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].