Clinical use of antimalarials recently regained large public attention related to a chain of events after an observational trial of COVID-19 patients in France  and a trial in China, which suggested antiviral activity by hydroxychloroquine (HCQ) on the basis of limited information . Subsequently, governmental agencies, politicians and certain medical societies rushed to recommend antimalarial, especially HCQ in the treatment of COVID-19 . Antimalarials are one of the oldest conventional synthetic disease-modifying antirheumatic drugs, which were introduced for rheumatic diseases 70 years ago. It appears remarkable that despite a highly regulated world of drug approvals, antimalarial may again undergo possible repurposing in the area of infectious diseases based on empiric approaches. However, there are many open questions. While we are aware of the value of antimalarial for comorbidities, including reduced rates of infections , why was their presumed unique antiviral potency overlooked so far? What are the mechanisms of such presumed antiviral action? What is the appropriate antiviral dose and related safety? In the absence of a comprehensive clinical development program, it is particularly challenging the explain the mechanism(s) of antiviral functions of antimalarial. In any case, although there is an obvious strong medical need for COVID-19 therapies, the clinical value of antimalarial in this context deserves rigorous scientific evaluation before the medical community can celebrate a renaissance in a hitherto unchartered territory to combat corona virus infection. This review will summarize current insights and challenges.
Antimalarials have a variety of functions characteristically related to their direct interferences with lysosomal degradation and autophagolysosomes [5▪] but also indirect functions, related to modulation of the host's immune response. As such, antimalarial can interfere in vitro with several intracellular pathogens, such as malaria, amebiasis, RNA viruses including rabies, polio, hepatitis A and C, influenza, Chikungunya, Dengue, Zika, Lassa, Hendra, Nipah, Ebola, HIV as well as DNA viruses such as hepatitis B or Herpes simplex virus (reviewed by ). To which extent the net result of these in-vitro efficacies and the immunomodulation of the immune response impact on clinical outcomes are unknown.
POTENTIAL EFFECTS OF ANTIMALARIALS IN COVID-19 INFECTION
There are different possibilities how antimalarial may have an effect on certain viral infections, such as COVID-19 (Fig. 1):
- (1) Direct antiviral effects by blocking viral entry to the host cell
- (2) Inhibiting viral replication and survival in the host cells
- (3) Dampening an exaggerated acute immune response by the host (a ‘flatten the immune curve’ function).
These functions are not mutually exclusive but emphasize the potential pleiotropic role of antimalarial in virus–host interactions. Another interesting example of pleiotropic drugs considered to have additional antiviral capacity are heparins  making it difficult to dissect this from anticoagulatory effects.
Direct antiviral effects by blocking viral entry to the host cell
The proposed mechanism for blocking viral entry to the host cell is that it changes the terminal glycosylation of angiotensin converting enzyme-2 (ACE2) [8▪▪], the binding receptor for viral entry of severe acute respiratory syndrome (SARS)-CoV-2 and SARS, presumably resulting in reduced infectivity (Fig. 1a).
The reports that terminal glycosylation of ACE2 is impaired by chloroquine goes back to a study of Vincent et al. in 2005 [8▪▪]. These authors show that chloroquine did not change the levels of cell-surface ACE2 expression but the terminal glycosylation by changing posttranslational modifications. Whether this change in glycosylation also results in a reduced binding of SARS-CoV or even SARS-CoV-2 is not known [8▪▪]. This pioneering study could demonstrate that chloroquine has dose dependent antiviral efficacy in a pre as well as postinfection model in vitro which allowed the conclusion that these drugs have value for prophylactic as well as therapeutic purposes. Notably, the same study compared the effects of chloroquine with NH4Cl (ammonium chloride) which showed even higher antiviral potency but suggest that both substances may have shared unspecific activities based on their weak alkaline properties. As such, they can increase endosomal pH required for virus endocytosis and influence certain processes, including glycosylation.
SARS-CoV and SARS-CoV-2 share 72% identity in amino acid sequences, and molecular simulation reveals highly similar tertiary structures in the receptor binding domain of the spike protein and ACE2 . However, the higher affinity of SARS-CoV-2 to the ACE2 receptor makes it possible that changes in terminal glycosylation may not impact SARS-CoV-2 binding to the ACE2 receptor. The relevance of this mechanism for SARS-CoV-2 thus remains very theoretical and unproven.
All publications that report in-vitro entry inhibition use Vero E6 cells, a cell line derived from African green monkey kidneys but it has not been shown in primary cell cultures of human lung tissue. Whether the effect is similar remains speculative. Within the lung ACE2 has been recently found to be predominantly expressed in a transient secretory cell type  displaying the primarily infected cell type. While the concentration of HCQ was low in lung tissue in animal studies , human pharmacokinetiks modeling recently reported substantial accumulations of HCQ in the lungs with higher anti-SARS-CoV-2 efficacy than chloroquine . In-vitro studies of COVID-19 infection studying remdesivir as well as chloroquine blocked virus infection at low-micromolecular concentrations (remdesivir EC50 0.77 um; chloroquine EC50 1.13 um)  in a dose dependent manner. Chloroquine showed EC90 values of 6.9 um expected to be clinical achievable under 500 mg daily. However, whether the concentration of HCQ and chloroquine in the actual site of action is sufficiently high for inhibiting virus entry remains to be fully delineated.
Another mechanism that would interfere with the uptake of the virus to the host cell is that chloroquine suppresses phosphatidylinositol binding clathrin assembly protein expression, which is necessary for clathrin-mediated endocytosis  and thereby prevents endocytosis-mediated uptake of SARS-CoV-2 . Computer modeling identified that baricitinib may provide sufficient activity to interfere with this entry process .
Inhibiting viral replication and survival in the host cells
Inhibiting viral replication by HCQ and chloroquine has been proposed to be related to an increase of endosomal pH resulting in failure to transport virions to the ultimate releasing site . A study by Liu et al. showed colocalization of virus particles with either the early endosomal protein endosome antigen 1 or the endolysosomal protein late endosomal–lysosomal protein 1 by fluorescence microscopy. In the presence of chloroquine or HCQ, SARS-CoV-2 was identified in early endosomes but not in lysosomes which may indicate impaired processing of the virus. A more direct proof for reduced virus release from the cell was found by diminished real time-PCR virus detection in the supernatant. These two related findings have already been reported for Ebola virus and SRAS-CoV providing evidence that transport of virus to the endolysosomes is a rate-defining step in viral release .
Immunomodulatory effects by antimalarial: ‘flatten the immune curve characteristics’
Therapeutic management of most virus infections, including COVID-19 is supportive and symptom driven. Recent pandemics, such as SARS, Middle East respiratory syndrome (MERS) and SARS-CoV-2 carry high mortality due to respiratory failure from acute respiratory distress syndrome (ARDS). In a number of patients, acute virus infection results in an exaggerated innate immune response with cytokine release syndrome or secondary hemophagocytic lymphohistiocytosis (sHLH) consistent with fatal hypercytokinemia .
Experiences in rheumatology with HCQ and chloroquine led to their categorization as immunomodulators. The underlying mechanisms may have value for the host response in acute virus infections. In this regard, one of their best described effects is to inhibit cytokine release from various immune cells by reducing Toll-like receptor (TLR) signaling [5▪] (Fig. 1b). Unlike most other observations on mechanisms of antimalarial this effect has been shown in clinical studies of rheumatoid arthritis patients, where long-term HCQ treatment reduced IL-1 and IL-6 levels . In systemic lupus erythematosus, IFNα, a key type I interferon also induced by virus infection was reduced by antimalarial . These effects on cytokine release have been reported during long-term use of chloroquine and HCQ. Closer to an acute condition, antimalarials have been shown to inhibit high-mobility group protein B1 release in an animal model of lethal endotoxemia and sepsis in vivo and have therefore been suggested to treat cytokine storm in sepsis . Clinical studies are lacking for the use of HCQ or chloroquine in sHLH or sepsis, therefore benefits in these entities remains unclear.
Neither of the COVID-19 studies have reported the effect of HCQ/chloroquine on cytokine levels. Given the slow pharmacokinetics of antimalarial treatment and the clinical course of COVID-19 with a frequent aggravation more than day 10 after infection early treatment initiation with HCQ has been recommended  as well as preexposition prophylaxis which is currently addressed by ongoing clinical trials .
CLINICAL STUDIES OF CHLOROQUINE/HYDROXYCHLOROQUINE IN COVID-19 INFECTIONS
There is a clear gap between compelling in-vitro data and early uncontrolled clinical studies suggesting antiviral effects against COVID-19 of HCQ or chloroquine (Table 1), whereas controlled study data are very limited (Table 1) or not yet available (refer to the Ongoing trials section). Notably, no randomized clinical trial (RCT) has provided robust evidence of antiviral activity for either chloroquine or HCQ. Thus, available research appears to be hypothesis-generating at best. Nevertheless, antimalarial may have clinical value either as combination partner of antiviral strategies or as immunomodulator permitting adequate control over immune activation during acute infection. Clinical outcome would be the net result of these two presumed activities and remains subject of ongoing studies. Regarding side effects cardiac arrhythmias have been reported for COVID-19 patients under HCQ  but whether this is caused by HCQ or the underlying virus infection affecting also coronary arteries resulting in cardiac injury  is not yet clear. Doses of HCQ used in COVID-19 are also substantially higher than used in rheumatological patients. A recent systematic literature review of antimalarial in rheumatology described cardiac arrhythmias related to HCQ as a very rare complication of which the frequency is not known from clinical trials .
Controlled studies of hydroxychloroquine/chloroquine in COVID-19
A few studies evaluated chloroquine or HCQ in randomized trials including a first report of ‘more than 100 Chinese patients suffering from COVID-19’  with very limited information that led to the recommendation by Chinese officials to consider HCQ for prevention and treatment of COVID-19. Further trial data are listed in Table 1. Most studies used higher dosages of HCQ than recommended for patients with rheumatic conditions (up to 5 mg/kg) but achieved plasma concentrations (range 0.5–1.3 μmol/l) considered clinically active. There is overall insufficient evidence from randomized, controlled, double-blind studies, including evaluations among non-Asian patients.
Several uncontrolled trials addressed the clinical value of antimalarial almost simultaneously with the emergence of the SARS-CoV-2 spread likely related to prior studies (refer to the ANTIMALARIALS IN OTHER VIRUS INFECTIONS section). Guatret et al. reported on a case series of COVID-19 patients who received HCQ at 600 mg/day, combined HCQ/azithromycin and a control group. Patients under HCQ, especially the combination treatment showed faster viral clearance . Substantial limitations, such as confounding factors of treatment in the control group, different baseline viral loads, handling of patients lost to follow-up, lack of focus on outcome have been raised and precluded solid conclusions . Another small study with 11 patients could not confirm rapid viral clearance of COVID-19 under the combined HCQ/azithromycin treatment . Mahevas et al. retrospectively reported 181 patients with SARS-CoV2 pneumonia in four French hospitals with a daily HCQ dose of 600 mg compared with patients who standard of care. Seventeen patients in the HCQ group also received azithromycin. 27.4% in the HCQ group and 24.1% in the non-HCQ group developed ARDS within 7 days. Eight patients needed to discontinue HCQ because of ECG abnormalities. These contradictory data even from uncontrolled studies do not support use of HCQ  for the general treatment of SARS-CoV-2.
Various trials are currently ongoing to address the potential benefit of antimalarial treatment under certain conditions. Most studies use HCQ (33 ongoing and recruiting, another 47 not yet recruiting, two enrolling by invitation). Chloroquine is less frequently tested (three ongoing and recruiting, 17 not yet recruiting, ClinicalTrials.gov accessed 15 April 2020). These studies differ in concomitant medications as well as in the timing of treatment and disease severity and can be classified into (examples listed in brackets):
- (1) Studies that have a preemptive (prophylactic) approach for example in healthcare workers
- (a) Only one dose compared with placebo (NCT04308668)
- (b) High and low-dose arm (NCT04329923)
- (2) Studies that compare HCQ or chloroquine to placebo in different severities of SARS-CoV-2 infections
- (a) Mild (NCT04329923)
- (b) Moderate (NCT04345692, NCT04341870)
- (c) Severe (NCT04325893)
- (3) Studies that use HCQ or chloroquine in combination with other immunosuppressants or antiviral drugs
- (a) Tocilizumab (NCT04332094)
- (b) Sarilumab (NCT04341870)
- (c) Azithromycin (NCT04329832)
- (d) Lopinavir/ritonavir (NCT04307693)
- (e) Oseltamivir (NCT04303299)
It is expected that these studies comprising unprecedented large numbers of patients during an acute virus infection will provide answers about the benefit/risk evaluation of antimalarial during this acute virus infection and a potential place in the treatment algorithm, including dosing, timing, beneficial combinations.
ANTIMALARIALS IN OTHER VIRUS INFECTIONS
Prior to the interest in using antimalarial against SARS-CoV-2, preclinical suggestions and clinical observations in other virus infections suggested their use as they target intracellular pathogens. For only very few nonviral infections, there is sufficient evidence of their efficacy, such as malaria , especially chloroquine sensitive strains . For Q fever with Coxiella burnetii and Whipplei's disease caused by Tropheryma whipplei, HCQ in combination with doxycycline is often recommended but have not been studied by randomized controlled trials.
With regard to viral infections, antimalarial have been evaluated for certain infections. Comprehensive reviews have summarized the value of HCQ against viruses and antimalarial drugs against virus infections [36–38]. Notably, there is a consistent gap between convincing antiviral activity on preclinical grounds, while available clinical studies did not provide convincing efficacy.
Severe acute respiratory syndrome-CoV and Middle East respiratory syndrome
The SARS and MERS outbreaks in 2003 and 2012 have been much more limited than the current COVID-19 outbreak with around 8000 cases for SARS and 2000 cases for MERS worldwide  compared with almost 2 million cases of COVID-19 worldwide (15 April 2020). Consequently, randomized controlled trials investigating the role of antimalarial were not conducted for SARS and MERS. Experimental evidence for the antiviral effects of chloroquine and HCQ against SARS and MERS is comparable with reports related to SARS-CoV-2 virus. Chloroquine and HCQ led to reduced replication in cell cultures for SARS and MERS [40–42] as well as inhibited cell entry of SARS by altered glycosylation of ACE2 [8▪▪]. For MERS dependent on dipeptidyl peptidase 4 as entry receptor  and the cellular proteases [e.g. type II transmembrane serine protease (TMPRSS2)] , it is not known how chloroquine or HCQ are able to interfere.
Early studies suggested that HCQ may inhibit HIV replication and exert with beneficial effects on the immune system. Among them, immunomodulatory and anti-inflammatory properties of antimalarial have been quoted especially with regard to T cells . In-vitro effects of antimalarial on HIV replication have been attributed to an increase in endosomal pH thereby changing posttranslational modification of gp120 . Early clinical studies reported some benefit [47,48] and a downregulation of TLR-mediated signal transduction as well as a decreased production of IL-6 . Mechanistic antiviral activity against HIV by HCQ and chloroquine has been shown in several small studies, including reduction in HIV-1 RNA copy numbers and reduction of CD4+ T-cell activation .
With regards to clinical outcome, compelling evidence of antimalarial efficacy against HIV is still lacking. The AIDS Clinical Trials Group A5258 carried out a randomized, double-blind, placebo-controlled study in 33 HIV-1-infected participants off antiretroviral therapy and 37 patients on antiretroviral therapy. Chloroquine modestly reduced immune activation in participants under antiretroviral therapy . Another trial with 1499 patients (randomized, controlled, open-label, phase III trial) evaluated standard of care with chloroquine prophylaxis compared with no prophylaxis in HIV-positive patients. There was no improvement in CD4 T-cell recovery, lymphoid and myeloid immune activation or inflammatory markers . Another study with 400 mg/day HCQ over 48 weeks reported that in HIV-infected patients not taking antiretroviral therapy, the use of HCQ compared with placebo did not reduce CD8 cell activation but did result in a greater decline in CD4 cell count and increased viral replication . Thus, most studies in HIV found mechanistic hints of chloroquine or HCQ against HIV but the clinical studies did not provide convincing evidence for their efficacy.
Inhibition of influenza A replication has been observed in cell cultures upon the addition of chloroquine . Like for SARS and other viruses, the in-vitro effect was pH dependent as the same effect could be achieved by ammonium chloride , whereas in-vivo prevention of H1N1 or H3N2 could not be found by Vigerust et al. However, contradictory reports show that chloroquine can enhance H1N1 replication in a cell line of human lung epithelial cells . A large, double-blind, placebo-controlled trial in 724 patients using 500 mg chloroquine/day for 1 week, then once a week to complete 12 weeks failed to prevent infections with influenza .
There are two negative RCTs evaluating chloroquine in dengue fever. In a Vietnamese study with 153 patients, chloroquine did not reduce the durations of viremia and NS1 antigenemia in dengue patients but fever cleared earlier . Another randomized clinical study of 19 patients, there was no difference between chloroquine treated patients and controls in the duration of disease, disease intensity or days of fever despite reduced pain .
In a randomized controlled study of 27 patients, chloroquine treatment was not different from standard of care in acute chikungunya infection  (mean duration of fever, arthralgia or viremia decrease) as well as viral clearance.
In-vitro data suggested also anti-Ebola activity by chloroquine. However, the clinical trials were inconsistent and variations of plasma concentrations may have influenced outcome .
- (1) For chloroquine and HCQ direct antiviral activities in vitro have been reported for several viruses, including SARS-CoV-2 . This activity appears to depend on rather unspecific interferences with lysosomal processing (including downstream abnormalities, such as change in glycosylation patterns etc.) which is pH sensitive, as ammonium chloride resulted in similar findings when studied for certain viruses.
- (2) Chloroquine and HCQ also act as immunomodulators with pleiotropic effects on reduced cytokine production, inhibition of TLR signaling and interferences with lysosomal processing involved in antigen presentation. These functions have been adapted in the treatment of rheumatic diseases and may exert better control of exaggerated immune activation in viral infections (flatten the immune curve).
- (3) In contrast to in-vitro data, results from controlled clinical studies available to date have provided insufficient evidence that antimalarial exert convincing clinical activity in acute virus infections, including SARS-CoV-2. According to NIH guidelines (https://covid19treatmentguidelines.nih.gov), use of antimalarial in SARS-CoV-2 outside of clinical trials is not recommended.
- (4) However, antimalarial interfere with endolysosomal and autophagosome processing, which represent cross-roads of immune activation during infections, autoimmunity and cancer. The detailed understanding of these mechanisms may provide the basis for selective targeting of extrinsic (virus) versus intrinsic (cancer, autoimmune) activation paths.
The authors would like to thank the Clinical Scientist Program of the Berlin Institutes of Health who provided grant support to E.V.S. and the Deutsche Forschungsgemeinschaft who provided grant support to T.D. (projects Do491/7-4, 10-1, 11-1 and Transregio130 TP24).
Financial support and sponsorship
Deutsches Rheumaforschungszentrum (DRFZ) Berlin is funded by the Leibniz Society.
Conflicts of interest
There are no conflicts of interest.
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