The world is facing a frightening pandemic due to coronavirus disease 2019 (COVID‐19)
caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) with thousands
of severe infections and fatalities. Since no therapy has proven effective, an extraordinary
race is taking place to identify an effective and safe treatment able to limit the
disease progression and severity.
Two nonrandomized open‐label trials conducted in France and China
1
,
2
established the effectiveness of hydroxychloroquine alone or combined with azithromycin
in decreasing nasopharyngeal viral load and carriage duration in patients with COVID‐19,
although evidence to support clinical benefits remained low. Thereafter, many studies,
still unpublished in peer‐reviewed journals for the majority, showed contrasting results
and revealed potential safety hazards (Table 1).
3
,
4
,
5
,
6
,
7
,
8
,
9
,
10
,
11
To date, multiple trials aiming at investigating chloroquine or hydroxychloroquine
at various dose regimens to treat COVID‐19 (N = 81) or prevent the disease in high‐risk
populations (N = 19) are cited on clinicaltrials.gov (accessed April 21, 2020). Interestingly,
only 14 trials (17%) will investigate the azithromycin‐hydroxychloroquine combination.
Table 1
Emerging List of Studies Investigating Chloroquine/Hydroxychloroquine With or Without
Azithromycin to Treat COVID‐19
Authors
Country
Design
N
Time in the Disease Course and Infection Severity
Groups and Dose Regimen
Main Resultsa
Gautret et al
1
published
France
Uncontrolled noncomparative observational study
80
Early (d 5)
Mild
1 group, HCQ (200 mg ×3/d, 10 d) + AZ (500 mg on d 1 followed by 250 mg/d, 4 d)
Clinical improvement, rapid discharge, rapid fall in nasopharyngeal viral load, negative
viral culture on d 5 in almost all patients
Chen et al
2
China
Randomized open‐label parallel‐group trial
62
Unknown
Moderate
2 groups, HCQ (200 mg ×2/d, 5 d) vs no HCQ
Significant clinical improvement based on body temperature recovery and cough remission
times and increased recovery from pneumonia
Chen et al
3
published
China
Randomized open‐label controlled trial
30
Early (d 6)
Mild
2 groups, HCQ (200 mg ×2/d, 5 d) vs no HCQ
No reduction in the percentage of negative SARS‐CoV‐2 nucleic acid of throat swabs,
the time from hospitalization to virus nucleic acid negative conservation, temperature
normalization, and radiological progression
Molina et al
4
published
France
Uncontrolled noncomparative observational study
11
Unknown
Moderate
1 group, HCQ (600 mg/d, 10 d) + AZ (500 mg/d, d 1 and 250 mg/d, d 2‐5)
Positive SARS‐CoV‐2 RNA in 8/10 patients (80%, 95% confidence interval, 49%‐94%) at
d 5‐6 after treatment initiation
Magagnoli et al
5
United States
Retrospective cohort study
368
Unknown
Moderate
3 groups, HCQ vs HCQ+AZ vs no HCQ (dosages not available)
No reduction in mechanical ventilation
Increased overall mortality in HCQ group
Mahévas et al
6
France
Retrospective cohort study
181
Early (d 7)
Moderate
2 groups, HCQ (600 mg/d within 48 h after admission) vs no HCQ
No reduction in ICU transfer or death, death within 7 d and ARDS within 7 d
Million et al
7
France
Uncontrolled noncomparative observational study
1061
Early (d 6)
Mild
1 group, HCQ (200 mg ×3/d, 10 d) + AZ (500 mg on d 1 followed by 250 mg/d, 4 d); analysis
of the patients who took HCQ + AZ during at least 3 d
Significant reduction in mortality in comparison to patients treated with other regimens
in all Marseille public hospitals
Barbosa et al
8
United States
Retrospective cohort study
63
Unknown
Moderate
2 groups, HCQ vs No HCQ (dosages not available)
Increased need for escalation of respiratory support and no benefits on mortality,
lymphopenia, or neutrophil‐to‐lymphocyte ratio improvement
Tang et al
9
China
Randomized open‐label controlled trial
150
Delayed (day 16)
Mild to moderate
2 groups, HCQ (1200 mg/d for 3 d followed by 800 mg/d; total duration: 2 wks [mild/moderate]
or 3 wks [severe]) vs no HCQ
No differences in the overall 28‐d negative conversion rate, the negative conversion
rate at d 4, 7, 10, 14, or 21, the improvement rate of clinical symptoms within 28 d,
the normalization of C‐reactive protein and the blood lymphocyte count within 28 d
Increased adverse events
Borba et al
10
published
Brazil
Randomized double‐blinded parallel phase IIb trial
Safety‐oriented study
81
Early (d 7)
Moderate
2 groups, high‐dose CQ (600 mg ×2/d, f10 d, or total dose 12 g) vs low‐dose CQ (450
mg for 5 d, ×2/d only on the first day, or total dose 2.7 g)
No differences in clinical outcome
However, high‐dose CQ with potential safety hazards (QTc prolongation), especially
when taken concurrently with AZ and oseltamivir
Chorin et al
11
United States
Retrospective cohort study
Safety‐oriented study
84
Unknown
Moderate to severe
HCQ + AZ (dosages not available)
QTc prolongation >500 ms in 11% patients; no torsade de pointes
ARDS, acute respiratory distress syndrome; AZ, azithromycin; CQ, chloroquine; HCQ,
hydroxychloroquine; ICU, intensive care unit; SARS‐CoV‐2, severe acute respiratory
syndrome coronavirus 2.
Results as presented in the study, considering that the majority has not been peer
reviewed yet.
John Wiley & Sons, Ltd.
This article is being made freely available through PubMed Central as part of the
COVID-19 public health emergency response. It can be used for unrestricted research
re-use and analysis in any form or by any means with acknowledgement of the original
source, for the duration of the public health emergency.
Drug 1: Hydroxychloroquine
Hydroxychloroquine is a cheap and readily available decades‐old drug with immunomodulatory
properties used to treat autoimmune rheumatic diseases. Its multitude of anti‐inflammatory
and immune‐regulatory effects continue to puzzle medical experts worldwide. Pharmacological
pathways mainly include the blockage of Toll‐like receptor–mediated signaling (Toll‐like
receptor‐7 and ‐9, the endosomal innate immune sensor capable of detecting single‐stranded
RNA), the modulation of complement‐dependent antigen/antibody reactions, the activation
of T‐regulatory cells, and the inhibition of proinflammatory cytokine production such
as interleukin‐6, tumor necrosis factor‐α and interferon‐γ.
12
Therefore, hydroxychloroquine immediately appeared attractive to attenuate the inflammatory
response directed against SARS‐CoV‐2 that results in the cytokine storm, which is
held responsible for severe COVID‐19 presentations.
The processes of SARS‐CoV‐2 replication with the resulting pulmonary epithelial and
endothelial cell injury and angiotensin‐converting enzyme‐2 (ACE2) downregulation
and shedding rapidly result in exuberant inflammatory responses, evidenced by increasing
plasma concentrations of various cytokines including interleukin‐6. Hydroxychloroquine‐mediated
inhibition of interleukin‐6 production has been well established in vitro on peripheral
blood mononuclear cells stimulated by phytohemagglutinin or lipopolysaccharide.
13
Its benefit in counteracting the inflammatory rheumatologic disease activity is well
correlated in vivo with the resulting lowering effects on most cytokines and proinflammatory
markers.
14
Therefore, since interleukin‐6 plays a key starter role in COVID‐19–related cytokine
storm, hydroxychloroquine rapidly appeared, at least theoretically, as a potential
immunomodulatory anti–COVID‐19 drug, if administered early enough in the disease time
course.
The 4‐aminoquinoline compounds are active in vitro against a range of viruses with
different suggested mechanisms of action. Recently, hydroxychloroquine‐attributed
anti‐SARS‐CoV‐2 activity was established in vitro (50% effective concentration [EC50]
= 0.72 μmol/L at a multiplicity of infection of 0.01 [100 PFU/well] in Vero cells
for 2 hours) and found more potent than chloroquine (EC50 = 5.47 μmol/L in the same
conditions).
15
However, the mechanisms of hydroxychloroquine‐attributed anti–COVID‐19 activity remain
presumptive (Figure 1).
Figure 1
Suggested mechanisms for the antiviral and immunomodulatory activities of hydroxychloroquine
and azithromycin in COVID‐19 highlighting possible synergic effects between the 2
drugs if prescribed in combination (adapted from Savarino et al,
34
with permission). Possible hydroxychloroquine‐attributed effects include (1) interference
with ACE2 glycosylation and reduction of viral binding, (2) endosome and lysosome
alkalization limiting viral uncoating and assembly, (3) alteration of antigen processing
and MHC class II–mediated autoantigen presentation, (4) disruption of RNA interaction
with TLRs and nucleic acid sensors, (5) inhibition of proinflammatory genes transcription,
(6) inhibition of T‐cell activation, and (7) inhibition of cytokine production. Possible
azithromycin‐attributed effects include (1) interference with ACE2 and reduction of
viral binding, (2) endosome and lysosome alkalization limiting viral uncoating and
assembly, and (3) role of lysosomal P‐glycoprotein that enhances intralysosomal concentrations
of azithromycin. ACE2, angiotensin‐converting enzyme 2; AZ, azithromycin; COVID‐19,
coronavirus 2019 disease; HCQ, hydroxychloroquine; IL, interleukin; MHC, major histocompatibility
complex; P‐gp, P‐glycoprotein; RNA, ribonucleic acid; SARS‐CoV‐2, severe acute respiratory
syndrome coronavirus 2; TNF‐α, tumor necrosis factor‐α; TLR, Toll‐like receptor.
In addition to the previously reported immunomodulatory action, hydroxychloroquine
may alter ACE2 glycosylation, blocking SARS‐CoV‐2 interaction with its membrane receptor
and subsequently the virus/host cell membrane fusion.
16
Consistent with the hypothesis of ACE2 interaction, chloroquine was shown to inhibit
quinone reductase‐2, a structural neighbor of UDP‐N‐acetylglucosamine‐2‐epimerases,
involved in sialic acid biosynthesis. Although deserving deeper investigations, this
molecular mechanism is considered to mediate antimalarial drug activity in vitro on
various viruses such as HIV, SARS‐CoV‐1, and orthomyxoviruses.
16
,
17
However, when discussing any potential benefits of interacting with the ACE/ACE2 system,
we should acknowledge that the clinical effects of ACE inhibitors in patients with
COVID‐19 still remain controversial. These drugs may appear attractive to treat cardiovascular
diseases by reducing pulmonary inflammation; however, their use may be accompanied
by enhanced ACE2 expression that facilitates the viral invasion.
18
Interestingly, as a weak base, hydroxychloroquine increases the intracellular pH,
mainly in the acidic organelles such as endosomes/lysosomes (pH 4.5) where it intensively
accumulates. Moreover, hydroxychloroquine alkalinizes the phagolysosomes (pH ∼6.5;
known as the lysozomotropic activity) and may thus inhibit the viral cleavage mediated
by pH‐dependent proteases, disrupt the fusion process and stop the viral replication.
These last effects complete the wide pharmacological target spectra of hydroxychloroquine
in COVID‐19.
Finally, in antigen‐presenting cells, hydroxychloroquine prevents antigen processing
and subsequently T‐cell activation, differentiation, and cytokine production.
19
,
20
At the molecular level, hydroxychloroquine acts by (1) altering the digestion pattern
of the antigenic peptides, (2) retarding the major histocompatibility complex class
II α and β chains from forming a stable compact complex with the antigenic peptide
due to the diminished degradation of the nonpolymorphic invariant chain, and (3) altering
the recycling of α‐β‐peptide complexes from the cell surface.
19
,
20
To date, there is no definitive evidence that all these potential antiviral activities
could be achieved by the usual hydroxychloroquine doses (400‐600 mg daily) that are
considered clinically safe, as initially suggested.
15
The exact clinically effective hydroxychloroquine dose is still undetermined. Given
the weaknesses of Gautret's
1
and Chen's
2
studies along with the negative results of the recently released US veterans study
5
(Table 1), improving effectiveness by increasing hydroxychloroquine doses has been
questioned by different modeling approaches including a physiologically based pharmacokinetic
study
15
and a pharmacokinetic/virology/QT model.
21
Both studies concluded that hydroxychloroquine doses >400 mg twice daily for at least
5 days are needed to ensure efficacy on viral load decline and cardiac safety. Moreover,
the last study highlighted that suboptimal dosing is not efficient on viral load resulting
in wasted time and resources. Several trials like the PATCH study (see clinicaltrials.gov)
are currently investigating higher doses up to 600 mg twice daily, mainly for the
sickest patients. Another major issue also remains the time in the disease course
at which the treatment is initiated since, based on the reported antiviral or immunomodulatory
mechanisms of action, expected benefits may be reached only if hydroxychloroquine
is started early.
Drug 2: Azithromycin
Azithromycin is a macrolide antimicrobial agent with established antiviral properties
in vitro and anti–SARS‐CoV‐2 activity (EC50 of 2.12 μmol/L). Several mechanisms have
been proposed to explain its antiviral effects (see review; Figure 1).
22
Similarly to hydroxychloroquine, azithromycin is highly trapped in the subcellular
acidic organelles such as lysosomes,
23
causing even more severe impairment of acidification. Additionally, azithromycin is
responsible for a global amplification of the host's interferon pathway‐mediated antiviral
responses. Finally, it may alter SARS‐CoV‐2 entry by interfering between its spike
protein and host ACE2 receptor. Very recently, an energetics‐based modeling provided
high binding affinity for azithromycin at the interaction point between SARS‐CoV‐2
spike and ACE2, whereas hydroxychloroquine appeared ineffective to directly inhibit
this interaction.
24
Clinical studies have shown the ability of azithromycin to reduce the viral load with
demonstrated benefits on patient outcome including accelerated recovery (influenza
A infection), reduced respiratory morbidity (respiratory syncytial virus and SARS
infections) and improved mortality (Middle East respiratory syndrome–coronavirus infection).
22
Several trials in addition to those testing the hydroxychloroquine‐azithromycin combination
are currently ongoing to investigate the anti–COVID‐19 benefits of azithromycin alone
or in combination with other drugs, focusing as end points not only on viral load
but also on clinical outcomes.
Interestingly, azithromycin is a known substrate of P‐glycoprotein (ABCB1), a member
of the adenosine triphosphate–binding cassette transporters superfamily, highly expressed
and oriented from the cytosolic to the internal side of the lysosome membrane.
25
Therefore, we hypothesized that azithromycin accumulates more intensively under the
ABCB1 trapping effect inside the lysosomes and like hydroxychloroquine, reduces lysosomal
acidity and exhibits its antiviral properties. Consistently with our hypotheses, synergistic
in vivo and in vitro properties have been attributed to the azithromycin/chloroquine
combination, used to protect against malaria and treat sexually transmitted infections.
26
,
27
Similarly, the antiviral synergy of the azithromycin/hydroxychloroquine combination
was observed in vitro at concentrations achieved in vivo and detected in pulmonary
tissues
28
and was shown to accelerate viral clearance in humans in comparison to hydroxychloroquine
alone.
1
Thus, azithromycin ABCB1‐dependent lysosomal sequestration plus hydroxychloroquine
is very likely an optimal combination to hamper the low‐pH–dependent steps of viral
replication and limit COVID‐19 progression. Future clinical studies investigating
the azithromycin/chloroquine combination have to focus on clinical outcome improvement
and not only viral load reduction, although this target may also be interesting to
limit interhuman contagiousness.
Risks‐Benefits of Combining the 2 Drugs
Fears may emerge from increased risks of QT interval prolongation (resulting from
the human Ether‐à‐go‐go‐Related Gene potassium channel blockage), torsade de pointes,
and cardiovascular death.
29
Despite potential risks acknowledged to be limited if either drug is prescribed singly,
30
,
31
drug‐drug interaction resulting from their coadministration as well as patients’ advanced
age, preexisting comorbidities, and COVID‐19–related myocardial and kidney injuries
represent challenging conditions. Interestingly, a recent multinational, network cohort,
and self‐controlled case series study supported the safety of short‐term hydroxychloroquine
treatment but highlighted the risks of heart failure and cardiovascular mortality
when combining hydroxychloroquine with azithromycin, potentially due to synergistic
effects on QT length.
32
Data from recent COVID‐19 trials clearly reported increased adverse events with hydroxychloroquine,
especially at high doses and in combination with azithromycin.
9
,
10
,
11
Additionally, extending prescriptions to mildly infected patients is also at risk
of misuse and overdose. Clinical toxicologists remember the 1982 suicide outbreak
in France following the publication of Suicide: A How‐To Guide, which promoted chloroquine
ingestion to complete suicide,
33
resulting in a major crisis of fatalities attributed to chloroquine poisonings.
In patients with COVID‐19 treated with the azithromycin/hydroxychloroquine combination,
physicians should be cautious when coprescribing QT interval–prolonging drugs (enhanced
toxicity) or P‐glycoprotein substrates/inhibitors (reduced effectiveness). Nevertheless,
abandoning azithromycin may importantly limit hydroxychloroquine‐attributed effectiveness.
Therefore, well‐designed randomized, double‐blind and placebo‐controlled clinical
trials are awaited to evaluate the exact synergy/toxicity balance of this potentially
lifesaving combination. Appropriate statistical hypotheses should be formulated when
designing the study so that the alternative hypothesis can be inferred upon the rejection
of the null hypothesis. Observational studies are not sufficient in nature to meet
regulatory requirements and decide if a treatment with potentially serious toxicity
should be advocated. Pharmacovigilance departments and poison control centers should
be alert to collect all useful toxicological exposure data and trends to guide public
health response.
In conclusion, both hydroxychloroquine and azithromycin are friends and foes, when
considering the balance issue between the expected synergistic effectiveness to clear
the virus from the body and the safety concerns to avoid possible risks of cardiotoxicity
when the 2 drugs are combined.
Conflicts of Interest
The authors declare no conflicts of interest.