Azithromycin in viral infections

Summary Azithromycin (AZM) is a synthetic macrolide antibiotic effective against a broad range of bacterial and mycobacterial infections. Due to an additional range of anti‐viral and anti‐inflammatory properties, it has been given to patients with the coronaviruses SARS‐CoV or MERS‐CoV. It is now being investigated as a potential candidate treatment for SARS‐CoV‐2 having been identified as a candidate therapeutic for this virus by both in vitro and in silico drug screens. To date there are no randomised trial data on its use in any novel coronavirus infection, although a large number of trials are currently in progress. In this review, we summarise data from in vitro, murine and human clinical studies on the anti‐viral and anti‐inflammatory properties of macrolides, particularly AZM. AZM reduces in vitro replication of several classes of viruses including rhinovirus, influenza A, Zika virus, Ebola, enteroviruses and coronaviruses, via several mechanisms. AZM enhances expression of anti‐viral pattern recognition receptors and induction of anti‐viral type I and III interferon responses. Of relevance to severe coronavirus‐19 disease (COVID‐19), which is characterised by an over‐exuberant innate inflammatory response, AZM also has anti‐inflammatory properties including suppression of IL‐1beta, IL‐2, TNF and GM‐CSF. AZM inhibits T cells by inhibiting calcineurin signalling, mammalian target of rapamycin activity and NFκB activation. AZM particularly targets granulocytes where it concentrates markedly in lysosomes, particularly affecting accumulation, adhesion, degranulation and apoptosis of neutrophils. Given its proven safety, affordability and global availability, tempered by significant concerns about antimicrobial stewardship, there is an urgent mandate to perform well‐designed and conducted randomised clinical trials.


| INTRODUCTION
Azithromycin (AZM) is a second-generation, broad-spectrum, synthetic macrolide antibiotic used since the early 1980s 1,2 to treat a wide range of bacterial and mycobacterial infections of respiratory and skin infections. It is therefore on the WHO list of essential medications, 3 and manufactured on a large scale globally. Its antibacterial activity derives from its ability to bind to the 50S ribosomal subunit, inhibiting protein synthesis. 4 It also has an intriguing range of anti-viral and anti-inflammatory properties, and is now being investigated as a potential candidate treatment for viruses including SARS-CoV-2, which causes coronavirus-19 disease (COVID- 19). It has been used as a treatment in previous coronavirus diseases during the epidemics of severe acute respiratory syndrome (SARS) in 2003 and Middle East respiratory syndrome (MERS) 5 in 2012, but to date there are no randomised trial data on its use in any novel coronavirus infection. Its proven safety, affordability and global availability make it an attractive candidate for repurposing as a treatment for COVID-19.
Given the expected massive global impact of COVID-19, particularly in low-to-middle income countries, it is important not only to develop therapies that treat the virus successfully, but also to ensure that these therapies are readily implementable at all levels of development and economy. 6 This review summarizes the current understanding of the anti-viral and anti-inflammatory effects of AZM, with a view to

| MECHANISMS OF ANTI-VIRAL EFFECTS
A range of human in vitro and in vivo studies provide evidence of anti-viral activity of macrolides across a broad range of viral species and families (Table 1). Some studies suggest improved symptom resolution and reduction, [17][18][19][20][21][22] although not all studies have observed these effects. 23 ninefold reduction in viral shedding, respectively. 8 The use of AZM alone increased viral-induced interferons (IFNs) and interferonstimulated gene (ISG) mRNA expression and hence production of these gene products. 7,8 In the latter study, while viral replication was suppressed, AZM did not suppress pro-inflammatory responses.
In vivo data from the AMAZES study, the largest clinical trial of a long-term macrolide in airways disease, showed a striking 40% reduction in asthma exacerbations with AZM. 29 The mechanism is unknown, and would be consistent with an anti-viral effect, although metagenomic analyses suggest an antibacterial effect reducing This is of relevance to coronaviruses as type I IFN inhibit replication of both SARS-CoV 37 and SARS-CoV-2 38 in vitro.
RV replication was also inhibited by the macrolides erythromycin 39

| Mechanism of effects in influenza A
In a randomised trial in patients with influenza A receiving oseltamivir, 5 days' adjunctive AZM 500 mg daily was associated with more rapid reductions in plasma concentrations of IL-6, IL-8, IL-17, CXCL9, soluble tissue necrosis factor (TNF) and C-reactive protein (CRP). 16  infection. 43 However, the effect was not sustained, and not associated with a change in virus-induced weight loss, a sensitive measurement of influenza pathology. Another study found AZM reduced lung viral titres at day 6 post infection, though the effects were not additive to that achieved with oseltamivir in terms of survival, viral titres or cytokine levels, 46 and so these data remain conflicting. 47 In a separate influenza study, AZM decreased total leukocyte accumulation in lung tissue and BAL, with the largest reduction being in neutrophils, and associated with decreased inflammatory mediators.

| Mechanism of anti-viral effect in Zika virus
In a drug screen of 2177 compounds against the flavivirus Zika, AZM reduced viral proliferation and virus-induced cytopathic effects in glial cell lines and human astrocytes. 11

| Anti-viral effects in Ebola
AZM was similarly evaluated in a drug screen for its efficacy as a therapy for Ebola. 14 While AZM demonstrated high in vitro potency (50% effective concentration [EC 50 ] = 5.1 μM) and low toxicity, when tested in an in vivo mouse model it did not consistently improve survival in mice or guinea pigs.

| Anti-viral effects in enteroviruses
Enterovirus A71 (EV-A71) causes hand, foot and mouth disease in young children. AZM and spiramycin (another macrolide) provided significant in vivo protection against EV-A71 infection in mice. 13 Spiramycin impaired EV-A71 viral RNA synthesis, and it is likely spiramycin and AZM work through a common mechanism, after viral entry, impairing viral RNA synthesis either directly or indirectly.   55 This combination has been tested in non-human primates, where a significant anti-viral effect was not seen in the five macaques which received AZM in addition to hydroxychloroquine. 56

| Immunomodulatory effects on phagocytes
AZM is rapidly absorbed after oral administration with a large volume of distribution 109 (Table 2). 70 One pathway for macrolide immunomodulation is through binding to macrophilin-12 inhibiting calcineurin and thus T cell activation, via the same mechanism as tacrolimus, 29 with consequent downstream inhibition of many immune cells including eosinophils and basophils. 68 Macrolides also inhibit mammalian target of rapamycin (mTOR) activity, also important in T cell activation and granulocyte differentiation, 115 suppressing cell proliferation and CD4 + T cell cytokine secretion. 116

| POTENTIAL CLINICAL UTILITY IN COVID-19
Beyond its anti-viral properties, the anti-inflammatory effects of AZM It is understandable therefore, that more than 80 clinical trials have been designed to test AZM efficacy in COVID-19 (Table S1).
These differ significantly from each other according to dosing regime, duration of therapy, whether being used in combination with hydroxychloroquine and, critically, according to the population being  Given the significant clinical utility of AZM as an antibiotic, the current rapid spread of antimicrobial resistance is of particular concern. Widespread use of AZM to treat viral infections runs an inevi-

DATA AVAILABILITY STATEMENT
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.