4.1 – In vitro data
In Vero E6 cells with a MOI of 0.002, azithromycin showed an EC50 of 2.12 µM, an EC90 of 8.65 µM and a 50 % cytotoxic concentration > 40 µM, with a selectivity index > 19[64]. On the contrary, in another study performed in Vero E6 cells with a MOI of 0.25 azithromycin alone did not show any antiviral activity[65]. However, the combination of hydroxychloroquine at 5 µM with azithromycin at 5 and 10 µM was found to be synergistic and significantly inhibited viral replication[65]. The different MOI among the two studies may have accounted for these differences. Anyway, caution is advised when interpreting these results given the different MOI, cell lines, incubation times and analytical methods[24].
Azithromycin used at conventional doses could achieve therapeutic concentrations in the lung to be effective against SARS-CoV-2[24,65]. Based on previous described pharmacokinetic andin vitro data, Cmax/EC50 ratios of 91.5 in alveolar macrophages or 4.3 in lung tissue could be achieved[24,65]. In the study of Andreani et al. authors concluded that the observed synergy was achieved at concentrations achieved in vivo in the lungs[65].
Other macrolides have also demonstrated in vitro antiviral activity against SARS-CoV-2, as bafilomycin A decreased the entry of pseudovirions by 99 % compared to the control group[11].
In Figure 1, the proposed antiviral and immunomodulatory mechanisms of action of azithromycin in the treatment of COVID-19 were described.
Azithromycin could act in SARS-CoV-2 binding to respiratory cells. Its intracellular accumulation led to an increase in the pH that may impair trans-Golgi network (TGN) and lysosome functions [12,45]. Poschet etal . found that the treatment of CF bronchial epithelial cells with 100 µM for 1 h and 1 µM of azithromycin for 48 h led to an increase in TGN pH from 6.1 ± 0.2 to 6.7 ± 0.1 [45]. Authors postulated that this increase in pH in TGN may alter glycosylation of hACE2 and other proteins [45]. Using molecular dynamic simulations, another direct antiviral mechanism of this macrolide was theorized [9]. Azithromycin resulted in a ganglioside-mimic given its similar volume and analogous chemical features than GM1. Since the spike protein of SARS-CoV-2 displays a ganglioside-binding site, azithromycin might inhibit SARS-CoV-2 infection by binding to this site. This would prevent the virus spike protein to reach gangliosides on the host plasma membrane that are involved in SARS-CoV-2 pathogenesis [9]. Ulrich etal . concluded that azithromycin may have antiviral activity against SARS-CoV-2 by interfering in the spike protein/CD147 interaction or CD147 expression[66].
Endocytosis and fusion process activation by lysosomes are essential for SARS-CoV-2 entry and infection [12]. The increase in the lysosomal pH by azithromycin may alter the endocytosis process [7,67,68]. Furthermore, the impairment of lysosomal proteases such as cathepsins and furin may also play a role [7,45,67,69]. Poschet et al found that 100 µM of azithromycin could normalize the excessive processing and activation of furin [45]. Given that SARS-CoV-2 has been shown to present a furin-like cleavage site in the spike protein, the reduction in the activation of furin by azithromycin could prevent the entry of SARS-CoV-2 into human epithelial cells [13,70].