Dealing with complicated infections and disorders, normally multiple medications are required that may cause drug-drug, besides drug-target interactions. Drug-drug interactions might be occurred at any one of the following three levels; i.e., pharmaceutical, pharmacodynamics, and pharmacokinetic. Such interactions may also have modulated (e.g. increase or decrease) the drug pharmaceutical action [12]. In the current study, we have investigated the pharmaceutical interactions of NTZ and AZT. As aforementioned, NTZ is a thiazolide anti-infective agent, while AZT is a macrolide antibiotic use to treat various bacterial and viral infections. Moreover, the combination of these two drugs have already been subjected in clinical trials for early treatment of COVID-19. Thus, persuaded drug-drug interaction study is very significant and immediate need. The potential interaction was established experimentally using UV/Vis, Fluorescence, FTIR and CD spectroscopy, biological assays, and complemented by in silico studies. The outcome revealed significant interaction between NTZ and AZT.
Interaction of nitazoxanide with azithromycin
Nitazoxanide and azithromycin alone and their 1:1 interaction were followed by UV/Vis spectroscopy. The spectrum of NTZ alone shows two absorption bands, a hyper chromic band at 346 nm followed by a hypochromic band at 436 nm (Fig. 2a). The band at 346 nm was due to the n → π* transitions of lone pair of electrons of the carbonyl group, while band at 436 nm corroborated to the internal charge transfer via thiazol ring from the 2-amino to 5-nitro group [26]. In contrast, AZT alone did not show any absorption band in the given range. After interaction was taken placed by mixing the two drugs (1:1 ratio) on constant stirring, the solution turns into the bright yellow color from a very light yellow solution (Fig. 2a; Insert). After interaction, the typical NTZ absorption bands were fully disappeared and a new hyper chromic band was appeared at 420 nm (Fig. 2a). After the reaction started, a time-dependent interaction was observed till 90 min with subtle increase in band intensity at 420 nm (Fig. S1).
The NTZ/AZT interaction was further validated in presence and absence of carrier protein HSA. HSA was allowed to interact with NTZ, AZT alone, their co-interaction and interaction with NTZ: ATZ complex. The results were initially followed by UV/Vis spectroscopy (Fig. 2b). Compare to HSA alone and its interaction with AZT revealed just a minor increase in intensity of absorption peak at 280 nm (tracing E and A). While in case of NTZ, a significant increase followed by a bathochromic shift from 346 to 420 nm (tracing E, B and D) was observed, and as expected suggested the strong binding [27]. Likewise, a similar interaction pattern of HSA: NTZ: AZT was observed, except with high peak intensity (tracing E and F). The quenching pattern in intrinsic fluorescence of HSA after interaction with AZT (minor), NTZ (high), and with NTZ: AZT interaction complex showed the most significant quenching than NTZ alone (Fig. S2a). Moreover, circular dichroism (CD) analysis of HSA alone shows a typical all alpha protein CD spectrum characterized by two negative peaks at 208 and 222 nm followed by a positive band at 196 nm. Conformational changes observed in terms of ellipticity (mdeg) and the calculated secondary structure of HAS (Fig. S2b; Insert) after interaction with two drugs alone and their interaction complex, suggesting the very differential binding, while prior drug-drug interaction significantly altered the protein binding and thus their biological functions [27, 28]. Conformational changes associated with HSA after drug(s) binding was also complemented with native gel electrophoresis as the results illustrated in Fig. S2c.
In order to measure the linearity of interaction between two drugs, the concentration of one drug kept constant, while the concentration of other drug was varied in a dose-dependent fashion. Firstly, the concentration of AZT was kept constant (100 µM) and concentration of NTZ was varied (0-100 µM). As the concentration of NTZ increases against AZT, the absorption intensity at 420 nm is also significantly increased. Even at highest concentration of NTZ (100 µM), it fully interacts with AZT and gives only a sharp peak at 420 nm (Fig. 3a). In contrast, at fixed concentration of NTZ (100 µM) and variable concentrations of AZT (0-100 µM) the absorption band at 346 nm was gradually decreased, while the absorption band of interaction complex at 420 nm was gradually increased in a concentration-dependent manner (Fig. 3b). At the highest concentration of AZT, the absorption band at 346 was totally disappeared and only hyper chromic band at 420 nm was observed. Since, confirmed a linear drug-drug interaction with significant regression and complex formation (Fig. S3a, b). Moreover, from the established linear regression lines, limit of detection and quantification (LOD/LOQ) can also be established and utilized for the both drugs detection as chemosensor. The LOD’s for NTZ and AZT were found to be 1.37 µM (R2 = 0.997) and 1.69 µM (R2 = 0.994), respectively (Fig. S3a, b).
Binding stoichiometry
The interaction between NTZ and AZT was also monitored by Job`s method of continuous variation and mole ratio methods. In Job`s method, the mole fraction of NTZ was varied but the concentration of NTZ and AZT was constant. Figure 4a, shows the Job`s plot where absorption at 420 nm was plotted against the mole fraction. It was seen that 2:1 binding stoichiometry (NTZ: AZT) was established. Mole ratio method was also performed to analyze binding stoichiometry. In this method, the mole fraction of NTZ was kept constant, while AZT was varied. Figure 4b, shows the absorption against the mole ratio (NTZ: AZT) and 2:1 binding stoichiometry was also observed, which complement the results obtained from Job`s plot.
Determination of binding constant (kb)
The binding constant between NTZ and AZT complex was also calculated by Benesi-Hildebrand formula:
1/ΔA = 1/Δε[AZT] + 1/Δε[AZT]kb × 1/[NTZ]
Where; ΔA = change in absorbance, Δε = change in absorption coefficient, [AZT] = concentration of AZT and [NTZ] = concentration of NTZ. The plot of 1/ΔA against 1/[NTZ] was found to be linear with coefficient of correlation i.e., R2 = 0.9969 (Fig. 4c). The binding constant (Kb) was calculated from the ratio of intercept/slope of 1/ΔA versus 1/[NTZ] plot and was found to be 8400 M− 1. Gibb’s free energy (ΔG°) of interaction of NTZ and AZT was also calculated by the given equation:
ΔG° = - RT ln kb
Where; R = ideal gas constant (Jmol− 1 K− 1), T = temperature (K). ΔG° for drug-drug interaction was found to be -22.4 KJmol− 1 indicating that the interaction is energetically favorable.
Binding mechanism
Fourier transform infrared (FTIR) spectroscopic measurements were performed to identify the potential functional groups that are involved in interaction. As illustrated in Fig. 5, the specific bands of NTZ (alone) appeared at 3358 cm− 1 (stretching of N-H bond), 3087 and 3094 cm− 1 (stretching of C-H and O-H bonds), 1772 and 1662 cm− 1 (stretching of C = C, C = O and C = N bonds and bending of N-H bond) and 1473 and 1364 cm− 1 (stretching and bending of C = O and C-N) [29]. Similarly, AZT (alone) illustrated principal bands at 3561, 3496 cm− 1 (stretching of O-H bonds), 1724 cm− 1 (stretching of C = O), 1344 cm− 1 (bending of C-H bonds), 1282, 1269, 1251 cm− 1 (stretching of C-O bonds), and 1046 cm− 1 (stretching of C-N bond), and corroborated well with previously reported data [30]. In contrast, the FTIR spectrum of the NTZ: AZT complex revealed that both drugs were involved in the interaction as the band intensities of both significantly reduced (Fig. 5).
Stability analysis of NTZ: AZT complex
In order to established the stability of NTZ: AZT interaction complex, two drugs alone and their complex was first subjected to pH stability. Fig. S4a, shows the effect of pH on the NTZ alone. Nitazoxanide is a pH sensitive drug, and at pH6.0 NTZ shows a hyperchromic band at 346 nm and a hypochromic band at 420 nm. When pH was decreased (< pH6.0), the only band appears at 346 nm, while band at 420 nm was completely lost. In contrast, when pH was increased (> pH6.0) the only band appeared at 420 nm and band at 346 nm was lost (Fig. S4a). Obtained results are very well in agreement with earlier studies [26]. On the other hand, AZT did not show any specific UV/Vis band and thus effect of variable pH does not produce any signal (Fig. S4c). Interestingly, NTZ: AZT interaction complex was found sensitive towards pH and at decreased pH (< pH5.0) a bathochromic shift is observed from 420 nm to 346 nm. In contrast, at high pH (~ pH10.0) hyper chromic effect was seen at 420 nm (Fig. S4b). While the hypochromic effect was seen at highly alkaline pH (> pH10.0). This behavior of NTZ: AZT complex was mainly associated with NTZ interaction and complex formation with AZT, and the change in the ionization state of NTZ at different pH.
Temperature is also a key parameter towards drug stability and their interactions. The interactions between two drugs was found to be quite stable towards temperature up to 80oC as no significant change have been observed in absorption maximum at 420 nm. While further increase in temperature (i.e., 80 to 100oC) decreases the intensity of absorption maximum up to 60% but without any significant change in peak position (Fig. S4d). This might because at high temperature, the interacting forces between two drug molecules were partially weakened. Likewise, the effect of ionic strength (i.e., 0.05 to 2 M NaCl) of the medium on NTZ: AZT complex was also measured spectroscopically. Results revealed that interaction between the two drugs were quite stable even at highest ionic concentration of salt i.e., 2 M NaCl (Fig. S4e).
Finally, in order to established the stability of the two drugs (alone) and their interaction complex (i.e., NTZ: AZT), identical concentrations (i.e., 100 µM) were incubated and spiked in the healthy fresh human plasma and urine samples. Significant drug-drug interaction was also observed in urine and plasma samples as compared to each drug alone (Fig. S5a, b). In order to complement, a pre-incubated NTZ: AZT interaction complex (i.e., complex of two drugs in DI) was also included as control and spiked in the human plasma and urine samples. Obtained results revealed no significant change in absorption maxima of the pre- and post-interaction complex of the two drugs, suggesting the high affinity for binding between the drugs even in presence of human urine and complex plasma matrix (Fig. S5a, b).
In silico studies
In order to established the interaction between two drugs, molecular docking analysis was also performed using MOE. The obtained results show that both drugs interact well with each other via two strong hydrogen bonds (Fig. 6). Chemically, NTZ is a combination of acetylsalicylic acid and 5-nitrothiazole-2-amine linked by an amide linkage. In contrast, AZT has two tetrehydro-2H-pyran rings in which one has ether, while other has amine linkage with the methyl groups and these two rings are attached with larger ring of macrolide by ether linkages (Fig. 1a, b). In NTZ: AZT complex, two tetrehydro-2H-pyran rings of AZT shows open hand like structure and their ester linkage with larger ring of macrolides is involved in interaction with amide group of NTZ by hydrogen bonding (Fig. 6).
Antimicrobial activities and cellular toxicity
When multiple drugs co-administrated in pharmacy practice, their chances of interaction with other drugs are increased substantially, as a result of interaction the pharmaceutical action of the drug might be changed [12, 31]. In order to validate this aspect, two drugs alone and their interaction complex were subjected for antibacterial and antibiofilm activities against some major human pathogens. Obtain results summarized in table 1 revealed very differential antimicrobial profiles, mainly depends on the type of pathogen tested. For example, the antibacterial potential of drug complex (NTZ: AZT) was well retained in comparison to NTZ alone, while significantly decreased compared to AZT against E. faecalis (ATCC 29212). Moreover, the minimum bactericidal concentrations (MBCs) were also reduced for several folds (Table 1). Likewise, ATZ was found inactive against S. aureus (ATCC 25923) as compared to NTZ alone, while the drug complex (NTZ: AZT) also shows substantially decreased activity. Moreover, NTZ alone was found inactive against K. pneumoniae (ATCC 700603) and A. baumannii (ATCC: 19606), except P. aeruginosa (NCTC 10662), while ATZ alone was found highly potent against all mentioned Gram-negative pathogens. In contrast, the antimicrobial activity of NTZ: AZT complex was significantly decreased, except P. aeruginosa (NCTC 10662). Cumulatively, antimicrobial potential of these two drugs were substantially reduced after interaction or NTZ: AZT complex formation (Table 1).
On the other hand, cellular toxicity established against the human fibroblast cells (ATCC CRL-2522) by using standard MTT assay revealed the IC50 values of 92.96 ± 0.33, 45.68 ± 5.53 and 144.54 ± 5.73 µg/mL for the NTZ, AZT and NTZ: AZT interaction complex, respectively. Significant reduction in cellular toxicity of NTZ: AZT complex compared to two drugs alone also depicted the strong interaction and biological impact on activity and bioavailability.
Under study two drugs are widely used alone and in combination with other drugs against COVID-19. In particular, NTZ and AZT combination are successfully in practice for the early management of COVID-19, while the results of these clinical trials in the other part of world are still awaited [32–34]. To the best of our knowledge, no drug-drug or drug-disease interaction was established before in COVID-19 scenario. As NTZ is a high affinity protein binding drug, since when co-administrated with other drugs having narrow therapeutic indices and high affinity for plasma protein binding, thus the chances of unbound drug in plasma will be significantly high [31, 35]. Despite, NTZ has a favorable drug interaction profile; unfortunately, no study regarding its interaction with other co-administered drugs was known, so far [36]. As these drugs are widely in used, since there is a significant need of the time to establish their interactions with other drugs as well as with different biological matrices [37].