JPT-06668; No of Pages 21

Pharmacology & Therapeutics xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Pharmacology & Therapeutics

journal homepage: www.elsevier.com/locate/pharmthera

Azithromycin: Mechanisms of action and their relevance for

clinical applications
Michael J. Parnham a,b,c,⁎, Vesna Erakovic Haber d, Evangelos J. Giamarellos-Bourboulis e,f, Gianpaolo Perletti g,h,
Geert M. Verleden i, Robin Vos i
a
Fraunhofer Institute for Molecular Biology and Applied Ecology, Project Group Translational Medicine and Pharmacology, Frankfurt am Main, Germany
b
Institute of Pharmacology for Life Scientists, Goethe University Frankfurt, Frankfurt am Main, Germany
c
Institute of Clinical Pharmacology, Goethe University Frankfurt, Frankfurt am Main, Germany
d
Fidelta d.o.o., Zagreb, Croatia
e
4th Department of Internal Medicine, University of Athens, Medical School, Athens, Greece
f
Integrated Research and Treatment Center, Center for Sepsis Control and Care, Jena University Hospital, Jena, Germany
g
Biomedical Research Division, Department of Theoretical and Applied Sciences, University of Insubria, Busto A., Varese, Italy
h
Department of Basic Medical Sciences, Ghent University, Ghent, Belgium
i
Respiratory Division, Lung Transplantation Unit, University Hospitals Leuven and Department of Clinical and Experimental Medicine, KU Leuven, Belgium

a r t i c l e i n f o a b s t r a c t

Keywords: Azithromycin is a macrolide antibiotic which inhibits bacterial protein synthesis, quorum-sensing and reduces
Azithromycin the formation of biofilm. Accumulating effectively in cells, particularly phagocytes, it is delivered in high
Macrolide antibiotic concentrations to sites of infection, as reflected in rapid plasma clearance and extensive tissue distribution.
Mechanisms of action Azithromycin is indicated for respiratory, urogenital, dermal and other bacterial infections, and exerts
Pharmacokinetics immunomodulatory effects in chronic inflammatory disorders, including diffuse panbronchiolitis, post-
Immunomodulation
transplant bronchiolitis and rosacea. Modulation of host responses facilitates its long-term therapeutic benefit
Clinical efficacy
in cystic fibrosis, non-cystic fibrosis bronchiectasis, exacerbations of chronic obstructive pulmonary disease
(COPD) and non-eosinophilic asthma.
Initial, stimulatory effects of azithromycin on immune and epithelial cells, involving interactions with
phospholipids and Erk1/2, are followed by later modulation of transcription factors AP-1, NFκB, inflammatory
cytokine and mucin release. Delayed inhibitory effects on cell function and high lysosomal accumulation
accompany disruption of protein and intracellular lipid transport, regulation of surface receptor expression, of
macrophage phenotype and autophagy. These later changes underlie many immunomodulatory effects of
azithromycin, contributing to resolution of acute infections and reduction of exacerbations in chronic airway
diseases. A sub-group of post-transplant bronchiolitis patients appears to be sensitive to azithromycin, as may
be patients with severe sepsis. Other promising indications include chronic prostatitis and periodontitis, but
weak activity in malaria is unlikely to prove crucial. Long-term administration of azithromycin must be balanced
against the potential for increased bacterial resistance. Azithromycin has a very good record of safety, but recent
reports indicate rare cases of cardiac torsades des pointes in patients at risk.
© 2014 Elsevier Inc. All rights reserved.

Abbreviations: AECOPD, acute exacerbations of COPD; AP-1, activator protein-1; ARDS, acute respiratory distress syndrome; AUC, area under the plasma concentration-versus-time
curve; BAL, broncho-alveolar lavage; BOS, bronchiolitis obliterans syndrome; CAD, cationic amphiphilic drugs; CAP, community acquired pneumonia; CBP, chronic bacterial prostatitis;
CF, cystic fibrosis; CFTR, CF transmembrane conduction regulatory protein; Cmax, peak plasma concentration; COPD, chronic obstructive pulmonary disease; COX, cyclo-oxygenase;
cPLA2, cytoplasmic phospholipase A2; DNFB, dinitrofluorobenzene; DPB, diffuse panbronchiolitis; DTH, delayed type hypersensitivity; ERK, extracellular signal-regulated kinase; FGF, fi-
broblast growth factor; GM-CSF, granulocyte–macrophage colony stimulating factor; GVH, graft versus host reaction; HR, hazard ratio; IL, interleukin; i.n, intranasal; i.p, intraperitoneal; i.t,
intratracheal; i.v, intravenous; JNK, c-Jun NH(2)-terminal kinase; LC3, microtubule-associated protein 1A/light chain 3; LOS, length of stay; LPS, bacterial lipopolysaccharide; LRTI, lower
respiratory tract infection; MAPK, mitogen-activated protein kinase; MDR1, multidrug resistance protein 1; MIC, minimum inhibitory concentration; MLSbK, macrolides, lincosamines,
streptogramin B and ketolides; MMP, metalloproteinase; MODS, multiple organ dysfunction syndrome; MPO, myeloperoxidase; MUC5AC, mucin 5AC; MV, mechanical ventilation;
NFκB, nuclear factor kappaB; NGU, non-gonococcal urethritis; NK, natural killer cells; NR, not reported; OR, odds ratio; p.o, oral; PG, prostaglandin; PI, phosphatidylinositol; PI3K,
phosphoinositide-3-kinase; PK, pharmacokinetic; PMNL, polymorphonuclear leukocytes; PS, phosphatidylserine; RCT, randomized controlled clinical trial; ROS, reactive oxygen species;
SAA, serum amyloid A protein; SMC, smooth muscle cell; SREBP, sterol regulatory element binding protein; STAT, signal transducers and activators of transcription; STD, sexually trans-
mitted disease; t1/2, plasma half-life; TLR, Toll-like receptor; Tmax, time to Cmax; TNFα, tumor necrosis factor alpha; Th, T helper cell; VAP, ventilation assisted pneumonia; Vd, volume of
distribution; VEGF, vascular endothelial growth factor.
⁎ Corresponding author at: Fraunhofer IME-TMP, Theodor-Stern-Kai 7, D-60596 Frankfurt am Main, Germany. Tel.: +49 69 6301 84234.
E-mail addresses: michael.parnham@ime.fraunhofer.de (M.J. Parnham), Vesna.ErakovicHaber@glpg.com (V.E. Haber), egiamarel@med.uoa.gr (E.J. Giamarellos-Bourboulis),
gianpaolo.perletti@uninsubria.it (G. Perletti), geert.verleden@uzleuven.be (G.M. Verleden), robin.vos@uzleuven.be (R. Vos).

http://dx.doi.org/10.1016/j.pharmthera.2014.03.003
0163-7258/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Parnham, M.J., et al., Azithromycin: Mechanisms of action and their relevance for clinical applications, Pharmacology &
Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.03.003
2 M.J. Parnham et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2. Antibacterial mechanisms of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
3. Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
4. Immunomodulatory activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
5. Clinical applications in respiratory infections and airway inflammation . . . . . . . . . . . . . . . . . . . . . 0
6. Infections of the urogenital tract and sexually-transmitted infections . . . . . . . . . . . . . . . . . . . . . . 0
7. Clinical application in sepsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
8. Other clinical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
9. Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
10. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

1. Introduction properties and immunomodulatory actions contribute to the effects

Azithromycin, a second generation macrolide, broad-spectrum
antibacterial, has received increasing attention in recent years because
of additional effects on host-defence reactions and chronic human
diseases. It is the prototype 15-membered lactone ring azalide, synthe-
sized in the early 1980s as a semi-synthetic derivative of erythromycin.
Discovered around the same time by researchers at Pfizer in the United
States (Bright & Hauske, 1984) and at PLIVA, Croatia (Kobrehel et al.,
1982), PLIVA patented first and licensed the compound to Pfizer.
With much improved pharmacokinetic properties over erythromycin,
azithromycin became the most widely used broad-spectrum antibacte-
rial in North America. Pfizer Inc's Arthur E. Girard and Gene Michael
Bright, together with PLIVA's Slobodan Djokic (posthumously) and
Gabrijela Kobrehel, received in 2000 the American Chemical Society's
award of “Heroes of Chemistry who have promoted human welfare in
the area of health” for their discovery of Zithromax® (azithromycin).
Azithromycin shares the same mechanism of antibacterial action
as other macrolide antibiotics (Allen, 2002), but accumulates more
effectively in phagocytes, thus being delivered in high concentrations
to sites of infection (Miossec-Bartoli et al., 1999; Wilms et al., 2006).
It also inhibits bacterial quorum-sensing and reduces formation of
biofilm and mucus production, which extend its range of antibacterial
actions (Tateda et al., 2001; Hoffmann et al., 2007). As an antibiotic,
azithromycin is indicated for respiratory, urogenital, dermal and other
bacterial infections, but has beneficial effects in chronic inflammatory
disorders such as diffuse panbronchiolits, bronchiolitis obliterans and
rosacea. Efficacy in these conditions is ascribed to immunomodulatory
effects on innate and adaptive immune responses. Modulation of host
response reactions also accounts, at least partially, for beneficial effects
in cystic fibrosis, non-cystic fibrosis bronchiectasis, bronchial obliterans
syndrome (BOS) and chronic obstructive pulmonary disease (COPD).
Azithromycin is well-tolerated and has a very good record of safety.
Although macrolides have a class warning for potential cardiac QT pro-
longation, azithromycin does not show this effect under experimental
conditions (Milberg et al., 2002). Until recently, only a handful of cases
of QT prolongation had been reported for patients treated with the
drug (Kezerashvili et al., 2007). This is mainly because azithromycin,
unlike other macrolide antibiotics, does not interact with CYP3A4,
despite a minor interaction with the anti-coagulant warfarin (Kanoh
& Rubin, 2010; Mergenhagen et al., 2013). Recently, evidence for
increased risk of QT prolongation with azithromycin has appeared, but
mainly in patients with greater susceptibility to adverse cardiac effects
(Giudicessi & Ackerman, 2013).
We review here the antibacterial actions and pharmacokinetics of
azithromycin, as well as its immunomodulatory effects and the
mechanisms involved, in comparison to other macrolide antibiotics.
We discuss the main clinical uses of azithromycin, drawing attention
to emerging indications and emphasising how its pharmacokinetic
observed.
2. Antibacterial mechanisms of action
2.1. Inhibitory actions
Azithromycin, like other macrolide antibiotics, inhibits bacterial
protein synthesis by binding to and interfering with the assembly of
the 50S large ribosomal subunit and the growth of the nascent poly-
peptide chain (Champney & Burdine, 1998; Champney et al., 1998;
Hansen et al., 2002). It binds at the polypeptide exit tunnel, close to
the peptidyl transferase center (PTC) on the 23S rRNA, but does not
inhibit PT activity, in contrast to larger macrocyclic antibiotics. The
basicity of azithromycin leads to faster penetration of the outer mem-
branes and more effective entrance into the bacteria, thereby enhancing
activity against Gram-negative bacteria (Dinos et al., 2001).
Binding sites on the bacterial ribosome for the structurally different
macrolides, lincosamines, streptogramin B and ketolides (MLSbK) over-
lap significantly, so that changes in a single ribosomal region simulta-
neously alter susceptibility to various MLSbK antibiotics.
Although ineffective as a bactericidal agent against Pseudomonas
aeruginosa at clinically relevant concentrations, azithromycin inhibits
the generation of both growth-stimulating, quorum-sensing com-
pounds, and alginate biofilm which protects the micro-organism from
antibiotic actions (Tateda et al., 2001; Hoffmann et al., 2007). Efficacy
against both P. aeruginosa virulence factor production and biofilm
formation, as well as its ability to decrease the minimum inhibitory
concentration (MIC) of anti-pseudomonas agents (Lutz et al., 2012)
are related to ribosomal protein synthesis inhibition (Kohler et al.,
2007).
Azithromycin also shows moderate activity against the malaria par-
asite, Plasmodium spp., exhibited as delayed killing, which is achieved
via the apicoplast (Dahl & Rosenthal, 2007). Clinical studies, however,
failed to demonstrate even equivalence of three-day treatment with
azithromycin to other antimalarials or drug combinations (van Eijk &
Terlouw, 2011).
2.2. Resistance phenotypes
Bacteria resist azithromycin in two ways: a) by changing the target/
binding site via methylation of key rRNA nucleotides or mutation of
some ribosomal components and b) by efflux pump activity, thereby
decreasing its intrabacterial accumulation.
Ribosome methylation is the most important resistance mechanism
for all MLSbK antibiotics (Sutcliffe & Leclerq, 2002). The MLSbK-II
phenotype, characterized by the presence of Erm methylases, is highly
resistant to all MLSbK antibiotics, including azithromycin, which
induces erm genes in Streptococci and in Staphylococci (Sutcliffe &

Please cite this article as: Parnham, M.J., et al., Azithromycin: Mechanisms of action and their relevance for clinical applications, Pharmacology &
Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.03.003
 M.J. Parnham et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx
Leclerq, 2002). In S. pyogenes there are three types of inducible
phenotype (a) iMLSbK-A, (b) iMLSbK-B, which is highly resistant to
azithromycin (MICN128 μg/mL) and (c) iMLSbK-C with a medium resis-
tance profile (MICs up to 16 μg/mL) against azithromycin (Giovanetti
et al., 1999; Giovanetti et al., 2002). In addition to methylation of
23rRNA, resistance can be achieved by mutation of various genes coding
for ribosomal components. This type of resistance has been observed
infrequently in S. pneumonia, S. pyogenes and H. influenzae, the most
common mutations occurring in domain V rRNA and proteins L4 and
L22 (Tait-Kamradt et al., 2000; Franceschi et al., 2004).
Mef transporter (efflux pump) expression typically results in a lower
level of specific resistance towards 14- and 15-membered macrolide
antibiotics (MICs 4–32 ug/mL) and this type of resistance is referred
to as the M phenotype (Leclercq, 2002).
Increased clinical use of macrolide antibiotics is linked with an
increase in pneumococcal macrolide resistance. The Alexander Project,
monitoring antibiotic resistance among respiratory pathogens, reported
a global rate of pneumococcal macrolide resistance between 16.5% and
21.9% for 1996 and 1997 (Felmingham & Gruneberg, 2000), increasing
to 24.6% from 1998 to 2000 (M. R. Jacobs & Johnson, 2003). Over this
period, macrolide-exceeded penicillin-resistance in 19/26 countries.
The PROTEKT US surveillance study reported similar findings. From
2000 to 2004, erythromycin resistance among S. pneumoniae isolates
remained high (Jenkins et al., 2008), mef(A) being the most common
macrolide resistance genotype, accounting for N60% of tested isolates
(Farrell & Jenkins, 2004). Although macrolide resistance rates have
subsequently stabilized, the prevalence of clonal isolates with a com-
bined erm(B) and mef(A) genotype and a high-level of macrolide and
multidrug resistance has increased.
Distribution of macrolide resistance genotypes varies between and
within countries. In the studies mentioned above, highest resistance
rates were in Asia and some European countries (France, Italy, Spain,
Belgium), being N70% and 25–60%, respectively. Macrolide resistance
was common among S. pneumoniae isolates (77.2–81.9%) in Japan
during PROTEKT years 1999–2004, the erm(B) genotype accounting
for most resistance (Inoue et al., 2008). In Portugal (1994–2002),
emergence of macrolide-resistant S. pneumoniae strains clearly correlat-
ed with increased azithromycin use (Dias & Canica, 2004).
In a recent study on commensal Staphylococcus aureus in Europe,
except for penicillin, the highest recorded resistance rate was to
azithromycin (from 1.6% in Sweden to 16. 9% in France) (den Heijer
et al., 2013). As usage of azithromycin and clarithromycin has increased
for indications beyond RTI, resistance has started to emerge in non-
respiratory pathogens. For instance, macrolide treatment of healthy
volunteers, in comparison to placebo, significantly increased the
proportion of macrolide-resistant oral streptococci for up to 180 days
after treatment (Malhotra-Kumar et al., 2007). Consequently, as with
all antibiotics, azithromycin should be administered for all relevant
conditions with due consideration of the associated benefits and risks.
3. Pharmacokinetics
Early pharmacokinetic (PK) studies on azithromycin, indicating low
plasma levels, were incorrectly interpreted as reflecting poor PK proper-
ties. Azithromycin is now known to have a large volume of distribution,
achieving high tissue concentrations and is efficiently delivered to sites
of infection. Compared with older generation macrolides, it is more
stable in acidic media and has a longer half-life, allowing for once a
day or even single dose treatment (Amsden, 1995, 2001). Moreover,
azithromycin is barely metabolized, with no active metabolites and
does not induce CYPs (Amsden, 1995; Nightingale, 1997).
3.1. Cellular accumulation
Concentrations of azithromycin achieved in tissues, are up to 100-
fold higher than those in plasma (Foulds et al., 1990; Shepard &
Please cite this article as: Parnham, M.J., et al., Azithromycin: Mechanisms of action and their relevance for clinical applications, Pharmacology &
Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.03.003
3
Falkner, 1990) as a result of high cellular accumulation, particularly in
phagocytes, in which at least 200-fold higher intracellular than extracel-
lular concentrations can be achieved (Bosnar et al., 2005; Stepanic et al.,
2011). Consequently, specific accumulation of azithromycin occurs at
sites of inflammation (Miossec-Bartoli et al., 1999; Wilms et al., 2006).
In drug-free medium, preloaded cells release azithromycin slowly,N80%
being retained, so cellular accumulation and retention account for the
long half-life of azithromycin in vivo (Bosnar et al., 2005; Stepanic
et al., 2011). Accumulation of azithromycin in polymorphonuclear
cells (PMNLs) acts as an innate targeted delivery system to the site of in-
fection. Degranulation of PMNs by bacteria then releases azithromycin
into the extracellular space, where it exerts its antibacterial activity.
Azithromycin also accumulates, to a less and varying extent, in other
cells such as epithelial cells, fibroblasts, lymphocytes, hepatocytes
(Bosnar et al., 2005; Gladue & Snider, 1990; Matijasic et al., 2012;
Stepanic et al., 2011). Fibroblasts, due to their wide distribution, have
been proposed as a potential reservoir for azithromycin, slowly releas-
ing or passing it to nearby phagocytes for transport to the site of infec-
tion (Gladue & Snider, 1990; McDonald & Pruul, 1991). Based on such
retention in epithelial lining and bronchoalveolar cells, macrolides,
including azithromycin, find veterinary use for the treatment of pneu-
monia (Villarino & Martin-Jimenez, 2013).
Most intracellular azithromycin is located in acidic organelles such
as lysosomes. Initially shown to concentrate in the cytoplasm and plas-
malemma of PMNLs and monocytes (Wildfeuer et al., 1993), the vast
majority of azithromycin in these cells is associated with the granular
fraction (Carlier et al., 1994; Miossec-Bartoli et al., 1999). Recent data
support its lysosomal localization in hepatocytes (Matijasic et al.,
2012). In keeping with lysosomal accumulation, azithromycin causes
lysosomal pH change and functional impairment in murine macro-
phages (Nujic et al., 2012a).
The exact mechanism of cellular accumulation remains unclear. One
possibility is passive transmembrane transport and trapping by proton-
ation in acidic organelles, as this can be inhibited simply by neutraliza-
tion of lysosomal pH, markedly increasing azithromycin efflux (Hand &
Hand, 2001). Moreover, macrolide cellular accumulation and retention
can be predicted by experimentally determined physicochemical
parameters and the calculated number of positively charged atoms
(Stepanic et al., 2011). Like other cationic amphiphilic drugs (CADs),
macrolides may degrade cell membranes, together with which the
drugs are carried to lysosomes (Baciu et al., 2006).
An active uptake mechanism, mediated by transporter protein(s), is
supported by dependence of accumulation on cell viability, tempera-
ture, pH and extracellular calcium concentration (Gladue et al., 1989;
Mtairag et al., 1995; Pascual et al., 1995). Macrolides also compete for
cell accumulation (Munic et al., 2010). They are inhibitors of ATP-
binding cassette transporters such as P-glycoprotein (multidrug
resistance protein 1, MDR1) (Wang et al., 2000; Yasuda et al., 2002;
Asakura et al., 2004; Munic et al., 2010), multidrug resistance associated
protein 2 (MRP2) (Sugie et al., 2004) and organic anion-transporting
polypeptide, OATP (Garver et al., 2008) and substrates for MDR1.
Ranking of macrolides for cellular accumulation is proportional to
cellular expression of MDR1, its overexpression decreasing macrolide
accumulation 2- to 80-fold, particularly with azithromycin and erythro-
mycin (Munic et al., 2010).
It seems likely that both passive and active mechanisms are in-
volved, variability in macrolide accumulation resulting from changes
in acidic organelles or in different transporter proteins.
3.2. Pharmacokinetic properties
Extensive data are available on the clinical PK of various formula-
tions of azithromycin, both under normal and pathologic conditions
(Table 1). Knowledge of the salient kinetic features is crucial to an
understanding of its therapeutic effects.
4 M.J. Parnham et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx

Azithromycin shows plasma protein binding of approximately 30%
and relatively low oral bioavailability (17–37%) (Foulds et al., 1990;
Luke & Foulds, 1997). Following a single standard 500 mg oral dose,
peak plasma concentrations of 0.2–0.4 mg/L are attained, with a Tmax
of 2–4 h that may be delayed significantly in elderly subjects (Coates
et al., 1991) (Table 1). Single oral doses of 1.5 g azithromycin attain
peak plasma concentrations as high as 1.46 μg/mL (Amsden, 1996).
The kinetics of azithromycin are best described by a 3-compartment
pharmacokinetic model (Ballow et al., 1998; Pene Dumitrescu et al.,
2013).
When administered as 500 mg extended, rather than immediate
release tablets, a threefold higher AUC0–24 and twofold higher Cmax
may be attained (Liu et al., 2011; Lucchi et al., 2008) (Table 2). Plasma
clearance of azithromycin is remarkably high (50 L/h), and the volume
of distribution (Vd) is approximately 30 L/kg (Singlas, 1995). Hence, low
plasma concentrations are the consequence of rapid blood-tissue distri-
bution. The plasma half-life of azithromycin is approximately 70 h or 50
h following oral or intravenous formulations, respectively (Lode et al.,
1996). This remarkable pharmacokinetic characteristic of azithromycin
allows infrequent dosing (in bacterial urethritis or cervicitis, single
administration may be sufficient), and ensures optimal patient compli-
ance and sustained exposure to the drug. In fact, the AUC0–24 after a
single 500 mg oral dose is 3 μg∙h/mL (Matzneller et al., 2013), reaching
as high as 8–11 μg∙h/mL after intravenous administration of 500 mg
(Luke & Foulds, 1997; Rodvold et al., 2003). After a standard dose of
500 mg/day for 3 days, much of the drug is present in blood leukocytes
(Matzneller et al., 2013; Pene Dumitrescu et al., 2013). On day 3, blood
concentration was double that in plasma and Vd to the periphery was
2980 L, compared with 439 L in plasma. After 30 days, drug concentra-
tions were 4-fold higher in blood than in plasma. In healthy volunteers,
much less drug is present in tissues (Matzneller et al., 2013). Release is
likely induced by inflammatory stimuli.
Azithromycin is mainly eliminated unchanged in the feces by both
biliary excretion and trans-intestinal secretion. Only about 6% (oral
dose) to 12% (i.v.) is recovered unchanged in the urine (Singlas,
1995). Using ileostomy fluid sampling and correction for the fraction
of i.v. azithromycin recovered in the fluid, approximately 45% of an
oral dose of 500 mg was unabsorbed after intestinal passage up to the
ileal stoma (Luke & Foulds, 1997). Thus, incomplete absorption, rather
than a first-pass effect, is the major cause of the sub-optimal bioavail-
ability of azithromycin (Luke & Foulds, 1997).
The inactive descladinose and the active 9a-N-desmethyl metabo-
lites are frequently recovered because of acidic degradation in the gut
(Luke & Foulds, 1997; Raines et al., 1998). Hepatic biotransformation
is marginal (Curatolo et al., 2011). Importantly, azithromycin is neither
metabolized by nor an inhibitor of CYP3A4 and therefore, does not
interact with many commonly used medications that are oxidatively
metabolized by CYP3A4 (Shakeri-Nejad & Stahlmann, 2006). Other
factors than drug metabolism are thought to account for serious adverse
effects seen with azithromycin (Fleet et al., 2013) (see Section 9).
PK parameters may be affected considerably by co-administration of
azithromycin with food, prolongation of Tmax, 50% reduction of Cmax,
and of AUC0–72h having been observed in fed, compared to fasting pa-
tients (Table 1) (Curatolo et al., 2011). Azithromycin pharmacokinetics,
in keeping with low hepatic metabolism, are not significantly affected
by mild or moderate hepatic impairment or class-A or -B liver cirrhosis
(Mazzei et al., 1993), nor by mild or moderate renal insufficiency
(Hoffler et al., 1995; Singlas, 1995). PK parameters are also virtually
unchanged in patients with diabetes (Ernst et al., 2000). Hence, no
dose modifications of azithromycin appear to be necessary in patients
suffering from these conditions. However, severe renal impairment
noticeably increases Cmax and AUC0–120 (http://www.medicines.org.
uk/emc/medicine/26131). Interestingly, in such patients on permanent
peritoneal dialysis due to oliguric end-stage renal failure (Kent et al.,
2001), the Cmax of azithromycin increased but Tmax appeared to be in
the normal range.
3.3. Tissue distribution
The distribution of azithromycin into inflamed tissues and compart-
ments has been extensively investigated. In bronchial washings and
lung tissue biopsy specimens (Di Paolo et al., 2002), in lungs, in sinus
mucosal tissue (Ehnhage et al., 2008; Fang et al., 2009), in middle ear
fluids in children with otitis media (Pukander & Rautianen, 1996;
Scaglione et al., 1999), in the aqueous humor and in conjunctival biopsy
specimens (Tabbara et al., 1998), in tissues lining periodontal pockets
(Gomi et al., 2007), and in inflammatory skin blisters (Ballow et al.,
1998), azithromycin concentrations were always higher than those in
plasma. This tissue accumulation offsets the sub-optimal absorption of
the drug. In contrast to many other antibiotics, macrolides penetrate
well into prostatic tissue and seminal fluids. For example, Cmax of
azithromycin and clarithromycin in human prostatic tissue after 500
or 750 mg oral dosing is 2.54 and 3.83 μg/ml, respectively, about 20-
and 2-fold higher than in plasma (Foulds et al., 1991; Giannopoulos
et al., 2001). This is important with regard to the use of azithromycin
in prostatitis (see Section 6.2).
In summary, azithromycin concentrates to a considerable extent in
many inflamed tissues and fluids. In this respect, its extensive uptake
into tissue-infiltrating leukocytes is a significant contributing factor
(see Sections 3.1 and 4.1.2).
3.4. Pediatric pharmacokinetics
The AUC0–∞ of azithromycin (2 g dose) is reduced in pregnant
women (Salman et al., 2010), and plasma half-life is similar in both
placenta and fetus (70 h), though considerably shorter in amniotic

Table 1
Azithromycin pharmacokinetic data extracted from a sample of studies published between the years 1996 and 2013.

(Luke & Foulds, 1997) (Amsden, 1996) (Rodvold et al., 2003) (Matzneller et al., 2013) (Ballow et al., 1998) (Coates et al., 1991)

Dose/route (mg) 500 IV 500 oral 1500 oral 500 oral 500 IV 500 oral 500 oral 500 oral young 500 mg oral elderly
(18–40 years) (65–85 years)
Cmax (μg/mL) 3.40 0.21 1.46 0.54 1 0.46 0.27 0.41 0.38 μg/mL
tmax (h) 0.7 2.5 – – – 2.92 – 2.50 3.80 h
AUC (μg∙h/mL) 11⁎ 1.27⁎ 13.1⁎⁎ 11.2⁎⁎ 8.2⁎ 3.05⁎ – 2.5⁎ 3 μg∙h/mL⁎
t1/2 (h) 50.2 – – – – – 78.6
Ke (h−1) 0.00138 – – – – – –
Clplasma (L/h) 46.6 – – – – – –
F (%) 17.8 – – – – – –
Vd (L/Kg) – 89.5 L/Kg 109.2 – – –

Cmax, peak plasma concentration; tmax, time- to Cmax; Ke, pharmacokinetic elimination constant; Cl, plasmatic clearance; F, oral bioavailability; Vd, distribution volume.
AUC, area under- (definite integral of-) the plasma concentration-versus-time curve
⁎ AUC0–24.
⁎⁎ AUC0–∞.

Please cite this article as: Parnham, M.J., et al., Azithromycin: Mechanisms of action and their relevance for clinical applications, Pharmacology &
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 M.J. Parnham et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx
Table 2
Pharmacokinetic data for azithromycin administered in different oral formulations or in fasting vs. fed patients.
(Lucchi et al., 2008)
Dose/route 500 mg immediate 2 g extended
release oral release oral
Cmax (mg/L) 0.39 0.94
tmax (h) 4 4
AUC0–24 (μg∙h/mL) 3.1 10
Clplasma (L/h)
Vd (L)
fluid (3 h) and umbilical cord serum (12 h) (Ramsey et al., 2003). The
serum half-life in extremely preterm infants is shorter (58 h) than at
full term or in adults. Importantly, preterm infants, treated with
10 mg/kg intravenous azithromycin, appear to be overexposed (about
3.2-fold) to the drug compared to older children (age: 0.5–2 years)
(Jacobs et al., 2005; Hassan et al., 2011) and clearance is low (0.18 L/h
in 1 kg pre-term neonate vs 0.98 L/h/kg in older children). This may
be attributed to the immature biliary excretion pathways in preterm
neonates (Hassan et al., 2011).
In pediatric subjects (mean 24 months) given an immediate-release
formulation of azithromycin (30 mg/kg), plasma concentrations and
drug exposure were higher than in adults (Liu et al., 2011), necessitating
a dose adjustment (Table 1). When an extended release formulation
was administered to children, the Cmax was still elevated compared to
adult subjects, but the AUC0–24 was lower (Liu et al., 2011).
4. Immunomodulatory activities
Early studies on macrolide antibiotics and host defense revealed
inhibitory effects on neutrophils in vitro, on experimental inflammation
(Culic et al., 2001) and particularly actions on cytokine release (Bartold
et al., 2013). While azithromycin was shown in the late 1980s to inhibit
inflammation and lysosomal enzyme release in arthritic rats (Carevic &
Djokic, 1988), subsequent studies were carried out predominantly with
erythromycin, clarithromycin and roxithromycin. All macrolide antibi-
otics inhibit various standard models of inflammation (Ianaro et al.,
2000; Culic et al., 2001; Ivetic Tkalcevic et al., 2012) and more recent
studies have sought to clarify immunomodulatory mechanisms in vivo
(Table 3) and at the molecular level.
4.1. Cellular functions
Modulation of host defense by azithromycin and other macrolide
antibiotics occurs through interaction with structural cells, such as
epithelial or endothelial cells, smooth muscle cells or fibroblasts, as
well as with leukocytes (macrophages, polymorphonuclear leukocytes
or neutrophils, mononuclear leukocytes or monocytes, T cells and
dendritic cells). Immunomodulatory effects on cellular functions of the
macrolide antibiotics as a class were reviewed a few years ago (Kanoh
& Rubin, 2010).
4.1.1. Structural cells
In epithelial cells, azithromycin maintains integrity and the
transepithelial resistance in the face of permeability induced by
virulence factors from P. aeruginosa, in conjunction with rearrangement
of tight junction adhesion molecules, including claudins (Asgrimsson
et al., 2006; Halldorsson et al., 2010). Increased cell integrity is also
accompanied by reduced mucin secretion (Imamura et al., 2004),
altered signal transduction (MAPK pathway) (see Section 4.2.4) and
attenuated expression of inflammatory (e.g. IL-6 and 8, MMP-1, 2, 9,
10 and 13, and GM-CSF), lipid metabolism and cell cycle pathways
(Gielen et al., 2010; Murphy et al., 2008b; Ribeiro et al., 2009). In
bronchial epithelial cells infected with rhinoviruses, azithromycin
stimulated interferon and interferon-stimulated gene expression, in
Please cite this article as: Parnham, M.J., et al., Azithromycin: Mechanisms of action and their relevance for clinical applications, Pharmacology &
Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.03.003
5
(Liu et al., 2011) (Muto et al., 2011) (Curatolo et al., 2011)
500 mg IR oral 2 g extended 2 g extended 500 mg oral fast dissolving
release oral release oral formula in fasting/fed patients
0.40 0.72 0.4/0.2
2 3.5 2/4
2.67 7.87
122 103
518 1830
association with reduced rhinovirus proliferation and release (Gielen
et al., 2010). Such activities undoubtedly contribute to therapeutic
benefit in respiratory infections.
In airway smooth muscle cells, azithromycin inhibits interleukin (IL)-
17-induced IL-8 and 8-isoprostane release, whereas dexamethasone
failed to attenuate the IL-8 production (Vanaudenaerde et al., 2007).
On the other hand, azithromycin and dexamethasone (more strongly)
both reduce fibroblast growth factor (FGF)-induced vascular endothelial
growth factor (VEGF) production through interaction with p38 mitogen-
activated protein kinase (MAPK)-signaling (Willems-Widyastuti et al.,
2013). Azithromycin also demonstrates an anti-proliferative and auto-
phagic effect (Stamatiou et al., 2009) (Section 4.2.2), and directly relaxes
pre-contracted airway smooth muscle cells (Daenas et al., 2006).
4.1.2. Leukocytes
In neutrophils, subsequent to its pronounced accumulation (see
Section 3.1), slow efflux of azithromycin prolongs its effects on the
cells in vitro and in vivo (Bosnar et al., 2005; Culic et al., 2002). This
extensive intracellular retention of azithromycin, for up to 28 days in
human neutrophils after standard dosing, leads to initial stimulation
of neutrophil degranulation and phagocytosis-associated oxidative
burst, further contributing to its antibacterial activity (Culic et al.,
2002). Acute inhibitory effects of azithromycin include down-
regulation of neutrophil chemokine production (IL-8, GRO-α, MPO);
late effects comprise attenuation of neutrophil oxidative burst re-
sponses, down-regulation of myeloperoxidase (MPO) production,
increased neutrophil apoptosis and attenuation of chemokine (IL-8)-
and leukotriene (LT)B4-dependent and chemokine-independent
neutrophil chemotactic responses by inhibition of transcription factors,
nuclear factor kappa-B (NFκ-B) and activator protein-1 (AP-1) (Culic
et al., 2001; Culic et al., 2002; Tamaoki et al., 2004; Tsai & Standiford,
2004). Azithromycin also inhibits prostaglandin (PG)E2-synthesis by
suppressing expression of prostaglandin synthetic enzymes (COX-1
and COX-2) in both lipopolysaccharide (LPS)-stimulated polymorpho-
nuclear leukocytes and monocytes (Miyazaki et al., 2003). In the latter,
azithromycin decreases tumor necrosis factor-α (TNF-α) and granulo-
cyte–macrophage colony stimulating factor (GM-CSF) production
(Khan et al., 1999).
In sputum cells (81.7% neutrophils, 12.9% monocytes/macrophages)
isolated from steroid-naïve patients with COPD, azithromycin and
other macrolides inhibited, in a concentration-dependent manner,
the release of a variety of inflammatory cytokines and chemokines
(Marjanovic et al., 2011). The macrolides were more broadly inhibitory
than the corticosteroid, dexamethasone, the PDE4 inhibitor, roflumilast,
and the p38 kinase inhibitor, SB203580. Azithromycin was less effective
than clarithromycin and roxithromycin, but its broad inhibitory activity
may provide corticosteroid-sparing effects in treatment of respiratory
inflammation (see Section 5.4). An extensive list of the effects of
azithromycin on cytokine production, generally, was published recently
(Bartold et al., 2013). Azithromycin inhibits neutrophilic inflammation
in vivo induced by LPS given by various routes (Table 3). Lung
neutrophilia induced by intranasal LPS is inhibited by azithromycin in
association with a reduction in lung GM-CSF and IL-1β, both of which
were also inhibited by azithromycin in macrophages in vitro (Bosnar
6 M.J. Parnham et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx

et al., 2009). Subsequently, the action on lung neutrophilia was localized
to the alveolar macrophages (Bosnar et al., 2011).
In alveolar macrophages, azithromycin attenuates LPS-induced
production and expression of pro-inflammatory cytokines through inhi-
bition of AP-1 (Bosnar et al., 2011; Meyer et al., 2009) (see Section 4.2.4)
and it inhibits arachidonate release and metabolism in LPS-stimulated
macrophages (Banjanac et al., 2012). Azithromycin increases phago-
cytosis, probably by upregulating the macrophage mannose receptor,
CD206 (Hodge et al., 2008) and makes lysosomes in macrophages
more resistant to oxidant challenge (Persson et al., 2012). Azithromycin
also attenuates T helper-1 cell (Th-1) responses following LPS or
interferon-γ (IFN-γ) stimulation of macrophages, shifting polarization
of activated macrophages towards the alternative/anti-inflammatory
M2-phenotype, which plays a role in directing Th-2 responses and coor-
dination of repair following inflammation (Murphy et al., 2008a;
Yamauchi et al., 2009). Enhancement of M2-like macrophage genera-
tion is observed in vivo after azithromycin pretreatment of mice with
a cystic fibrosis-like disease (Legssyer et al., 2006).
The modulation by azithromycin of monocytes/macrophages has
been studied in detail in classically activated human blood monocytes
primed with IFN-γ and activated with LPS (Vrancic et al., 2012).
Azithromycin, in a concentration-dependent manner, distinctively
inhibited the gene expression and/or release of M1 macrophage
markers (CCR7, CXCL11 and IL-12p70), but not TNFα or IL-6 and en-
hanced expression and/or release of M2 macrophage markers (IL-10
and CCL18), and the pan-monocyte marker CD163. Modulation of the
Toll-like receptor (TLR) 4 signalling pathway, NF-kB and signal trans-
ducer and activator of transcription-1 (STAT-1) activation were shown
to be involved. The inhibitory profile of azithromycin was similar to
that of the lysosomotropic drug chloroquine, indicating that lysosomal
accumulation of the macrolide played a crucial role (see Section 4.2.1).
In dendritic cells, azithromycin modulates differentiation and LPS-
induced maturation towards a regulatory phenotype (e.g. enhanced
IL-10) with increased phagocytic capacity (Polancec et al., 2012;
Sugiyama et al., 2007). Furthermore, azithromycin inhibits expression
of co-stimulatory (CD40 and CD86) and major histocompatibility
complex (MHC) class II molecules in LPS-stimulated dendritic cells,
and attenuates Toll-like receptor (TLR)-4 expression, IL-12 production
and allostimulatory capacity, suggesting potential for modulation of
allogeneic responses (Iwamoto et al., 2011, 2013). Animal studies
suggest that azithromycin may also modulate Th2 cell responses
in vivo, probably by effects on antigen-presenting cells (Table 3). In-
duction of a modulatory DC phenotype could explain findings in healthy
human volunteers that azithromycin enhanced proliferative and IL-2
receptor responses of blood mononuclear cells (MNC) to pokeweed
mitogen (Tomazic et al., 1993). An inhibitory effect on antigen-
presentation by DCs almost certainly explains the reduction by
azithromycin pretreatment of the serum titre of specific anti-PCV7,
IgG1 antibodies in mice immunized with polyvalent, polysaccharide,
pneumococcal conjugate vaccine (PCV7) (Fernandez et al., 2004). This
finding also indicates that treatment of individuals with azithromycin
should be avoided when an immunization procedure is planned.
In human natural killer (NK) cells stimulated with IL-15, azithro-
mycin inhibited cytotoxic function through down-regulation of perforin
expression (Lin et al., 2012). Inflammatory cytokine production was not
inhibited, but it was in the transformed NK cell line, NK-92.
It should be noted that the pleiotropic effects of azithromycin are not
limited to a single anatomic site or organ or by the presence of microbes.
For instance, azithromycin, like dexamethasone, inhibits the production
of the acute phase protein serum amyloid-A (SAA) during sterile inflam-
mation in mice (Glojnaric et al., 2007). SAA, like C-reactive protein
(CRP), is a pentraxin synthesized by hepatocytes after IL-1β or IL-6
stimulation, and can be inhibited indirectly by azithromycin through re-
duction of cytokine release at the inflamed site. Nevertheless, given the
strong accumulation of azithromycin in hepatocyte lysosomes, it may
also directly inhibit hepatic pentraxin and cyto-/chemokine synthesis
(Matijasic et al., 2012).
4.2. Cell signaling processes
The mechanisms of the immunomodulatory actions of macrolide
antibiotics, including azithromycin have been reviewed previously

Table 3
Recent studies on the anti-inflammatory/immunomodulatory effects of azithromycin in animal models in vivo.

Reference Model Azithromycin dosing Variables changed

(Tsai et al., 2004)
(Legssyer et al., 2006)
(Tsai et al., 2009)
(Yamada et al., 2013)
(Beigelman et al., 2010)
(Tong et al., 2011)
(Geudens et al., 2008)
(Ballard et al., 2007)
(Terao et al., 2003)
(Hao et al., 2013)
(Ivetic Tkalcevic et al., 2006)
(Bosnar et al., 2009; Bosnar et al., 2011) Murine i.n. LPS lung inflammation
(Fernandez-Robredo et al., 2013)
(Glojnaric et al., 2007)
(Beigelman et al., 2009)
(Ivetic Tkalcevic et al., 2012)
(Plesko et al., 2010)
(Iwamoto et al., 2013)
(Fernandez et al., 2004)
Murine P.aeruginosa lung infection s.c. from day 3 ↓ lung leukocytes, inflammatory cytokines,
not bacterial counts
CFTR-mutated mouse P.aeruginosa p.o. 4 wks ↓ lung leukocytes, MPO,
lung infection Pretreatment ↑ lung M2-like macrophages
Murine P.aeruginosa lung infection s.c. from days 1 to 5 ↓ lung bacterial counts, inflammatory cytokines,
↑ survival, lung efferocytotic macrophages
Murine MDR A.baumanii pneumonia s.c. from days 1 to 6 ↓ mortality, lung leukocytes, inflammatory
cytokines, not bacterial counts
Murine Sendai virus pneumonia s.c. from days 1 to 7 ↓ lung leukocytes, inflammatory cytokines, not viral load
Murine Bacillus pyocyaneus shock 1 h before ↑ survival
Murine lung ischemia–reperfusion p.o. 14 days ↓ lung leukocytes, inflammatory cytokines,
injury Pretreatment
Neonatal mouse lung hypoxia From days 1 to 10 ↓ mortality, lung leukocytes, inflammatory
cytokines,
Murine i.p. LPS inflammation i.g. 20 min ↓ lung NO, plasma NO & inflammatory cytokines,
Pretreatment ↓ plasma kynurenine & inflammatory cytokines,
Murine i.p. or i.v. LPS shock p.o. 30 min before ↓ plasma TNFα, mortality
p.o./i.p.4 h ↓ lung leukocytes, inflammatory cytokines
Pretreatment IL-1β from alveolar macrophages
Rat subconjunctival LPS 3 days topical pretreatment ↓ ocular inflammation
Murine sterile s.c. AgNO3 inflammation p.o. from 1 to 3 days ↓ plasma SAA & inflammatory cytokines,
Murine OVA asthma s.c. from 1 day before or after challenge ↓ lung inflammation, BAL leucocytes & Th2 cytokines
Murine acute OXA skin DTH Topical after challenge ↓ ear edema, leukocytes, IL-4
Murine chronic skin DTH, repeated OXA Topical after each challenge ↓ ear mast cells, IgE, IL-4, not leukocytes,
Murine DNFB colon DTH From before or after challenge ↓ colitis
Murine allogeneic GvH disease p.o. from day −2 to 2 ↑ graft resistance, due to effect on DCs?
Murine immunization to pneumococcal i.p. 1 h before ↓ specific anti-PCV7 IgG1 antibodies
vaccine PCV7

CFTR: cystic fibrosis transmembrane conduction regulatory protein; DNFB: dinitrofluorobenzene; GvH: graft versus host disease to transplanted bone marrow; i.g.: intragastric; i.n.:
intranasal; i.p.: intraperitoneal; i.v.: intravenous; MDR: multidrug resistant; MPO: myeloperoxidase; OVA: ovalbumin; OXA: oxazolone; p.o.: oral; s.c.: subcutaneous.

Please cite this article as: Parnham, M.J., et al., Azithromycin: Mechanisms of action and their relevance for clinical applications, Pharmacology &
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 M.J. Parnham et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx
(Kanoh & Rubin, 2010; Shinkai et al., 2008). Mainly based on studies on
mucus and cytokine production by airway epithelial and inflammatory
cells, the authors concluded that macrolide antibiotics, including
azithromycin, exert biphasic stimulatory then inhibitory effects on
MAPK kinase (ERK, JNK, p38) activity, with subsequent modulatory ef-
fects on NFκB and AP-1 transcription factor activities. Crosstalk between
ERK and p38 was suggested to cause mutually counteracting effects.
Azithromycin and to a less extent other macrolide antibiotics, interacts
with phospholipids with pronounced effects on lysosomal function and
these must be incorporated into any consideration of the intracellular
effects of azithromycin.
4.2.1. Interactions with phospholipids and lysosomes
Azithromycin and other macrolides readily penetrate into phospho-
lipid bilayers, the N′-3 of the desosamine being inserted into the hydro-
phobic domain and the macrocycle occupying a position at the
hydrophobic–hydrophilic interface (Montenez et al., 1996, 1999).
For azithromycin, this position allows the positively-charged amino
group in the macrocycle to neutralize the negatively charged groups
in the phosphate head of the acidic phospholipids, including phos-
phatidylinositol (PI), preferentially found at the inner surface of the
cell membrane bilayer (Montenez et al., 1999). These negatively
charged phospholipids bind signaling proteins to the inner plasma-
lemma and regulate many cellular functions, including phagocytosis
(Yeung & Grinstein, 2007). Consequently, neutralization by azithro-
mycin of the negative charge could conceivably initiate many cellular
effects.
PI contains high amounts of arachidonic acid, and in LPS-stimulated
macrophages, azithromycin, like an inhibitor of cytoplasmic phospholi-
pase A2 (cPLA2), inhibited arachidonate release and eicosanoid produc-
tion but not cPLA2 expression, probably preventing cPLA2 access to its
membrane substrates (Banjanac et al., 2012). Reported inhibition by
macrolide antibiotics, including azithromycin, of arachidonate release
and metabolism in LPS-stimulated neutrophils and mononuclear cells
(Miyazaki et al., 2003; Sato et al., 2007) was probably due to effects on
signaling further downstream from TLR4.
Interaction of azithromycin with phospholipid head groups
decreases their mobility and the fluidity and elasticity of the plasma
membrane (Fa et al., 2007; Tyteca et al., 2003), accounting for many
effects of the macrolide on cellular functions. These include altered
membrane insertion of markers into macrophage membranes and
slowing of their trafficking into lysosomes; inhibition of fluid phase en-
docytosis, but not phagocytosis, of macro-molecules; down-regulation
and delayed recycling of surface transferrin receptors, but not Fcγ re-
ceptors (Tyteca et al., 2002, 2003). Moreover, anti-inflammatory effects
of a range of synthetic macrolide derivatives on macrophages were
correlated with their phospholipid binding properties and number of
positively charged centers (Munic et al., 2011).
Extensive lysosomal accumulation of azithromycin results from
proton-driven concentration of its dicationic form in the acidic (pH 5)
lysosomal milieu. Here, azithromycin, like other cationic amphiphilic
drugs (CAD), inhibits lysosomal phospholipase A1 by blocking phos-
pholipid substrate access, causing phospholipidosis, the accumulation
of phospholipids in lysosomes (Kosol et al., 2012; Van Bambeke et al.,
1996). Phospholipidosis, in turn, correlates with phospholipid binding
by a range of macrolide derivatives (Munic et al., 2011).
Annother mechanism of action may also account for phospho-
lipidosis. Following phosphorylation of their mannose residues in the
Golgi apparatus, lysosomal enzymes are transported to endosomes by
the multifunctional mannose-6-phosphate/insulin growth receptor II
receptor, MPR300 (Kornfeld, 1992). Here, the acidic endosome environ-
ment causes enzyme release and transfer to lysosomes, while MPR300
is returned to the Golgi apparatus. All phospholipidosis-inducing CAD
tested, including erythromycin, prevent MPR300 recycling from the
endosomes to the Golgi apparatus, causing lysosomal depletion and ex-
tracellular secretion of lysosomal enzymes (Ikeda et al., 2008). Such an
Please cite this article as: Parnham, M.J., et al., Azithromycin: Mechanisms of action and their relevance for clinical applications, Pharmacology &
Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.03.003
7
action of azithromycin on fibroblast MPR300 (and/or on membrane
phospholipid fluidity) may account for its inhibition of the pinocytosis
of macromolecules and their transport from the plasma membrane to
endo/lysosomes (Tyteca et al., 2001). Acute effects on MPR300 may
also explain the paradoxical increase by azithromycin of lysosomal hy-
drolase activity in fibroblasts (Gerbaux et al., 1996) and the early release
and prolonged depletion of circulating neutrophil lysosomal enzymes
after azithromycin treatment of human volunteers (Culic et al., 2002).
In this context, inhibition of neutrophil lysosomal enzyme release by
neutrophils was the first anti-inflammatory effect of azithromycin
ever to be described (Carevic & Djokic, 1988). Interestingly, Niemann–
Pick C2 protein (NPC2), which transports cholesterol, glycolipids and
lysophosphatidic acid out of lysosomes, is also transported to endo/
lysosomes from the Golgi apparatus by MPR300 (Willenborg et al.,
2005), so azithromycin could potentially affect extra-lysosomal lipid
transport. In fact, expression of both NPC1 and NPC2 and of various
lipid and cholesterol metabolism genes in human epithelial cells was
increased by incubation with azithromycin (Ribeiro et al., 2009). Since
lysophosphingolipids, like azithromycin (see Sections 4.1.2 and 4.3.5),
modify macrophage differentiation (Yamamoto et al., 2011), they
could mediate the actions of azithromycin.
4.2.2. Autophagy
Autophagy is a process whereby ubiquitin-tagged or Hsp-
chaperoned, often damaged, cytosolic components or intracellular
pathogens are engulfed in autophagosomes and transported to
lysosomes for degradation (Randow & Munz, 2012). Fusion of
autophagosomes with lysosomes is mediated by the delipidated
autophagy protein, microtubule-associated protein 1A/1B-light
chain 3 (LC3), which binds to phospholipids from apopototic parti-
cles phagocytosed by leukocytes, thereby facilitating the engulf-
ment of the resulting efferocytotic vesicles by lysosomes (Florey
& Overholtzer, 2012). In smooth muscle cells (SMC), azithromycin,
at a clinically relevant concentration (10 μM), induced autophagy
and inhibited actively proliferating cells (Stamatiou et al., 2009).
This autophagy-mediated inhibition of SMC proliferation may
contribute to respiratory effects of azithromycin.
In contrast, in fibroblasts and human macrophages, azithromycin
blocks autophagy, measured by accumulation of lipidated LC3 (LC3-
II), by reducing acidification of autophagosomes and lysosomes
(Renna et al., 2011). Particle-induced generation of TNFα and intra-
cellular mycobacterial killing by the macrophages was inhibited. In
cystic fibrosis (CF) patients treated for 6 months with azithromycin,
mycobacterial killing was also reduced and LC3-II increased (Renna
et al., 2011). The clinical significance of this finding is questionable
because, in a nested case–control study on chronic azithromycin
treatment in a total of 27,112 CF patients, the longer the treatment
with azithromycin, the less likely were patients to have non-
tuberculous mycobacterial infections (Binder et al., 2013).
Azithromycin effects on autophagy may depend on the intracel-
lular site of action. Accumulation of azithromycin in muscle cells is
many-fold less than in fibroblasts and leukocytes, in which accumu-
lation is predominantly lysosomal (Matzneller et al., 2013). Effects
on SMC, thus, may involve surface membrane effects, while in the
other two cells, azithromycin may target predominantly lysosomal
degradation processes. Macrolides bind with low affinity to
valosin-containing protein (VCP), a crucial mediator of intracellular
protein degradation and autophagy (Ju & Weihl, 2010; Nujic et al.,
2012b; Tresse et al., 2010) which may account for some long-term
effects of azithromycin on autophagy.
Recently, azithromycin, clarithromycin and erythromycin
were found to sensitize to cytotoxic and pro-apoptotic effects
of bortezomib by blocking autophagic flux in myeloma cells
(Moriya et al., 2013), suggesting a potential use for macrolides
in oncology.
8 M.J. Parnham et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx
4.2.3. Apoptosis
Apoptosis is a crucial non-inflammatory process for terminating the
life of cells, particularly immunocompetent cells. Interactions exist be-
tween apoptosis and autophagy in that the proteins, autophagy protein
5 (Atg5) and autophagy inducing protein, Beclin-1 are involved in both
processes (Fimia & Piacentini, 2010). It is, therefore, not surprising that
effects of azithromycin have been observed on apoptosis.
In SMC, azithromycin initially induces autophagy (Stamatiou et al.,
2009) and then apoptosis after prolonged incubation (Stamatiou et al.,
2010). At concentrations of 100 μM or higher, azithromycin induces
apoptosis in neutrophils (Koch et al., 2000), an action shared by many
antibiotics (Healy et al., 2002); in human lymphocytes by inhibiting ex-
pression of Bcl-xL (Ishimatsu et al., 2004; Mizunoe et al., 2004); and in
NK cells (Lin et al., 2012). Induction of apoptosis is thus related to
prolonged, extensive accumulation, particularly at high doses, as seen
with circulating human neutrophils (Culic et al., 2002). This likelihood
is supported by studies on monocytes and macrophages.
Despite accumulating several hundred-fold in macrophages,
azithromycin does not readily induce apoptosis in these cells. To the
contrary, in human bronchial alveolar lavage (BAL) macrophages, lyso-
somal membrane permeabilization and ROS-induced cell death were
inhibited by long-term azithromycin treatment, an action confirmed
in vitro (Persson et al., 2012). Macrophages from azithromycin-treated
patients, in fact, show enhanced ability to phagocytose apoptotic cells.
First observed in patients with chronic obstructive pulmonary disease
(COPD), azithromycin in vitro restored to normal the reduced ability
of alveolar macrophages from COPD patients to phagocytose apoptotic
neutrophils (Hodge et al., 2006). Administered for 12 weeks to COPD
patients, the macrolide also enhanced alveolar macrophage effero-
cytotic capacity and mannose receptor (CD 206) expression, together
with a decrease in epithelial cell apoptosis (Hodge & Reynolds, 2012;
Hodge et al., 2008). This enhancement of macrophage efferocytosis is
now attributed to the ability of azithromycin to promote the expression
of the M2 macrophage phenotype (Section 4.1.2).
4.2.4. MAP kinases and AP-1
Kanoh and Rubin (2010) stated in their review that“there have been
so many different reported effects of macrolides on cell signaling path-
ways that attempts to put these actions together as one mechanism is
difficult. In fact, all macrolides seem not to have similar ranges or de-
grees of activity.” In a majority of the reports, clarithromycin was the
macrolide tested, so it is important to consider azithromycin separately.
The clearest indication for involvement of the Erk1/2 MAP kinase
signalling pathway has been obtained from studies on mucin produc-
tion by airway epithelial cells. In these cells, azithromycin inhibited
mucin 5 AC (MUC5AC) secretion and the increase in Erk1/2 phosphory-
lation induced by some, but not all stimuli (Ishimotoet al., 2009; Luo
et al., 2011; Morinaga et al., 2009). Subsequent transcription of the
MUC5AC gene, in H. influenzae-or EGF-stimulated cells, was inhibited
by azithromycin through inhibition of AP-1 activation (Araki et al.,
2010; Nie et al., 2012). The same signaling pathway is linked with inhib-
itory effects of azithromycin on IL-8 production in epithelial cells
(Srivastava et al., 2011) and on human neutrophil chemotaxis (Tsai
et al., 2004).
Subsequent inhibition of AP-1 binding to DNA by azithromycin has
been associated with inhibition of IL-8 production in CF airway cells
(Cigana et al., 2006); inhibition of proliferation in human vascular
SMCs, (Miller et al., 2000); suppression of IL-12p40 expression and
generation by M1-type macrophages (Yamauchi et al., 2009); and
inhibition of IL-1β production in LPS-stimulated macrophages (Bosnar
et al., 2011). Clearly, for inhibition of mucin and cytokine production
and of neutrophil chemotaxis, the ERK1/2, AP-1 signaling pathway is
an important target of azithromycin. The effect on AP-1 binding, though,
is probably indirect as basal, unstimulated AP-1 binding and IL-8
production is not affected by azithromycin (Cigana et al., 2006; Miller
et al., 2000).
Please cite this article as: Parnham, M.J., et al., Azithromycin: Mechanisms of action and their relevance for clinical applications, Pharmacology &
Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.03.003
4.2.5. NFκB pathway
In no study on MUC5AC secretion by airway epithelial cells was an in-
hibitory effect of azithromycin observed on NFκB activation, despite
clear NFκB activation by all stimuli (Araki et al., 2010; Morinaga et al.,
2009; Nie et al., 2012). However, apart from CF cells, a clear correlation
exists, in epithelial cells, between inhibition of IL-8 generation, NFκB ac-
tivation and DNA binding (Cigana et al., 2006; Matsumura et al., 2011;
Saint-Criq et al., 2012). This (indirect) NFκB inhibition involves an
action on the receptor signal-transmitting Rho GTPase, Ras-related C3
botulinum toxin substrate 1 (Rac-1) (Matsumura et al., 2011). Thus,
while azithromycin acts on epithelial mucin by targeting AP-1, inhibi-
tion of IL-8 involves both a direct action on AP-1 and indirect on NFκB
activation, apparently preceded by inhibition of membrane Rac-1,
supporting an action of azithromycin close to the phospholipid cell
membrane.
It is unclear whether NFκB is a potential target for azithromycin in-
hibition of leukocyte cytokine production (Aghai et al., 2007; Ikegaya
et al., 2009). Several macrolide antibiotics, though, inhibit NFκB activa-
tion in a variety of cells in vitro (Kanoh & Rubin, 2010), particularly
when the incubation period is long (Shinkai et al., 2008) and upstream
signaling has occurred. Moreover, in classically activated human mono-
cytes, the macrolide only reduced NFκB activation at relatively high
concentrations (50 μM) (Vrancic et al., 2012). Since both AP-1 and
NFκB are both dependent on upstream signaling processes, many of
the observations on their inhibition by azithromycin may be secondary
to direct cell membrane effects of the macrolide (see Section 4.2.1).
4.2.6. Gene expression
An overview of changes induced by azithromycin, at clinically rele-
vant concentrations, was gained from human airway epithelial cells
stimulated with CF lung supernatant (Ribeiro et al., 2009). In confirma-
tion of other studies, genes for mucin production, MMPs, CXCL3, CXCL6,
IL-7, tissue plasminogen activator (tPA) and cell cycle were down-
regulated. Genes for CXCR7, IL-1ra, IL-6 signal transducer, transferrin
receptor (CD71) and VEGFα were upregulated. IL-8 expression and se-
cretion was increased only by short-term (6 h) incubation. In keeping
with effects of azithromycin on lipid metabolism, the expression of
many proteins regulating lipid and cholesterol metabolism were up-
regulated. Prominent among these were SREBP1 (sterol regulatory ele-
ment binding protein 1) and SREBP2, master activating transcription
factors which control the expression of many other genes for cholesterol
metabolism. Effects of azithromycin on membrane phospholipids
and SREBP1/2 may be linked, SREBP1 phosphorylation and down-
regulation of cell cycle genes being regulated by effects on MAP kinases.
In summary, direct effects of azithromycin on charged phospholipds
in plasma and lysosomal membranes seem to be initial targets of the an-
tibiotic, with associated changes in the expression of lipid metabolising
genes, such as SREBP, autophagy and apoptosis. Effects on Rac-1, MAPK
kinases and other enzymes activated by membrane changes probably
determine downstream effects on transcription factors AP-1 and NFκB,
depending on the cell type and membrane stimulus (Fig. 1). Additional
direct effects of azithromycin on signaling molecules cannot be ruled
out. However, only valosin containing protein (VCP), a protein involved
in autophagy, has been identified as a low affinity (KD = 1.8 × 10−4 M)
azithromycin binding protein (Nujic et al., 2012b). No high affinity bind-
ing proteins for azithromycin were detected.
4.3. Role of state and phase of inflammation
Results of several investigations indicate that the immunomodulato-
ry effects of azithromycin and other macrolide antibiotics vary accord-
ing to the presence and phase of an inflammatory response.
4.3.1. Distinction between non-inflamed and inflammatory conditions
The activation state of immune cells determines their response
to azithromycin. Thus, the macrolide antibiotics, as a class, stimulate
 M.J. Parnham et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx
neutrophil and macrophage responses in healthy rodents, but generally
inhibit inflammation and leukocyte responses in experimental inflam-
matory models (Culic et al., 2001). This dichotomy was clearly seen in
a study in which pretreatment with azithromycin had no effect on
lung inflammatory cytokines, but increased IL-10 concentrations in
healthy mice, while in mutant Δ508 CF mice, azithromycin inhibited
lung inflammation and pro-inflammatory cytokines, but had no effect
on IL-10 (Legssyer et al., 2006). This may have been due to effects on
predominantly resident macrophages in healthy and M1-like macro-
phages in inflamed CF mice.
This difference between healthy and inflamed conditions is also seen
in humans. Standard dosing of healthy volunteers with azithromycin for
3 days stimulated blood neutrophil degranulation and oxidative burst,
but not in similarly treated COPD patients (Culic et al., 2002; Parnham
et al., 2005). In untreated endothelial cells, azithromycin increases
(Millrose et al., 2009), whereas in LPS-stimulated epithelial cells or neu-
trophils, macrolides suppressed adhesion molecule expression (Khair
et al., 1995; Lin et al., 2000). It seems likely that administered early dur-
ing a bacterial infection, azithromycin could promote host defense by
activating naïve leukocytes and endothelial cells, unprimed by cyto-
kines. At a later stage, when cells are activated by inflammatory stimuli,
immunomodulation by azithromycin would lead to inhibition of inflam-
mation and promotion of its resolution (Parnham, 2005).
4.3.2. Time dependency of effects
The concept of a biphasic, time-dependent immunomodulatory
action of azithromycin and other macrolide antibiotics is supported by
data in several publications. Initial stimulatory and later enhancing
effects have been observed with clarithromycin on IL-8 production
and Erk1/2 activation in bronchial epithelial cells (Shinkai et al., 2006)
and with erythromycin in vitro or roxithromycin given to mice on
mononuclear cell IL-1 and IL-2 production (Kita et al., 1990; Konno
et al., 1992). Similarly, in Candida albicans infected mice and in healthy
human volunteers, a merely transient stimulation of neutrophil chemo-
taxis was seen with erythromycin (Fernandes et al., 1984), a finding also
obtained with azithromycin in healthy human volunteers (Culic et al.,
2002). In the latter study, initially enhanced blood neutrophil activation,
2.5 h after azithromycin, changed to a significant reduction after 1–
28 days.
Early, mainly stimulatory effects of azithromycin, especially on
neutrophils, are probably mediated by interactions with phospholipids
and Erk1/2, followed by later modulation of transcription factors AP-1
and NFκB. Subsequent delayed inhibitory effects on cell function are
likely related to lysosomal accumulation of the macrolide, with disrup-
tion of protein and lipid transport through the Golgi apparatus and ulti-
mate effects on surface receptor expression, including macrophage
phenotype changes and autophagy (see Section 4.2) (Fig. 2).
4.3.3. Resolution of inflammation
Since azithromycin can drive the generation of an anti-inflammatory
M2 macrophage phenotype (Section 4.1.2), it is pertinent that it also
promotes the resolution of experimental inflammation. In Δ508 CF
mice with LPS lung inflammation, azithromycin pretreatment inhibited
inflammation 48 h after LPS, when lung IL-10 levels were increasing, i.e.
during the resolution phase (Legssyer et al., 2006). This effect on the
resolution process was further clarified in murine, self-limiting,
zymosan-induced, peritoneal inflammatory cell infiltration, which
peaks at 48 h and is inhibited by azithromycin pretreatment (Navarro-
Xavier,et al., 2010). Administered at 72 h, azithromycin inhibited
remaining leukocyte infiltration and also enhanced the ratio of M2/M1
macrophages in the peritoneal exudate. In contrast to other anti-
inflammatory drugs, azithromycin did not decrease the modulatory
cytokine, IL-10, in exudates. These data strengthen the proposal that
apart from acute anti-inflammatory effects on neutrophils, the major
target for the immunomodulatory effects of azithromycin is the
Please cite this article as: Parnham, M.J., et al., Azithromycin: Mechanisms of action and their relevance for clinical applications, Pharmacology &
Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.03.003
9
mononuclear phagocyte, promoting an inflammation-resolving M2
macrophage phenotype or a regulatory DC phenotype.
5. Clinical applications in
respiratory infections and airway inflammation
The unique imunomodulatory properties of azithromycin, its broad
antibacterial spectrum and exceptional pharmacokinetics, resulting
in extensive and sustained tissue penetration, largely account for its
disease-modifying effects in infectious or non-infectious inflammatory
airways diseases. These coincident properties also explain its indication
for upper and lower respiratory tract infections (e.g. acute bacterial
sinusitis, community-acquired pneumonia), in which macrolides exert
therapeutic benefits not solely explainable by antibacterial activities
(Amsden, 2005).
5.1. Upper and lower respiratory tract infections
With a broad antibacterial spectrum and oral bioavailability,
macrolides are the second most commonly used antibiotic class in
many European countries (Adriaenssens et al., 2011), exceeded only
by penicillins. Based on their efficacy against the main respiratory
pathogens (S. pneumoniae, S. pyogenes), macrolides are often used for
empirical treatment of respiratory tract infections. They are also active
against other bacteria including Streptococcus pneumoniae, Mycoplasma
pneumoniae, Chlamydia spp., Legionella pneumophila, Niesseria
gonorrhoeae, Bordatella pertussis, Campylobacter jejuni, Treponema
pallidum, Ureaplasma urealyticum, Corynebacterium diphtheria and
Listeria monocytogenes. Azithromycin has an even broader spectrum,
being active against Haemophilus influenza, Moraxella catarrhalis, non-
tuberculosis mycobacteria, Bartonella henselae, Rhodococcus equi,
Toxoplasma spp., Cryptosporidium spp. and Plasmodium species
(Blondeau et al., 2002). H. influenzae, M. catarrhalis, S. pneumoniae,
as well as Chlamydophila pneumonia and M. pneumonia are particularly
susceptible. Studies in mice indicate that even with multidrug re-
sistant bacterial strains, clinical benefit may result from the immuno-
modulatory actions of the drug (Yamada et al., 2013).
Azithromycin does not inhibit growth of Pseudomonas aeruginosa
in vitro, but shows efficacy in vivo mainly because of inhibition of
quorum-sensing and biofilm (Hoiby, 2011) (see Section 2). Given
prophylactically, azithromycin, therefore, reduces the incidence
of ventilation-associated pneumonia, particularly in patients with
rhamnolipid-positive isolates (van Delden et al., 2012), confirming
previous findings in P. aeruginosa-induced lung inflammation in mice
(Table 3). Promotion by azithromycin of macrophages with a regulatory
M2-like phenotype in animals (Feola et al., 2010; Hoffmann et al., 2007)
suggests that immunomodulation also contributes to the benefit seen
clinically in P. aeruginosa infection.
Clinical experience with azithromycin in the treatment of upper
and lower respiratory tract infections has been reviewed previously
(Mulholland et al., 2012; Panpanich et al., 2008; Wierzbowski et al.,
2006). Accumulation in leukocytes at inflamed sites means that
oral dosing either at 500 mg daily for 3 days or as a single dose of 1–
1.5 mg is often sufficient.
Azithromycin is effective in microbial eradication in patients
with presumed bacterial lower respiratory tract infections (LRTI),
community-acquired pneumonia and acute bacterial sinusitis (Henry
et al., 2003; Lakos et al., 2012; Panpanich et al., 2008; Yanagihara
et al., 2009). In acute sinusitis, there are few randomized studies of
azithromycin, but the clinical and bacteriologic efficacy and safety of
azithromycin is generally good and comparable to or even slightly
better than that of amoxicillin/clavulanate (Marple et al., 2010) or
levofloxacin (Murray et al., 2005). Azithromycin therapy was better
tolerated, with less frequent adverse reactions and lower cost, com-
pared to amoxicillin/clavulanate (Henry et al., 2003; Karpov, 1999). A
common clinical finding is the coexistence of sinusitis, periodontal
10 M.J. Parnham et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx

Phospholipid
bilayer penetration Lysosomal
enzyme &
Inflammatory
ROS release
stimulus
Neutralizes negative
PLs, reduces FA release,
decreases membrane fluidity

Lipid Rac-1?
Surface remodeling
molecule MAPKs (ERKs)
recycling
Lysosomal
accumulation Phospholipidosis NFκB SREBP1
AP-1
Increased Lysosome
phagocytosis? +LC3-II
High concs
+VCP
+ MPR
Autophagy - Bcl
Enzymes
Apoptosis

Lipid Golgi
transport
Cytokine, mucin and
ER inflammatory gene expression
depending on stimulus

Fig. 1. Effects of azithromycin on cell signaling. Azithromycin penetrates the cell membrane bilayer and stabilizes the membrane, reducing fluidity. The cationic moiety of azithromycin
comes into close proximity to the negatively charged phospholipids in the inner leaflet of the membrane, neutralising their charge. This results in reduced fatty acid release and also to
liberation of enzymes bound by electrostatic charge to the membrane. Lipid remodeling leads to modulation of signaling pathways, particularly of MAPK kinases, such as ERK1/2 (possibly
involving Rac-1) with subsequent inhibition of the activation of transcription factors including AP-1 and NFκB. The signaling pathway most affected is likely to be dependent on the
particular cell, its activation state and the stimulus by which it is activated. Lysosomal enzyme release and initial induction of the oxidative burst of neutrophils are a consequence of
the actions on membrane lipids. Membrane lipid remodelling also disrupts surface molecule recycling and probably phagocytosis. Molecules dependent on negatively charged
phospholipids are also affected, including LC3-II, with subsequent influences on lysosomal uptake and autophagy. The latter may also be influenced, with time, by low affinity binding
of azithromycin to VCP. Azithromycin accumulates in lysosomes, modulating MPR transport of enzymes and lipids and through lipid remodeling in the lysosome membranes ultimately
leads to phospholipidosis. Apoptosis may be induced by high concentrations of azithromycin, possibly by compromising the action of the apoptosis inhibiting Bcl molecules. [Ø indicates
inhibition].

disease and/or bronchiectasis (Brook, 2006), which may all benefit
simultaneously from azithromycin therapy (Sections 5.3 and 8.2).
Immunomodulation by macrolides has been proposed repeatedly to
contribute to decreased length of hospitalization and increased survival
in CAP and CAP-related sepsis (Amsden, 2005; Giamarellos-Bourboulis
et al., 2008; Metersky et al., 2007; Restrepo et al., 2009; Tessmer et al.,
2009). Recent meta-analyses indicate that while macrolides do decrease
mortality among hospitalized CAP patients, in RCTs or in guideline-
concordant studies a difference from other antibiotics was not evident
(Asadi et al., 2012; Asadi et al., 2013; Skalsky et al., 2013). As discussed
later for sepsis (Section 7), disease severity may determine sensitivity to
antibiotics. A recent study on 187 patients with pneumonia due to
S. pneumoniae, clearly confirmed that azithromycin significantly
reduces mortality rate (Shorr et al., 2013). This study differed from
previous studies in being limited to a single causative agent.
Azithromycin effectively reduces respiratory symptoms in chronic
or recurrent respiratory tract infections due to so-called atypical bacte-
ria, such as chronic Chlamydia pneumoniae or Mycoplasma pneumoniae
(Branden et al., 2004; Esposito et al., 2005; Mulholland et al., 2012).
Long-term, low-dose azithromycin, though, lacked efficacy in children
with acute respiratory syncytial virus bronchiolitis (Kneyber et al.,
2008; Pinto et al., 2012) or in chronic rhinosinusitis (Videler et al.,
2011). Nevertheless, an increasing number of small scale studies sug-
gest that azithromycin and other macrolides are of benefit in viral infec-
tions because of their immunomodulatory properties (Min & Jang,
2012). There is considerable interest, particularly in Japan, in using
macrolides to prevent the cytokine storm which occurs in patients dur-
ing respiratory viral epidemics (Bermejo-Martin et al., 2009). Certainly,
azithromycin inhibits and stimulates the resolution of lung inflamma-
tion in mice infected with mouse parainfluenza virus type 1 (Sendai
virus) without affecting viral load (Beigelman et al., 2010). It may well
also enhance anti-viral defences, since in contrast to erythromycin and
telithromycin, azithromycin significantly increases interferon genera-
tion and interferon-stimulated gene expression, reducing virus replica-
tion and growth in human bronchial epithelial cells infected with
rhinoviruses 16 and 1B (Gielen et al., 2010).

Please cite this article as: Parnham, M.J., et al., Azithromycin: Mechanisms of action and their relevance for clinical applications, Pharmacology &
Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.03.003
 M.J. Parnham et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx
5.2. Diffuse panbronchiolitis
Initial reports on clinical immunomodulation by macrolides ap-
peared in the late 1980s and 1990s, when it became clear that the
prognosis of patients with diffuse panbronchiolitis (DPB) improved dra-
matically after implementing long-term treatment with macrolides.
This was first documented with erythromycin, demonstrating signifi-
cant reduction in broncho-alveolar lavage (BAL) fluid of IL-1β, IL-8
and neutrophils (Sakito et al., 1996). Sputum volume in DPB is reduced
and lung function improved by macrolides, with marked reduction of
neutrophil activation and lung infiltration, of pro-inflammatory cyto-
kines and MMPs. This benefit is seen despite the relatively high inci-
dence of resistance to macrolides in Japan (Kobayashi et al., 1993;
Kudoh, 2004). Because of the established treatment of DPB with other
macrolides in Japan, azithromycin has been used less frequently, but
was recently reported to be effective and safe for long-term therapy
(Li et al., 2011).
Treatment of DPB is the most striking example of the introduction of
macrolide-therapy to chronic respiratory diseases. However, the experi-
ence in DPB is almost exclusively based on non-randomized, controlled
clinical trials (RCTs) or retrospective studies (Kudoh et al., 1998; Li et al.,
2011; Yang et al., 2013). Drawing on this experience in DPB, long-term,
low-dose azithromycin therapy was subsequently introduced in CF
(Saiman et al., 2003), non-CF bronchiectasis (Anwar et al., 2008),
COPD (Blasi et al., 2010; Hodge et al., 2008; Parnham et al., 2005),
asthma (Piacentini et al., 2007), infectious bronchiolitis (Naidoo &
Bryant, 2009) and BOS following lung or hematopoietic stem cell
transplantation (Gerhardt et al., 2003; Gottlieb et al., 2008; Vos et al.,
2010). Clinical evidence for immunomodulatory effects and applica-
tions of azithromycin in inflammatory airway disorders is briefly
discussed here based on landmark and important clinical trials.
Fig. 2. Proposed time course of azithromycin actions on infection and chronic inflammation.
Please cite this article as: Parnham, M.J., et al., Azithromycin: Mechanisms of action and their relevance for clinical applications, Pharmacology &
Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.03.003
11
5.3. Cystic fibrosis and non-cystic fibrosis bronchiectasis
As in DPB, pulmonary neutrophil infiltration predominates in
patients with CF and non-CF bronchiectasis and this encouraged
many investigators to study azithromycin in this indication. In an RCT
in 251 patients chronically infected with P. aeruginosa, standard 3 day
azithromycin treatment weekly for 24 weeks significantly improved
FEV1 and reduced risk of exacerbations (Saiman et al., 2003). Sub-
sequently, when treatment was extended to 12 months, FEV1 remained
unchanged in patients not infected with P. aeruginosa, although exacer-
bation and cough were significantly decreased (Clement et al., 2006;
Saiman et al., 2010). While pulmonary function, thus, only improves
on azithromycin treatment for up to 6 months and not thereafter, re-
duced exacerbations and reduced S. aureus counts, presumably related
to immunomodulation, are consistent findings (Southern et al., 2011).
The FEV1 evolution is explained by a decrease in FEV1 during the initial
inflammatory phase, which then increases with decreasing inflamma-
tion after azithromycin is initiated, FEV1 subsequently reaching a
steady-state or plateau phase (depending on % predicted for each
individual). Azithromycin is generally considered an important part
of the treatment armamentarium for CF, particularly in patients chron-
ically infected with P. aeruginosa (Spagnolo et al., 2013; Suresh Babu
et al., 2013).
The role of azithromycin in non-CF bronchiectasis is now well
established with three recent RCTs. In the EMBRACE trial, compared to
placebo, azithromycin decreased event-based exacerbation rates in
adult patients with non-CF bronchiectasis, whereas changes in FEV1
from baseline and in SGRQ total score during the 6-month treatment
period did not differ (Wong et al., 2012). The BAT trial confirmed that
daily use of azithromycin for 12 months results in a lower rate of in-
fectious exacerbations but azithromycin also increased FEV1 by 1.03%
12 M.J. Parnham et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx
per 3 months in comparison to a decrease of 0.10% per 3 months in the
placebo group (Altenburg et al., 2013). Macrolide resistance rate was
88% in azithromycin-treated individuals and 26% in the placebo group.
In children with non-CF bronchiectasis, once-weekly azithromycin for
up to 24 months also decreased pulmonary exacerbations, but again
with increased carriage of azithromycin-resistant bacteria (Valery
et al., 2013). Overall, these RCTs confirm the beneficial effects with
azithromycin or erythromycin described in prior, smaller, retrospective
studies in non-CF bronchiectasis (Anwar et al., 2008; Serisier & Martin,
2011). Although not addressing the underlying etiology of the disease,
azithromycin may be a therapeutic option in patients with frequent
exacerbations (Spagnolo et al., 2013; Suresh Babu et al., 2013).
5.4. Chronic obstructive pulmonary disease and asthma
Short-term azithromycin has been an alternative to first-line antibi-
otics in the treatment of acute exacerbations of COPD (AECOPD) for sev-
eral years (Butorac-Petanjek et al., 2010; Yamaya et al., 2012). The
benefit achieved is greater than with other therapies such as corticoste-
roids or roflumilast (Spagnolo et al., 2013). Long-term azithromycin
both decreased frequency of exacerbations and enhanced quality of
life in AECOPD, though with a small incidence of hearing decrements
(Albert et al., 2011). Azithromycin given for several months to patients
with COPD also increased efferocytosis in airway macrophages, in-
dicating an immunomodulatory component to its efficacy (Hodge &
Reynolds, 2012).
Azithromycin ameliorates bronchial hyperresponsiveness and air-
way neutrophil infiltration in some children with asthma (Piacentini
et al., 2007), but failed to reduce rates of severe exacerbations and
LRTI in adult patients with severe asthma (Brusselle et al., 2013). Corti-
costeroids have limited efficacy in COPD or neutrophilic asthma and in a
subgroup analysis of patients with non-eosinophilic severe asthma,
azithromycin significantly lowered rates of severe exacerbations and
LRTI requiring treatment with antibiotics (Brusselle et al., 2013).
Modulation of neutrophilic inflammation probably accounts for these
findings in asthma since azithromycin inhibits airway inflammation in
a non-infectious, ovalbumin-induced mouse model of allergic asthma
(Beigelman et al., 2009) (see Section 4.3.3).
Despite broad inhibitory activity in experimental studies (see
Section 4.1), efficacy of azithromycin in clinical airways inflammation
is limited. Clinical bronchodilatory effects have not been reported.
Azithromycin has a place in the treatment of COPD exacerbations, but
other than possibly contributing to corticosteroid-sparing in highly
selected patients, its potential role in the treatment of asthma remains
unclear (Spagnolo et al., 2013; Suresh Babu et al., 2013).
5.5. Post-transplant bronchiolitis
In lung transplant recipients with post-transplant bronchiolitis,
azithromycin for 3–6 months reduced local airway inflammation (cyto-
kines, neutrophils), oxidative stress and matrix remodelling (Verleden
et al., 2006, 2011a,b). It attenuates both neutrophilia and plasma levels
of multiple pro-inflammatory chemokines (Federica et al., 2011). These
findings with azithromycin have even led to the definition of novel
phenotypes of post-transplant chronic lung allograft dysfunction,
based on factors such as severity and degree of neutrophil infiltration
(Gottlieb et al., 2008; Vanaudenaerde et al., 2008a,b; Vos et al., 2010).
Interestingly, prophylactic azithromycin treatment following lung
transplantation resulted in significantly higher chronic rejection-free
survival and lower airway neutrophilia and systemic CRP levels in
comparison to placebo (Vos et al., 2011). This again confirms that
azithromycin attenuates inflammation resulting from allo- and non-
allo-immunologic events in the lung. Long-term RCTs are needed to
establish the routine use of azithromycin in this condition.
Please cite this article as: Parnham, M.J., et al., Azithromycin: Mechanisms of action and their relevance for clinical applications, Pharmacology &
Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.03.003
5.6. Other respiratory conditions
Macrolides (i.e. clarithromycin) appear to modulate cryptogenic
organizing pneumonia, radiation-related bronchiolitis obliterans
organizing pneumonia (Mann et al., 2005; Stover & Mangino, 2005)
and pulmonary fibrosis (Wuyts et al., 2010). This is not surprising
given that in these conditions, the distal airways and alveoli initially
may be involved in an inflammatory process.
In summary, numerous pulmonary disorders sharing common
innate inflammatory mechanisms can potentially benefit from the
anti-inflammatory and immunomodulatory properties of azithromycin.
Azithromycin may, therefore, prove to be a new milestone in the treat-
ment of chronic pulmonary disorders, always taking into account the
increase in macrolide resistance. In this respect, future development of
non-antibacterial, anti-inflammatory macrolide derivatives may be
crucial (Bosnar et al., 2012; Kobayashi et al., 2013; Mencarelli et al.,
2011; Tomaskovic et al., 2013) (Erakovic Haber et al., in press).
6. Infections of the urogenital
tract and sexually-transmitted infections
Over 90% of an oral dose of azithromycin is excreted via the hepato-
biliary system (Ballow et al., 1998; Foulds et al., 1990; Singlas, 1995).
Since very little drug is found unchanged in the urine, azithromycin is
not suitable for diseases involving significant bacteruria. Due to its opti-
mal systemic distribution and tissue penetration, though, azithromycin
accumulates up to 100-fold in a variety of tissues of urological interest
(Foulds et al., 1990; Foulds et al., 1991).
6.1. Urethritis
Their considerable penetration into human cells makes macrolides
particularly suited for treatment of infections caused by obligate intra-
cellular pathogens, like Chlamydia spp. For several years, azithromycin
has been the first-line drug for treatment of primary non-gonococcal
urethritis (www.uroweb.org/gls/pdf/15_Urological_Infections.pdf),
mainly caused, in both sexes, by Chlamydia trachomatis, Ureaplasma
urealyticum or Mycoplasma genitalium.
Essential clinical evidence was provided in 1995 for this indication
in men (Stamm et al., 1995). A single 1 g oral dose of azithromycin,
for empirical treatment of acute non-gonococcal urethritis syndrome,
was as effective as a standard 7-day course of twice-daily 100 mg doxy-
cycline in achieving clinical cure. Microbiological and overall clinical
cure rates with azithromycin were 83% and 81%, respectively (Stamm
et al., 1995).
A meta-analysis of RCTs confirmed the therapeutic equivalence of
the two regimens for chlamydial urethritis in men and women (Lau &
Qureshi, 2002). However, patient compliance to doxycycline for 7
days is often hampered by gastrointestinal side effects (Thorpe et al.,
1996). Since poor compliance is detrimental to resolution of the infec-
tion and may increase resistance, in our opinion, the 100% compliance
with a single-dose azithromycin regimen may minimize such risks.
Whereas azithromycin and doxycycline are equally active against
chlamydial infections, the macrolide achieved higher rates of eradica-
tion and clinical remission in patients with non-gonococcal urethritis
(NGU) caused by Mycoplasma genitalium (Falk et al., 2003; Mena et al.,
2009).
In women, chlamydial cervicitis/urethritis is highly prevalent, and
may occur concurrently with onset of preterm labor, preterm rupture
of membranes, spontaneous abortion, fetal death, postpartum endome-
tritis, salpingitis, with risks to the neonate (Pitsouni et al., 2007). A sin-
gle 1 g dose of azithromycin eradicates the infection in over 95% of cases
and is equivalent in terms of efficacy and symptom resolution to one-
week, once-daily 100 mg doxycycline (Thorpe et al., 1996). A meta-
analysis (Pitsouni et al., 2007), involving 587 pregnant women, demon-
strated high and equivalent efficacy of azithromycin, erythromycin and
 M.J. Parnham et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx
amoxicillin against chlamydial cervicitis/urethritis. However, azithro-
mycin was associated with fewer gastrointestinal adverse events,
fewer total adverse events, and better compliance.
6.2. Prostatitis
Chronic bacterial prostatitis (CBP) is a difficult-to-eradicate biofilm
disease, dramatically affecting quality of life and requiring long-term
antibacterial therapy for weeks or even months. Eradication of causative
pathogens is hampered by poor penetration of most antibacterial agents
into the prostate gland. Fluoroquinolones are currently the agents of
choice, due to adequate prostatic penetration, but recent RCTs show
that macrolides are becoming a primary therapeutic option for CBP
(Kolumbic Lakos et al., 2011; Magri et al., 2007, 2010; Skerk et al.,
2002, 2003, 2004, 2006). The rationale is the excellent penetration
and high concentration of this macrolide (see Sections 3.1 and 3.2) in
prostatic tissue and secretions, together with its powerful anti-biofilm
and immunomodulatory activities. Consequently, azithromycin is
recommended as a therapeutic option for CBP in specific cases by
the European Urological Association (www.uroweb.org/gls/pdf/15_
Urological_Infections.pdf). The clinical and preclinical evidence
supporting azithromycin as a valuable treatment option for CBP has
been reviewed in detail (Perletti et al., 2011).
6.3. Other sexually transmitted diseases (STDs)
Macrolides and azithromycin in particular, are used to treat a num-
ber of STDs including syphilis, granuloma inguinale (Donovanosis)
and chancroid. In the latter two diseases, azithromycin is recommended
as first-line therapy (http://www.cdc.gov/std/treatment/2010/toc.
htm).
6.4. Glomerulonephritis
Crescentic glomerulonephritis is a severe renal complication of
infectious endocarditis, frequently caused by Gram-positive cocci or
Gram-negative Bartonella species (Kannan & Mattoo, 2001). The condi-
tion is not a urinary tract infection per se, but rather derives from im-
mune activation by bacterial antigens. Bartonella endocarditis immune
complexes are deposited in the glomeruli cause necrotizing crescentic
glomerulonephritis. Interestingly, this form of glomerulonephritis may
improve with antibiotic therapy (Bookman et al., 2004). Resolution of
glomerulonephritis and normalization of serum creatinine levels was
achieved in several cases by aggressive therapy with azithromycin and
a parenteral cephalosporin (ceftriaxone) or by tobramycin/doxycycline
combination (Bookman et al., 2004). The efficacy of azithromycin
against facultative intracellular Bartonella may be explained by good
intracellular penetration of the macrolide (Massei et al., 2005).
7. Clinical application in sepsis
Sepsis is an overwhelming inflammatory host response to a bacterial
stimulus. Several animal studies have shown a significant anti-
inflammatory effect of azithromycin (Table 3) or clarithromycin in ex-
perimental sepsis. Pretreatment of mice with azithromycin improved
survival and attenuated plasma levels of TNFα in LPS-induced shock
(Ivetic Tkalcevic et al., 2006; Tong et al., 2011) and additional immuno-
modulatory effects have been described for clarithromycin. Adminis-
tered i.v. immediately after cecal ligation and puncture in rabbits,
clarithromycin reduced apoptosis in circulating lymphocytes and
production of TNFα by circulating monocytes, especially within the
first 4 h after treatment (Atmatzidis et al., 2011). Pretreatment with
clarithromycin, but not levofloxacin, also inhibited lung inflammation
and adhesion molecule expression due to high pressure ventilation in
mice (Amado-Rodriguez et al., 2013). Similar clinically relevant studies
on azithromycin have yet to be performed.
Please cite this article as: Parnham, M.J., et al., Azithromycin: Mechanisms of action and their relevance for clinical applications, Pharmacology &
Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.03.003
13
It is estimated that severe sepsis and septic shock, mainly due to CAP,
annually affect 377 of 100,000 members of the general population;
mortality is 35–50% making sepsis a leading cause of death, more com-
mon than malignancies and heart disorders (Kotsaki & Giamarellos-
Bourboulis, 2012). Current guidelines recommend intravenous antibiot-
ic treatment as early as possible, because with most antibiotic classes,
effectiveness decreases with time and sepsis severity (Dellinger et al.,
2013). Several retrospective analyses of prospectively collected data
have assessed the outcome of monotherapy over combination therapy
of severe CAP. The primary endpoint of these analyses was whether
inclusion of a macrolide (azithromycin, clarithromycin or erythromycin,
where recorded) in the combination affects mortality. The summary of
the seven studies published after 2006 is shown in Table 4. Four studies
indicated a significant decrease in risk of death with the intake of one
macrolide (Martin-Loeches et al., 2010; Metersky et al., 2007; Restrepo
et al., 2009; Tessmer et al., 2009); two showed a significant decrease in
duration of hospitalization (Ambroggio et al., 2012; Wilson et al., 2012);
and only one failed to show any benefit (Karhu et al., 2013).
Two prospective, RCTs have evaluated the impact of macrolide
administration on the outcome of severe infections. In both trials, the
macrolide was clarithromycin, but in view of the striking outcomes,
they are discussed here as indicators of the potential use of other
macrolides, such as azithromycin. The hypothesis was that clarithro-
mycin, administered at a dose of 1 g within 1 h on consecutive days,
should reach maximum serum levels (10 μg/mL) sufficient to modulate
circulating monocyte function. In the first study (Giamarellos-
Bourboulis et al., 2008), 200 patients with ventilator-associated pneu-
monia (VAP) received placebo or clarithromycin for three days. Isolated
bacterial pathogens were multidrug-resistant species of Acinetobacter
baumannii, of Pseudomonas aeruginosa and of Klebsiella pneumoniae,
not included in the antimicrobial spectrum of clarithromycin. The
macrolide significantly reduced by 5.5 days, time until resolution of
VAP (p = 0.011); by 6.5 days, time until weaning from mechanical
ventilation (p = 0.049); and the relative risk of death by septic shock
and multiple organ dysfunction (MODS; from 19.00 with placebo to
3.78 with clarithromycin; p = 0.048). No difference in incidence of
serious adverse events was seen.
Immunoparalysis develops during severe sepsis and drives towards
multiple organ dysfunction syndrome (MODS) and a lethal outcome
(Boomer et al., 2011). Crucially, clarithromycin treatment was accom-
panied by a reversal of immunoparalysis, most prominently 4 days
after start of treatment and among patients with septic shock and
MODS. Compared with placebo, clarithromycin decreased the serum
IL-10/TNFα ratio; increased monocyte CD86 expression, a marker
of antigen-presentation efficiency; and increased IL-6 production by
LPS-stimulated monocytes (Spyridaki et al., 2012). Reversal of immuno-
paralysis was unexpected as attenuation of inflammatory responses in
patients would have been anticipated, based on the results of animal
studies with macrolides (Table 3). It seems that, like azithromycin,
clarithromycin can modulate monocyte function, depending on the
inflammatory environment (see Section 4.1.2). Alternatively, these
findings, at least in part, may represent an epiphenomenon arising
from improvement in the clinical course of sepsis following treatment.
In the second RCT study (Giamarellos-Bourboulis et al., 2013), 600
patients were enrolled with sepsis due to primary or secondary Gram-
negative bacteremia, acute pyelonephritis or acute intrabdominal
infections, all with microbiologically-proven or clinically-suspected
Gram-negative bacteria insensitive to clarithromycin. Treatment was
extended to four days and the findings of the first RCT were confirmed.
Mortality due to septic shock and MODS was reduced by clarithromycin
to 53.6% compared to 73.1% with placebo (p = 0.020). Furthermore,
among patients with severe sepsis/shock, median time to infection
resolution was 6 days with clarithromycin and 10 days with placebo
(p = 0.037) and was accompanied by a significant shortening of hospi-
talization time and reduction of costs. Treatment with clarithromycin
did not impact the safety of the patients.
14 M.J. Parnham et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx

Current management of sepsis remains largely supportive. Therapy
targets the underlying infection and aims to support the function of
failing organs. All agents developed over the last two decades that
inhibit exaggerated pro-inflammatory responses have failed to prolong
survival or were even harmful. The fact that clarithromycin was able to
reverse the immunoparalytic state arising during severe sepsis, in an
RCT in patients with VAP (Spyridaki et al., 2012), raises hopes that
macrolides, including azithromycin, may offer a significant advance in
the management of sepsis. Their efficacy in this condition may help
explain the benefit of macrolides in patients with CAP. The good efficacy
of azithromycin in severe inflammatory cases of post-transplantation
bronchiolitis (see Section 5.5), suggests that there may be similarities
in the mechanisms involved in effects of macrolides in the two
conditions.
The mechanism of action is considered to be a combination of anti-
bacterial, immunomodulatory and biofilm-inhibiting effects, associated
with the prolonged accumulation of azithromycin in inflamed tissues
(Wang, 2010). The improved safety of azithromycin in comparison to
the current recommended adjuvant antibiotic treatment with amoxi-
cillin and metronidazole is a further added incentive to the introduction
of the macrolide.
Azithromycin is widely used to treat gingival overgrowth, a side-
effect of cyclosporin administration. Only 3 RCTs have been carried
out using a short treatment period, but clear reduction of tissue over-
growth was reported in all studies and use of azithromycin for this
purpose is recommended (Hirsch et al., 2012).
8.3. Ophthamology

8. Other clinical applications Azithromycin is used in ophthalmology for topical treatment of

8.1. Skin disease
Macrolide antibiotics, are widely used for the topical treatment of
acne and rosacea and their use for these and other skin disorders has
been reviewed (Alzolibani & Zedan, 2012). In several small studies,
oral azithromycin (for 2–4 weeks) provided significant improvement
in intractable rosacea. In one healthy volunteer-controlled study, stan-
dard dosing with azithromycin for 4 weeks to 17 patients with rosacea,
reduced inflammatory score and ameliorated the raised oxidave burst
(chemiluminescence) of leukocytes in biopsies from skin lesions
(Bakar et al., 2007). Since no microbiological analyses were carried
out, it is unclear whether the effects observed were due to anti-
inflammatory or antibacterial actions. Currently, rosacea is considered
to be a disease of dysregulated innate immunity. In the absence of
RCTs, the place of azithromycin in the treatment of rosacea remains to
be established.
8.2. Dentistry
bacterial conjunctivitis and for off-label use in chronic blepharitis
(inflammation of the eyelid) (Gebre et al., 2012). In the latter, applica-
tion of 1% azithromycin for 2 weeks reduced redness and swelling
and improved meibomian gland secretion (Luchs, 2010), probably
due to immunomodulatory effects, also observed in a study of
topical azithromycin in LPS-induced ocular inflammation in the rat
(Fernandez-Robredo et al., 2013).
8.4. Gastric motility
Erythromycin is a stimulator of gastric motility as a consequence of
agonist activity at motilin receptors (Peeters et al., 1989). Azithromycin
similarly stimulates gastric motility and in view of its better safety pro-
file to that of erythromycin, has been proposed as a treatment for gastric
motility disorders (Mertens et al., 2009; Rohof et al., 2012). Recently,
azithromycin has been shown to have similar potency to erythromycin
as an agonist at human motilin receptors, causing prolonged release of
acetylcholine from human gastric antrum muscle (Broad & Sanger,
2013).

Over the last 20 years, a number of studies, only a few them RCTs,
have reported beneficial effects of azithromycin in adjuvant treatment
of periodontal disease (Bartold et al., 2013; Hirsch et al., 2012). Micro-
biological studies indicate that azithromycin is highly effective against
Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans
(Bartold et al., 2013).
The balance of opinion is that azithromycin relieves infection and
inflammation and reduces pocket depth, particularly in aggressive
forms of periodontal disease, but further controlled studies are needed.
9. Safety
A concern about the use of macrolide antibiotics for long-term treat-
ment of chronic inflammatory diseases, is the possibility of increased
bacterial resistance, though the relationship between long-term use
and increased resistance is still uncertain (Cameron et al., 2012). A re-
cent meta-analysis of six RCTs on the effects of long-term azithromycin
in chronic lung diseases, revealed that while bacterial colonization de-
creased by 45%, there was a 2.7-fold increase in bacterial resistance (Li

Table 4
Summary of retrospective studied published after 2006 on the impact of macrolide administration on the outcome of community-acquired pneumonia (CAP).

Reference Setting Arms (n patients) Administered macrolide Effect of macrolide intake

(Ambroggio et al., 2012)
(Karhu et al., 2013)
(Martin-Loeches et al., 2010)
(Metersky et al., 2007)
(Restrepo et al., 2009)
(Tessmer et al., 2009)
(Wilson et al., 2012)
Hospitalized children
Severe sepsis due to CAP
Severe sepsis and MV due to CAP
Bacteremic pneumonia
Severe sepsis due to CAP
Hospitalized patients
Severe sepsis due to CAP
β-lactam (15809)
β-lactam + macrolide (4934)
β-lactam + quinolone (104)
β-lactam + macrolide (106)
β-lactam + quinolone (54)
β-lactam + macrolide (46)
Monotherapy (601)
Combination therapy (1408)
Non-macrolide (133)
Macrolide (104)
β-lactam (908)
β-lactam + macrolide (946)
β-lactam + quinolone (908)
β-lactam + macrolide (946)
NR Reduction of LOS by one day
among 31% of children
12–18 y (p b 0.0001)
Azithromycin or erythromycin No difference
NR Reduction of risk of death
(HR: 0.44, p: 0.03)
Azithromycin (n = 231) Risk of death after 30 days
Clarithromycin (n = 65) with macrolide intake 0.61
Erythromycin (n = 32) (p: 0.007)
NR Reduction of 90-day mortality
(12.5% vs 33.8%, p b 0.0001)
NR Independent predictor of lower
mortality at 14 days
(OR: 0.53, p: 0.031)
NR Reduction of mean LOS
(15.9 days vs 24.7 days,
p b 0.0001)

HR: hazard ratio; LOS: length of stay; MV: mechanical ventilation; NR: not reported; OR: odds ratio.

Please cite this article as: Parnham, M.J., et al., Azithromycin: Mechanisms of action and their relevance for clinical applications, Pharmacology &
Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.03.003
 M.J. Parnham et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx
et al., 2013). The benefit of long-term macrolide treatment of DPB pa-
tients considerably outweighs this concern, particularly since resistance
to macrolides is already high in Japan, where DPB is demographically
significant (see Section 5.2). In addition, the motilin-agonistic effects
of the antibiotic result in an increased incidence of adverse gastrointes-
tinal effects on prolonged treatment (Cameron et al., 2012). The devel-
opment of novel, non-antibacterial, non-motilin receptor binding,
immunomodulatory macrolides may overcome these concerns
(Cameron et al., 2012); Erakovic Haber et al., in press). A further safety
issue with long-term azithromycin treatment is the possible occurrence
in a small number of patients of hearing defects (Li et al., 2013).
For many years, azithromycin has been one of the safest broad
spectrum antibiotics. In contrast to other macrolide antibiotics, not
interacting with CYP3A4 means that it exhibits no metabolic inter-
actions with most other commonly used drugs. It is N20-fold less potent
an inhibitor of hERG potassium channels than other macrolide anti-
biotics (Giudicessi & Ackerman, 2013). However, in an observational,
non-randomized study of patients enrolled into a Tennessee Medicaid
program, patients receiving azithromycin had an incidence of cardio-
vascular death 2.88-fold higher than those taking no antibiotic, and
2.49-fold higher than in patients on amoxicillin (Ray et al., 2012). Sub-
sequently, the FDA introduced a black box warning about the potential
risk of fatal arrhythmias with azithromycin, advising health profes-
sionals to “consider the risk of fatal heart rhythms with azithromycin
when considering treatment options for patients who are already at
risk for cardiovascular events.” A further post-marketing safety surveil-
lance using seven population-based healthcare databases in Denmark,
Italy, and the Netherlands, identified azithromycin, among other medi-
cations, as a prime suspect for an association with drug-induced acute
myocardial infarction (Coloma et al., 2013). The US observational
study was criticized, though, because Medicaid patients generally
have a higher comorbidity than patients in the general population
(Giudicessi & Ackerman, 2013). Importantly, a large prospective study
in young to middle-aged Danish people at a low-risk of underlying
cardiovascular disease revealed that the risk of cardiovascular death
due to 5-day treatment was 15.4 in comparison to 85.4 per million
azithromycin courses in the Tennessee study (Svanstrom et al., 2013).
Thus, the risk of a cardiovascular event with azithromycin in otherwise
healthy patients is very small, but in patients at risk (i.e. idiopathic or
drug-induced baseline QT prolongation, familial long QT syndrome, or
multiple risk factors) azithromycin should be considered with care
and polypharmacy should be avoided (Giudicessi & Ackerman, 2013).
10. Conclusions
As a broad-spectrum antibacterial, azithromycin shares the same
mechanism of action as other macrolide antibiotics and its range of
activity is extended through inhibition of bacterial quorum-sensing
and biofilm. Accumulating more effectively than other macrolides in
cells, particularly circulating phagocytes, it is delivered in high concen-
trations to sites of infection. This important feature, combined with
the extended plasma half-life of azithromycin, often allows effective
single-dose administration for acute bacterial infections. Cellular accu-
mulation, accounting for rapid plasma clearance and extensive tissue
distribution, offsets the drug's moderate bioavailability.
The antibiotic effects of azithromycin are facilitated by its immuno-
modulatory actions on innate and adaptive immune responses. The
drug appears to exert a biphasic action which may serve to promote
initial host defence and later reduce bystander tissue injury and
promote inflammation resolution. Beneficial effects mediated by
immunomodulation are also seen in a variety of chronic inflammatory
disorders. These include DPB, post-transplant bronchiolitis (particularly
severe inflammatory forms) and rosacea. The ability of azithromycin to
modulate host response reactions contributes to beneficial effects in
CF, non-CF bronchiectasis, COPD and asthma. Currently, based on
Please cite this article as: Parnham, M.J., et al., Azithromycin: Mechanisms of action and their relevance for clinical applications, Pharmacology &
Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.03.003
15
comparative efficacy with other drugs, azithromycin is only recom-
mended for use in DPB, CF, non-CF bronchiectasis and bronchiolitis.
Early, mainly stimulatory effects of azithromycin on immune and
epithelial cells are probably mediated by interactions with phospho-
lipids and Erk1/2. Later modulation of transcription factors AP-1 and
NFκB, is accompanied by inhibition of inflammatory cytokine and
mucin release. Subsequent delayed inhibitory effects on cell function
are likely related to the high lysosomal accumulation of the macrolide,
with disruption of protein and lipid transport through the Golgi appara-
tus and ultimate effects on surface receptor expression, including
macrophage phenotype changes, and autophagy. These later changes,
particularly on macrophage and dendritic cell phenotype, appear to
underlie many in vivo experimental and clinical immunomodulatory
effects of azithromycin, contributing to resolution of acute infections
of the airways, genital tract and skin, as well as to reduction of exacerba-
tion frequency in chronic airway diseases.
Immunomodulation with long-term azithromycin to reduce exacer-
bations in COPD, CF and non-CF bronchiectasis patients must be
balanced against the potential for increased bacterial resistance. How-
ever, a sub-group of post-transplant bronchiolitis patients with pro-
minent neutrophil infiltration appears to be particularly sensitive to
long-term prophylactic therapy with azithromycin, as may be patients
with severe sepsis treated acutely with the drug. Other promising indi-
cations include chronic prostatitis, rosacea and periodontitis.
Although well-tolerated with a very good record of safety, azithro-
mycin has recently been associated with rare cases of torsades des
pointes in patients at risk of cardiac arrhymias and polypharmacy with
azithromycin is generally to be avoided.
Conflict of interest
GPP and RV declare that they have no conflicts of interest.
Acknowledgments
MJP has collaborated with Novo Nordisk and serves as an advisor to
Leo Pharma A/S and Xellia Pharmaceuticals ApS. VEH is an employee of
Fildelta d.o.o. and both MJP and VEH are previous employees of PLIVA
and GlaxoSmithKline. EGB has received unrestricted educational grants
from Abbott Hellas SA (program 70/3/7447 of the University of Athens).
He also serves as an advisor to AbbVie SA.
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