Current evidence on chloroquine and hydroxychloroquine and their role in the treatment and prevention of COVID-19

Abstract

Although chloroquine and hydroxychloroquine have not yet been shown to be safe or effective for the treatment or prevention of COVID-19, regulatory agencies in some countries have authorised their use in Coronavirus disease 2019 (COVID-19) due to the lack of available interventions. Several large clinical trials are currently underway to investigate these agents as potential therapeutic options for COVID-19. Previous research against similar pathogens that cause severe acute respiratory syndrome and Middle East respiratory syndrome has identified chloroquine and hydroxychloroquine as possible antiviral candidates against SARS-CoV-2. Despite promising pre-clinical evidence, data have thus far failed to confirm their efficacy, and recent studies suggest potential dose-related cardiotoxicity and mortality. Close monitoring for cardiac conduction abnormalities is advised with higher-than-approved doses. Additional, robust evidence from randomised controlled trials and meta-analyses are required to make informed risk-benefit assessments. Finally, the off-label prescription of these agents should be judiciously considered, and any such use should be conducted within clinical trials, or under the Monitored Emergency Use of Unregistered and Investigational Interventions framework.

Full text

Article title: Current evidence on chloroquine and hydroxychloroquine and their role in the treatment and prevention
of COVID-19
Authors: Rephaim Mpofu[1], Clifford Banda[2], Hannah Gunter[3], Enkosi Mondleki[4], Gayle Tatz[5], Phumla Sinxadi[6],
Karen Cohen[7], Karen Barnes[8], Marc Blockman[9]
Affiliations: University of Cape Town, Department of Medicine, Division of Clinical Pharmacology[1]
Orcid ids: 0000-0002-4732-5879[1], 0000-0002-0757-5259[2], 0000-0002-1892-4207[7], 0000-0002-5547-820X[8]
Contact e-mail: rephaim.mpofu@uct.ac.za
License information: This work has been published open access under Creative Commons Attribution License
http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited. Conditions, terms of use and publishing policy can be found at
https://www.scienceopen.com/.
Preprint statement: This article is a preprint and has not been peer-reviewed, under consideration and submitted to
AfricArXiv Preprints for open peer review.
Funder: None
DOI: 10.14293/111.000/000008.v2
Preprint first posted online: 15 July 2020
Keywords: COVID-19, chloroquine, hydroxychloroquine, SARS-CoV-2, MEURI, coronavirus

1
Abstract:
Although chloroquine and hydroxychloroquine have not yet been shown to be safe or effective
for the treatment or prevention of COVID-19, regulatory agencies in some countries have
authorised their use in Coronavirus disease 2019 (COVID-19) due to the lack of available
interventions. Several large clinical trials are currently underway to investigate these agents as
potential therapeutic options for COVID-19. Previous research against similar pathogens that
cause severe acute respiratory syndrome and Middle East respiratory syndrome has identified
chloroquine and hydroxychloroquine as possible antiviral candidates against SARS-CoV-2.
Despite promising pre-clinical evidence, data have thus far failed to confirm their efficacy, and
recent studies suggest potential dose-related cardiotoxicity and mortality. Close monitoring for
cardiac conduction abnormalities is advised with higher-than-approved doses. Additional,
robust evidence from randomised controlled trials and meta-analyses are required to make
informed risk-benefit assessments. Finally, the off-label prescription of these agents should be
judiciously considered, and any such use should be conducted within clinical trials, or under
the Monitored Emergency Use of Unregistered and Investigational Interventions framework.

2
Introduction
Several therapies have been described as a potential treatment of coronavirus disease 2019
(COVID-19). Among them is chloroquine, an antimalarial, anti-inflammatory and immuno-
modulatory drug, and its derivative, hydroxychloroquine. However, numerous questions have
been raised pertaining to the rationale, dosing regimen, and safety profile of these repurposed
drugs for COVID-19 treatment and prophylaxis. The World Health Organisation (WHO) and
other academic partners have launched large, multi-country, prospective clinical trials which
have included chloroquine or hydroxychloroquine to address some of these questions. The
hydroxychloroquine arm of the Solidarity study was recently stopped after a review of their
data and the recently announced results from the RECOVERY trial in the United Kingdom
showed that hydroxychloroquine was ineffective in the reduction of mortality of hospitalised
COVID-19 patients when compared with standard care1. The WHO statement noted that the
decision to stop hydroxychloroquine’s use in the Solidarity trial does not apply to the
evaluation of its role (or that of chloroquine) in pre- or post-exposure prophylaxis, or the
outpatient treatment of less severe COVID-19.
In this review, we discuss the pharmacokinetics, pharmacodynamics, safety profile, and
evidence for hydroxychloroquine and chloroquine use in the prevention and treatment of
COVID-19. We will also discuss the role of off-label prescription of these agents within an
ethical framework.
History of chloroquine and hydroxychloroquine
Quinine, the prototypical 4-aminoquinoline, was discovered in 1600 from the bark of the
cinchona tree as a cure for malaria2. Occupation of the Java plantations in World War II by the
Japanese army led to research to synthesise it in the United States. Chloroquine was first
synthesised in 1934 by Andersag in an effort to improve the safety and efficacy of quinine3,
and this became the drug of choice for the treatment of malaria until the late 1990s when
widespread resistance in Plasmodium falciparum limited its use. Hydroxychloroquine was
synthesised in 1946 and proposed as a safer alternative to chloroquine in 19554.
Hydroxychloroquine is an analogue of chloroquine in which one of the N-ethyl groups of
chloroquine is hydroxylated5. Hydroxychloroquine is now preferred for treating various
autoimmune conditions including rheumatoid arthritis, discoid lupus, and systemic lupus
erythematosus (SLE), in countries where it is available.

3
Pharmacokinetics of chloroquine
Absorption after oral administration is rapid and generally reliable (even in unconscious
patients)6. The total apparent volume of distribution is >100 L/kg, reflecting extensive tissue
binding7. Thus, the initial plasma concentrations are determined mainly by distribution
processes and not by elimination. This is important to understand dose-related adverse effects
such as cardiotoxicity. Toxicity can occur if absorption outpaces distribution, leading to
transiently toxic plasma concentrations. Due to its large volume of distribution, loading doses
are required to treat conditions like malaria, or potentially COVID-19, to ensure that
therapeutic concentrations, that would take weeks to achieve without a loading dose, are
achieved as rapidly and safely as possible.
Approximately 30-50% of an administered dose of chloroquine is transformed in the liver via
cytochrome P450 enzymes8. Upon absorption, chloroquine is rapidly dealkylated into the
pharmacologically active metabolites, desethylchloroquine, bisdesethylchloroquine and 7-
chloro-4-aminoquinoline9. Following single oral doses of chloroquine in healthy volunteers,
desethylchloroquine and bisdesethylchloride are rapidly detected, reaching plasma
concentrations of 40% and 10% of chloroquine concentrations respectively, which adds to the
pharmacological effect. Chloroquine has been shown to be a substrate of the CYP2C8,
CYP3A4 and CYP3A5 enzymes, as well as a potent inhibitor of CYP2D6 and CYP2D110, 11.
Furthermore, chloroquine and hydroxychloroquine are both inhibitors of the P-glycoprotein (P-
gp) transport system12. P-gp is an efflux transporter found most notably in the endothelial cells
of the gut lumen and blood-brain barrier. Metabolism of concomitantly administered drugs that
are substrates of CYP2D6 and/or P-gp may potentially be inhibited.
Mechanism of action of chloroquine
Chloroquine and its congener, hydroxychloroquine, are approved for first-line use as anti-
malarial and amoebicidal agents13, and are also used as anti-inflammatory adjuvants for
rheumatoid arthritis, discoid lupus and SLE14. These agents have been described to possess a
broad range of antiviral activities – Chloroquine’s mild antiviral action was initially
demonstrated against avian reticuloendotheliosis virus, as well as human immunodeficiency
virus-1. This is thought to be mediated through inhibition of pH-dependent endocytosis and
interference of viral glycoprotein glycosylation pathways15. In 2003, chloroquine and
hydroxychloroquine were investigated as possible treatments for the severe acute respiratory

4
syndrome (SARS) as SARS-CoV, a coronavirus which also involves pH-dependent
endocytosis, was found to be the aetiological agent11.
SARS-CoV-2 and other coronaviruses are a diverse group of enveloped, positive-sense strand
RNA viruses that can cause a wide spectrum of disease in their host species including
respiratory, enteral and neurological disease. Coronavirus particles contain four main structural
proteins: spike, membrane, envelope, and nucleocapsid proteins. SARS-CoV and SARS-CoV-
2 attachment to the host cell is initiated by binding of the spike protein to the host cell receptor,
angiotensin-converting enzyme-2 (ACE2)16. Following attachment, the virus fuses with the
host cell membrane through acid-dependent cleavage of the spike protein. This cleavage-
facilitated fusion allows for the release of viral contents into the host cell cytoplasm16, 17. This
process usually occurs within acidified endosomes without which viral internalization cannot
occur. The acidification of endosomal content to an average pH of 6.0-6.5 is primarily
generated by a vacuolar-type ATPase transporter, which uses ATP hydrolysis to drive protons
against their electrochemical gradient18. Chloroquine diffuses readily in its unprotonated form
across plasma cell membranes and cellular organelles like endosomes, lysosomes, or Golgi
vesicles. Upon entry into the acidic environment, the molecule becomes more protonated and
less lipophilic, which results in decreased diffusion and drug trapping19. This accumulation
increases the endosomal pH and prevents fusion and viral entry into the cell20. Chloroquine is
also thought to interfere with the glycosylation of cellular receptors, including ACE2
receptors10, 20. These 4-aminoquinolines may also have immunomodulatory properties that
affect the regulation of pro-inflammatory cytokines10 such as the inhibition of tumour necrosis
factor alpha and interleukin-6 production, which has been suggested as a mechanism for its
potential efficacy against the COVID-19 related acute respiratory distress syndrome11.
Safety profile of chloroquine
Chloroquine is generally well tolerated, with an established safety profile at the doses
recommended for its approved uses; for instance, SLE can be treated safely with 500 mg
chloroquine sulphate for 10 days, followed by a daily maintenance dose of 250 mg12, 14. Total
doses of 25 mg/kg over 3 days have also safely been used for the treatment of uncomplicated
malaria, and higher total doses are reportedly tolerated in children with uncomplicated
malaria21. Irreversible retinopathy has been described with higher doses and long-term use. The
risk of ophthalmological complications is less than 1% after 5 years of use at recommended

5
doses, with the risk doubling after 10 years of use22. Hydroxychloroquine is associated with
less ocular toxicity and is therefore preferred over chloroquine if available23. Haemolytic
anaemia in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency has been
reported in patients with concomitant use of primaquine and chloroquine, an effect which has
been mainly attributed to primaquine24. Monitoring for haemolysis in patients with known
G6PD deficiency and chloroquine use is therefore recommended. The spectrum of toxicity with
accidental or intentional overdosage is dependent on the dose, duration of treatment,
concomitant drugs as well as underlying renal function. The main concern with high dosages
is cardiovascular toxicity, which can present with conduction abnormalities, cardiomyopathy,
and hypotension with sudden collapse13. Conduction abnormalities include a prolonged QT
interval, impaired ventricular conduction that can result in widened QRS complexes, and
increased automaticity that can induce arrhythmias. Chloroquine causes dose-dependent
prolongation of the corrected QT (QTc) interval by potassium-channel blockade and
prolongation of the cardiac repolarization time. This can lead to fatal complications such as
sudden cardiac death25-28.
QTc interval prolongation can be prevented by avoiding use in those with genetic
predispositions (congenital long QT syndrome or family history of sudden death) or electrolyte
abnormalities (hypokalaemia, hypocalcaemia, and/or hypomagnesaemia)29. Additionally,
concomitant use with other medicines with QTc interval prolonging properties, including
macrolide and fluoroquinolone antibiotics, oseltamivir and certain antipsychotics, should be
avoided. Baseline and routine follow-up electrocardiograms with use of higher doses of
chloroquine are recommended30. Although generally safe and well-tolerated with appropriate
administration, chloroquine is potentially lethal in overdose, and incidents of self-medication
and consequent accidental poisoning with chloroquine in response to COVID-19 have recently
been reported in some countries including the United States of America and Nigeria31, 32.
Additionally, recent literature has suggested that chloroquine may be associated with
significant adverse effects when used in high dosages in the management of hospitalised
COVID-19 patients (Takla, preprint, 2020)33, as the dosages required to reach therapeutic
concentrations may increase the risk of adverse outcomes in this patient population30. Until
results on the safety of chloroquine for the treatment of COVID-19 from high-quality
randomised clinical trials (RCT) have been appraised, no specific recommendations on the

6
safety of chloroquine in the management of patients with SARS COV-2 can be made, but it is
advised that clinicians should avoid its use in patients with risk factors for QT prolongation.
Pre-clinical evidence supporting the use of 4-aminoquinolines
including chloroquine & hydroxychloroquine in coronaviruses
The first data demonstrating chloroquine’s antiviral activity against coronaviruses published in
2001 showed that this drug was able to inhibit cellular infection with HCoV-229E, a
coronavirus, in a time-dependent manner (Table 1)34. Other studies have also shown that
chloroquine effectively inhibited viral replication of SARS-CoV and MERS-CoV in-vitro35, 36.
In 2014, a mouse model study reported that chloroquine administered at doses of 15, 5, and 1
mg/kg improved the survival of new-born mouse pups infected with HCoV-OC43 infection
(100%, 92.9%, 33.3% survival respectively)37. This study provided some evidence that higher
doses may be required for successful treatment of coronaviruses. By contrast, a study
conducted by Barnard and colleagues was unable to demonstrate chloroquine as an effective
antiviral agent at doses up to 50 mg/kg in-vivo but simultaneously conducted in-vitro
experiments showed that chloroquine was an effective inhibitor of SARS-CoV replication. The
authors concluded that this discrepancy suggested that chloroquine may have insufficient
activity on its own as a therapeutic antiviral agent38.
Early in the COVID-19 pandemic, an in-vitro study demonstrated that chloroquine was also
effective against SARS CoV-2 at a low-micromolar concentration39. Hydroxychloroquine is
also thought to have similar action to chloroquine, but evidence regarding their relative potency
is conflicting40, 41. These data provided the rationale for Gautret et. al. to conduct a non-
randomised trial of hydroxychloroquine in the treatment of COVID-1942.
Clinical evidence for chloroquine and hydroxychloroquine use in
COVID-19
In an observational study, Gao J et al. reported that chloroquine was effective in the treatment
of COVID-19 in 100 Chinese patients compared to a control group, with a dosing regimen of
500 mg twice daily for 10 days43. The reasoning behind this dosing regimen is not well
documented44. By contrast, another observational study using data from a large medical centre
in New York City in the United States of America failed to show an association between

7
hydroxychloroquine administration and a decreased probability of intubation or death (adjusted
hazard ratio = 1.04, 95% Confidence interval [95% CI]: 0.82-1.32)45. It is important to note
that, although the analysis was adjusted for differences in baseline characteristics,
hydroxychloroquine-treated patients were more severely ill at baseline compared to the
standard care group45.
The earliest clinical trial data suggesting a possible benefit from hydroxychloroquine in
COVID-19 came from a non-randomised study conducted in France that was published in
March 2020, which found that 70% in the intervention arm had virological cure compared to
12.5% in the control group by day 6 (p<0.001) (Table 2)42. Despite significant limitations, this
study was a key catalyst which prompted further research into the efficacy and subsequent
emergency use authorisation (EUA) of these agents by the U.S. Food and Drug Administration
(FDA). Two small RCTs by Chen Z and Chen J subsequently reported conflicting results, with
Chen Z reporting improved clinical resolution with hydroxychloroquine use, while Chen J
showed similar swab conversion rates on day 7 between hydroxychloroquine and placebo
groups46, 47.
The first well-designed RCT that assessed hydroxychloroquine in COVID-19 found similar
probabilities of viral swab conversion by day 28 between the hydroxychloroquine (85.4%, 95%
CI: 73.8-93.8%) and standard of care arms (81.3%, 95% CI: 71.2-89.6%) in patients admitted
to hospital with mild to moderate COVID-1948. Unfortunately, the analysis was significantly
underpowered as enrolment was terminated early due to recruitment difficulties. Preliminary
results from the RECOVERY trial, a multi-intervention RCT, have so far only been reported
in a statement on 5 June 2020 which showed that 28-day mortality was similar between the
hydroxychloroquine (25.7%) and standard of care treatment arms (23.5%, hazard ratio [HR] =
1.11 [95% CI, 0.98-1.26]; p=0.10)49. As a result, enrolment to the hydroxychloroquine arm has
been stopped for futility. The authors state that “these data convincingly rule out any
meaningful mortality benefit of hydroxychloroquine in patients hospitalised with COVID-
19”49. These results are preliminary and will require a review of the full data once published,
but they provide significant evidence that hydroxychloroquine might not be a viable candidate
for treatment in hospitalised COVID-19 patients.

8
The role of chloroquine as post-exposure prophylaxis in COVID-19 is also being investigated
and results from one “pragmatic” RCT in which 821 participants were recruited through social
media and almost all data were self-reported by participants, showed that hydroxychloroquine
was not effective in preventing illness compatible with COVID-19 disease or confirmed SARS-
CoV-2 infection (only 3% were confirmed) in participants that had reported recent exposure50.
These data may indicate that post-exposure hydroxychloroquine use for prophylaxis might not
significantly alter the course of infection, although the long delay between perceived SARS-
CoV-2 exposure and the initiation of hydroxychloroquine (≥3 days in most participants)
suggests that the prevention of symptoms or progression of COVID-19 was being assessed,
rather than prevention of SARS-CoV-2 infection51. Other pre- and post-exposure prophylaxis
studies are currently ongoing52.
One of the critical issues in assessing the role of chloroquine or hydroxychloroquine in
COVID-19 is the dose that can be used to reach therapeutic concentrations without
compromising patient safety. Although numerous countries have incorporated the off-label use
of these drugs in their treatment policies, data guiding the rational dosing of these drugs in
COVID-19 are lacking. A systematic review found that at least 4 different treatment regimens
have been used thus far in studies assessing the use of chloroquine in COVID-19, with total
dosages ranging from 3 000 mg to 20 000 mg over the course of treatment (Takla, preprint,
2020)33. Pre-clinical evidence suggests that relatively high dosages may be required in order to
reach therapeutic tissue concentrations quickly53. This may be problematic, however, as a
recent publication reported higher rates of mortality when a high dosage regimen was compared
with a low dosage regimen30. ChloroCovid-19 was a double-blind RCT that aimed to assess
two treatment regimens: (a) 600 mg chloroquine base twice daily for 10 days (total dosage =
12 000 mg) or (b) 450 mg chloroquine base twice on day 0, followed by 450 mg once daily for
4 days (total dosage = 2 700 mg). The trial was terminated early after an unplanned safety
analysis showed a significant increase in mortality in the high dosage group (39.0% in the high
dosage group vs. 15.0% in the low-dosage group) with an odds ratio of 3.6 (95% CI: 1.2-10.6).
Additionally, a higher proportion of participants developed QT prolongation in the high dosage
group compared to the low dosage group; two of 41 patients given the higher dose regimen
developed ventricular tachycardia before death. Importantly, some patients were treated with
other QT-prolonging drugs in this trial such as azithromycin and oseltamivir30.

9
These data highlight a few key points: (1) Chloroquine has largely failed to show a clinical
benefit in hospitalized patients with COVID-19, (2) The dose potentially required for
therapeutic efficacy may be associated with unacceptable toxicity and increased mortality, and
(3) potential drug interactions require careful consideration in the cautious prescription of these
agents. A recently published, and subsequently retracted paper had reported an association
between hydroxychloroquine administration and increased mortality, which prompted WHO
to temporarily suspend the hydroxychloroquine arm from the Solidarity trial pending further
assessments54. The suspension was lifted after the available mortality data was reviewed, but
this arm was terminated on 17 June 2020 after a further review of available data showed that
hydroxychloroquine was not effective compared to the standard of care53, 55. Similarly, the
Medicines and Healthcare products Regulatory Agency (MHRA) has instructed UK clinical
trialists using hydroxychloroquine to treat or prevent COVID-19, to suspend recruitment of
further participants56. These latest developments, combined with the recently reported
mortality benefit with the use of dexamethasone57, will most likely see a reduction in
hydroxychloroquine use for the treatment of COVID-19 in hospitalised patients going forward.
Evidence to date indicates that hydroxychloroquine (and by extension chloroquine) may not be
effective in hospitalised COVID-19 patients, and possibly also not for post-exposure
prophylaxis. However, their role in the outpatient treatment of COVID-19 and pre-exposure
prophylaxis remain unresolved. Peer-reviewed results from ongoing RCTs are needed to
address these critical knowledge gaps.
Off-label, compassionate and emergency use of medicines in
pandemics – The role of chloroquine or hydroxychloroquine in COVID-
19
The COVID-19 pandemic has prompted important discussions regarding the off-label, and
compassionate, use of medicines. Numerous considerations are involved in the use of
therapeutic agents for indications that have not been investigated adequately, and this needs to
be conducted ethically and rationally. Off-label medication use is defined by the FDA as the
use of an approved medicine for an unapproved indication to treat a medical condition which
does not have any proven treatments, or where approved treatments have been unsuccessful58.
Compassionate use of medicines, on the other hand, also known as expanded access, involves

10
the use of an unapproved, investigational/experimental drug59. Compassionate use can be
applied when a patient has a serious condition whose life is immediately threatened by their
condition, and no comparable or satisfactory alternative intervention is available. Importantly,
the potential benefit should justify the potential risks of treatment59.
The EUA regulation allows the FDA to facilitate the availability of an unapproved medical
product or device in a health-related emergency to diagnose, treat, or prevent serious or life-
threatening conditions60. Since its establishment in 2004, the FDA has issued a limited number
of EUAs in response to public health emergencies such as Zika disease, Ebola, Middle East
Respiratory Syndrome (MERS), and the Avian flu. In response to COVID-19, the FDA has
issued EUAs to allow the use of hydroxychloroquine and remdesivir for the treatment of
hospitalized patients61. On 15 June 2020 however, the FDA revoked the EUA for
hydroxychloroquine, citing a lack of efficacy and ongoing safety concerns62. In South Africa,
the health products regulatory authority (SAHPRA) approved an application by a
pharmaceutical company in March 2020 to donate 500 000 chloroquine tablets for use in
COVID-1963. However, the regulatory authority also cautioned practitioners against the
irrational and overzealous prescription of chloroquine due to the lack of evidence in COVID-
19, and because the depletion of available drug stock may limit its access for other, approved
indications63. Numerous regulatory bodies, including SAHPRA, have advocated that
chloroquine should be reserved for use within the context of clinical trials; at the minimum, it
should be administered within a well-monitored setting, such as that defined by the Monitored
Emergency Use of Unregistered and Investigational Interventions (MEURI) framework64, 65.
The MEURI framework specifies that it may be ethical to treat patients with an experimental
intervention outside a clinical trial context in certain circumstances66. This framework requires
that: (i) treatment with a potential intervention is approved by local authorities and
appropriately qualified ethics committees, (ii) adequate resources are available to ensure that
risks are minimized, (iii) informed consent is obtained from the patient or an appropriate proxy,
(iv) the emergency use of the intervention is monitored, and (v) data are collected and shared
in a timely manner with the medical and scientific community66. The MEURI framework,
which has similar principles to the concept of compassionate use, was developed by the WHO
during the Ebola pandemic as a means to provide some form of treatment for an illness for
which no proven treatment existed, while simultaneously providing a method for data
collection to contribute to the body of research. Such regulatory policies are important as they

11
enable the healthcare sector to utilize the best available interventions in an emergency within
an approved, legal and ethical setting, which can improve access to potentially, life-saving
interventions. Withholding interventions when there is plausible evidence of therapeutic
benefit could be argued as unethical, and this may justify the use of an unapproved therapeutic
agent. However, this should not override the need for robust evidence generated from RCT or
other appropriate study designs such as adaptive trial designs. South Africa is currently
involved in various clinical trials, including the Solidarity trial, which aims to compare the
safety and effectiveness of various agents against COVID-19 including remdesivir;
lopinavir/ritonavir with, or without, interferon beta 1a; and previously, chloroquine or
hydroxychloroquine67.
The continued use of chloroquine and hydroxychloroquine within a MEURI framework
requires periodical review as more data on its efficacy and safety become available. Available
evidence concerning chloroquine or hydroxychloroquine use has largely failed to demonstrate
a significant benefit in the treatment of COVID-19, and safety concerns have been raised.
Additionally, the widespread use of these agents has also resulted in varying, often
unintentional consequences, including supply shortages for other conditions that they are
approved for, such as rheumatoid arthritis and SLE68. Bearing these facts in mind, it is
important to exercise caution when considering the use of these agents in the management of
COVID-19 outside of clinical trials until further, conclusive, evidence becomes available.
Conclusion
The COVID-19 pandemic has required a united effort to identify therapeutic agents that may
be useful for treatment or prophylaxis. Previous research on SARS and MERS have identified
4-aminoquinolines such as chloroquine and hydroxychloroquine as agents with potential, but
weak, antiviral activity against SARS-CoV-2. Despite promising pre-clinical evidence
suggesting potential efficacy against COVID-19, clinical data have thus far failed to show that
these agents are effective in hospitalised patients. Additionally, potentially life-threatening
cardiotoxicity has been associated with higher doses, and the small, potential benefit (if any)
from these agents currently do not appear to justify these risks associated with these drugs.
Various gaps in knowledge that still need to be addressed include their role in pre- and post-
exposure prophylaxis, outpatient treatment of COVID-19, as well as the appropriate dosage
and timing whenever these agents are used. Robust evidence from RCTs and meta-analyses are

12
required to make informed assessments for the early treatment or prophylaxis of COVID-19
that consider the potential benefit and harm. Finally, the off-label prescription of these agents
should be judiciously considered and should be limited to a clinical trial environment, or in
exceptional cases, used within the MEURI framework.
Conflicts of interest
The authors have no conflicts of interest to disclose.
Acknowledgements
None to declare.
Funding
No funding sponsorship grants were used for the development of this manuscript.
Abbreviations
95% CI – 95% confidence interval
ACE2 – Angiotensin-converting enzyme 2
CC50 – 50% Cytotoxicity concentration
CCID50 – 50% cell culture infective dose
COVID-19 – Coronavirus disease 2019
EC50 – 50% Effective concentration
EUA – Emergency use authorisation
FDA – U.S. Food and drug administration
G6PD - glucose-6-phosphate dehydrogenase
HIV-1 – Human immunodeficiency virus-1
Hpi – Hours post-infection
HR – Hazard ratio
IC50 –50% inhibitory concentration
MERS – Middle East respiratory syndrome
MERS-CoV – Middle East respiratory syndrome coronavirus
MEURI - Monitored Emergency Use of Unregistered and Investigational Interventions
framework
MHRA - Medicines and Healthcare products Regulatory Agency

13
P-gp – P-glycoprotein
QTc - Corrected QT interval
RCT – Randomised controlled trial
SAHPRA – South African health products regulatory authority
SARS – Severe acute respiratory syndrome
SI – Selectivity index
SLE – Systemic lupus erythematosus
WHO – World Health Organization

14
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Table 1: In-vitro studies for the use of chloroquine in SARS-CoV/SARS-CoV-2
Reference
Study design
Treatment
Main findings
Blau et al.
[34]
• Cell culture model with
MRC-5 lung epithelial
cells
• HCoV-229E challenge
• CQ 50 µM
• Bafil omycin A
• 28 ± 1.4% 229E antigen expression (-1 to 1 hour post infection [hpi]) – CQ
• 98 ± 0.8% 229E antigen expression (1 to 3 hpi) – CQ
• 80 ± 0.9% 229E antigen expression (8 to 12 hpi) – CQ
Keyaerts,
2004
[35]
• Cell culture model with
Vero E6 cells
• SARS-CoV challenge
• CQ 0.1, 1, and 10 µM
• IC
50
= 8.8±1.2 µM; CC
50
= 261.3±14.5 µM; SI = 30
• Dose-dependent increase in cell viability at 0.1, 1, and 10 µM
• Decreased antiviral effect with prolonged time to addition of CQ
De Wilde
[36]
• Cell culture model
• SARS-CoV, MERS-CoV
challenge and HCoV-
229E-GFP
• Numerous compounds
including CQ from 0-50 µM
• SARS-CoV: EC
50
= 3.0±1.1 µM; CC
50
= 58.1±1.1 µM; SI = 19.4
• MERS-CoV: EC50 = 4.1±1.0 µM; CC50 > 128 µM; SI > 31
• HCoV-229E-GFP: EC50 = 3.3±1.2 µM; CC50 >50 µM; SI >15
Keyaerts,
2009
[37]
• HCoV-OC43 challenge
• HRT-18 cells
• Female pregnant
C57BL/6 mice treated
with CQ for 2 days
before labour
• In-vitro: CQ at various
concentrations
• Murine: CQ 1, 5, 15 mg/kg
subcutaneously, or no CQ
treatment groups
• In vitro: EC
50
= 0.306±0.091 µM; CC
50
= 419.0±192.5 µM; SI: 1,369
• Murine: 100%, 88%, 83% & 0% new-born pup survival when given 15, 5, 1
mg/kg CQ and no CQ respectively, with a significant difference in survival
between the 15mg/kg compared to the 5 mg/kg (p=0.0237) and 1 mg/kg
treatment groups (p<0.0001)
Barnard et
al.
[38]
• Cell culture model with
Vero 76 cells
• SARS-CoV challenge
(four strains)
• Cytopathic effect
inhibition assay
• Mouse i nfection study
with BALB/c mice
• In-vitro: Numerous
antivirals, including CQ, CQ
monophosphate and CQ
diphosphate at four or eight
half log10 dilutions
• In-vivo: Intraperitoneal (i.p.)
or intranasal (i.n.) 1, 10 and
50 mg/kg CQ administration,
beginning 4h pre-virus
exposure
• CQ: IC
50
= 1-5 µM; CC
50
= 10-20 µM; SI = 2-20
• CQ monophosphate: IC50 = 4-6 µM; CC50 = 20-30 µM; µM; SI = 3-8
• CQ diphosphate: IC50 = 3-8 µM; CC50 = 10-30 µM; SI = 2-10
• 50 mg/ kg i. n. reduced viral lung titres from 5.4 ± 0.5 to 4.4 ± 1.2 in log10
CCID50/g at Day 3 – not statistically significant. 1, 10 mg/kg and i.p.
administration did not reduce viral titres
Wang et al.
[39]
• Cell-culture model with
Vero E6 cells
• SARS-CoV-2 challenge
• Numerous antivir als,
including CQ at various
concentrations
• EC
50
= 1.13 µM; EC
90
= 6.9 µM; CC
50
> 100 µM ; SI > 88.50
Liu et al.
[41]
• Cell-culture model with
Vero E6 cells
• Dose response analysis at
multiplicities of infection
of 0.01, 0.02, 0.2, and
0.8
• CQ at various concentrations
• HCQ at various
concentrations
• CC
50
(CQ) = 273.20 μM; CC
50
(HCQ) = 249.50 μM;
• EC50 (CQ) = 2.71, 3.81, 7.14, and 7.36 μM at all respective MOIs
• EC50 (HCQ) = 4.51, 4.06, 17.31, and 12.96 μM at all respective MOIs
• The differences in EC50 values between CQ and HCQ were statistically
significant at an MOI of 0.01 (P < 0.05) and 0. 2 (P < 0.001)

20
Table 2: Studies exploring the use of chloroquine and hydroxychloroquine in prophylaxis and treatment of COVID-19
Reference
Study design
Population
Intervention
Comparator
Main findings
Borba et al.
[30]
Randomized, double-
blind
Hospitalized adults in Brazil
with SARS-CoV-2 inf ection.
N= 81 (41 in high dosage
group, 40 in low dosage
group).
CQ base 600mg
twice daily for 10
days. Concomitant
administration of
azithromycin and
oseltamivir was
allowed.
HCQ base 450 mg twice on day 0,
followed by 450 mg once daily for 4 days
(total dose = 2700 mg). Concomitant
administration of azithromycin and
oseltamivir was allowed.
Mortality was 39.0% (16/41 patients) in
the high dosage group compared t o
15.0% (6/40 patients) in the low dosage
group (odds ratio = 3.6; 95% CI: 1.2-
10.6). The high dose CQ arm had more
patients with QTc>500ms (7/37, 18.9%)
than the low dose arm (4/36, 11.1%).
89.6% received oseltamivir, 100%
received azithromycin
Gautret et al.
[42]
Non-randomized,
open-label
Hospitalized adults in France
with confirmed COVID-19.
N=42 (26 HCQ, 16 control)
HCQ sul phate 200
mg three times daily
for 10 days.
Azithromycin was
added depending on
clinical presentation.
Standard of care without HCQ.
Azithromycin was added depending on
clinical presentation
Viral titres were significantly reduced in
the HCQ arm on day 6 - 70% compared
to 12.5% PCR negative, p=0.001). Of
those that received HCQ, 100% that
azithromyci n achieved viral clearance
compared to 57.1% that did not receive
azithromycin (p<0.001)
Geleris et al.
[45]
Observational,
prospective
Hospitalized adults in the
USA with confirmed
COVID-19. N=1376 (811
HCQ, 565 control).
HCQ 600 mg twice
daily on day 1, then
400 mg daily for 4
days. Azithromycin
and sarilumab were
added depending on
the clinician
discretion and
medical condition.
All other treatment except HCQ.
346 patients (25.1%) had a composite
endpoint event of intubation or death, and
there was no significant association
between HCQ use and intubation or
death. HR = 1.04 (95% CI: 0.82-1.32)
Chen Z et al.
[46]
(pre-print)
Randomized,
controlled trial
Hospitalized adults in China
with confirmed COVID-19
and mild illness. N= 62 (31
HCQ, 31 control)
Patients with severe disease
excluded
HCQ sul phate 200
mg 12 hourly orally
for 5 days plus
standard of care
(Oxygen therapy,
antiviral agents,
antibacterial agents,
and immunoglobulin.
Corticosteroids were
optional)
Standard of care only: Oxygen therapy,
antiviral agents, antibacterial agents,
and immunoglobulin. Corticosteroids
were optional.
Fever resolution time was improved in
the HCQ group (mean ± SD): 2.2 ± 0.4
vs. 3.2 ± 1.3 days. Cough resolution time
was significantly reduced in the HCQ
group (2.0 ± 0.2 vs. 3.1 ± 1.5 days).
Progression to severe disease was
reduced in the HCQ group (0/31 vs 4/31).
Proportion of patients with improved CT
results on day 6 was significantly higher
in the HCQ group: 25/31 in HCQ group
and 17/31 in control group (p=0.0476)
Chen J et al.
[47]
Randomized,
controlled trial
Hospitalized adults in China
with confirmed COVID-19.
N= 30 (15 HCQ, 15 control).
HCQ 400 mg daily
orally for 5 days plus
other treatments:
Other treatments: inhaled interferons,
abidor, umifenovir, and
lopinavir/ritonavir
Time to body temperature normalization
was similar

21
Patients with severe disease
excluded
inhal ed interferons,
umifenovir, and
lopinavir/ritonavir
(median [Interquartile range]): 1 [0 to 2]
day in HCQ group vs. 1 [0 to 3] day in
the control group.
SARS‐CoV‐2 nucleic acid conversion
rate on pharyngeal swab similar at Day 7:
negative in 13/15 in HCQ group, and
14/15 in control group (p>0.05).
Tang et al.
[48]
Randomized, open-
label
Hospitalized adults in China
with confirmed COVID-19.
N= 150 (75 HCQ, 75
control)
HCQ sul phate
loading dose of 1200
mg daily for thr ee
days followed by a
maint enance dose of
800 mg daily for 2-3
weeks plus
supportive and
symptomatic
treatment. No
antivirals permitted.
Supportive and sym ptomatic treatment.
No antivirals permitted.
The probability of negative conversion
by 28 days in the HCQ group was 85. 4%
(95% CI: 73.8% to 93.8%), similar to the
standard of care group (81.3%, 95% CI:
71.2% to 89.6%). The difference between
groups was 4.1% (95% CI: -10.3% to
18.5%).
Horby and
Landray
[49]
Randomized, double-
blind, adaptive trial
design
Hospitalized adults and
children with confirmed
COVID-19 (n=1542 in HCQ
arm, 3132 in usual care arm
at the time of early
discontinuation)
HCQ sul phate 2.4 g
loading dose over 24
hours, then 800 mg
daily for 9 days.
Simultaneously
randomized to
possibly receive
convalescent plasma
Usual standard of care. Simultaneously
randomized to possibly receive
convalescent plasma
Similar 28-day mortality rate (25.7%
HCQ vs. 23.5% usual care; HR = 1.11
[95% CI: 0.98 to 1.26]; p=0.10). No
evidence of beneficial effects on hospital
stay duration or other outcomes found
either.
Boulware et
al.
[50]
Randomized, double-
blind
Participants wit h household
or occupational exposure to
confirmed Covid-19 where
the risk of transmission was
considered moderate to high.
Conducted in the United
States of America. N=821
(414 HCQ, 407 placebo)
HCQ sul phate 800
mg once, then 600
mg 6-8 hours later,
then 600 mg daily for
4 more days
Placebo (folate) tablet regimen prescribed
identically to the intervention group
Incidence of new illness compatible with
Covid-19 or confirmed SARS-CoV-2
infection was similar between
participants receiving HCQ (49/414
[11.8%]) and those receiving placebo
(58/407 [14.3%]). The absolute
difference was −2.4% (95% CI: −7.0 to
2.2; p = 0.35)