison with two drugs alone revealed significant interaction which was nicely complemented by molecular docking studies. Interaction was also successfully demonstrated in presence of carrier protein HSA and by spiking the two drugs in real samples of human plasma and urine.