5 April 2020 - The antiviral potential of chloroquine: 130 years-old and still active?

Le Comité de crise Covid-19 de la Société Française de Virologie - Association Loi 1901

Correspondance : Bureau de la Société Française de Virologie - contact@societe-francaise-de-virologie.fr

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Something blue, something old

Chloroquine is a group 4 aminoquinoline, mainly known for its anti-malarial effect. It is derived from methylene blue, a synthetic dye, which ability to selectively destroy the parasite while preserving surrounding cells was first discovered by Dr Paul Ehrlich in 1891 (reviewed in [1]). One structural analog, quinacrine, showing a better efficacy, replaced quinine for treatment of malaria when it came to market in 1931. However in 1934, the ultimate structural modification leading to chloroquine (CQ) was obtained by the German chemist Johan “Hans” Andersag, presenting a highly superior anti-parasitic efficacy and the advantage to not color the skin and the eyes in yellow…

The serendipitous discovery of the anti-inflammatory properties of CQ occurred during world word II. Clinical benefits were evidenced only from 1955, after the addition of a beta-hydroxyl chain to CQ which reduced CQ toxicity by two- to three-fold, for a comparable efficacy. This compound, hydroxychloroquine (HCQ), is currently prescribed for long-term treatment of inflammatory or auto-immune diseases such as rheumatoid arthritis, juvenile idiopathic arthritis, systemic lupus erythematosus, or Sjögren’s syndrome, at daily doses higher than those used for antimalarial treatment.

Toxicity

Headaches, nauseas and vomiting, heart palpitations, and, less frequently, retinopathy and cutaneous hyperpigmentation are the most frequent undesired side-effects of CQ/HCQ treatments in approved clinical indications. A study conducted in 2010 on 4000 patients treated for rheumatoid arthritis or lupus erythematous showed that HCQ toxicity was significantly correlated with treatment duration (i.e. cumulated dose effect), and that patient’s daily dose, age or weight were not predictive factors [2]. A later study, including about 2000 patients treated for rheumatoid arthritis for 5 years or more, reported an increase of the average incidence of toxic events by 7.5% within this cohort, that varied according to the daily dose [3] The prevalence of retinopathy in patients treated for 10 years with the usually prescribed average dose of 4-5 mg/kg per day was low, about 2 %, in agreement with the 1st study [3].

HCQ toxicity has also been tested in cancer patients, alone or in combination with other molecules, in at least seven therapeutic trials. All studies reported innocuity and a good two-month tolerance of a daily HCQ dose of 400 to 600 mg, and up to 1200 mg in two studies (reviewed in [4]).

Structure and chemical properties

CQ and HCQ are racemic compounds, i.e. chiral molecules which synthesis results in two stereoisomers existing as mirror images of each other (also called enantiomers). No preferential usage of one over the other CQ or HCQ enantiomer was observed in vitro against most Plasmodium strains, which is not the case for other CQ derivatives (reviewed in [5]). Per Os CQ absorption does not seem to be stereoselective either (about 75% of a dose is absorbed by the stomach membrane and is potentially responsible for the nauseas). However, the fixation of CQ to some plasma components was shown to be partially stereoselective, with repercussions on the respective distribution and volume concentration of enantiomers in blood and tissues.

At neutral pH, CQ/HCQ exists as a mixture of three forms: non protonated, mono- and di-protonated. Because of the positive charge of the last two forms, only the non-protonated one can cross the plasma membrane.

Biological and cellular properties

CQ and HCQ have a selective affinity for some blood cells, such as thrombocytes and granulocytes, but also for albumin and acid glycoprotein alpha-1 (or orosomucoïde), two globular proteins of the acute inflammatory phase. The peak of plasma concentration is reached between 4 and 72 hours post-administration, while urinary elimination is slow, sometimes up two several months after the end of the treatment [6].

Inside the cell, CQ/HCQ exhibits a selective affinity for lysosomes, which are scavenger vesicles containing specific enzymes in charge of intracellular molecule degradation, such as hydrolases, which are only active at acidic pH. Sequestration of the CQ protonated form in this compartment increases lysosomal pH, inactivating its function [7-9]. This cellular interaction is the basis of CQ efficacy against malaria. The hemoglobin from host erythrocytes is the major nutrient source of the parasite. The parasite processes hemoglobin in its food vacuoles, which are functionally close to cellular lysosomes and active at acidic pH. By raising the pH in these vacuoles, chloroquine Inhibits this process. Accumulation of unprocessed hemoglobin then leads to apoptosis and parasite elimination [10].

The autophagy and apoptosis pathways are up regulated in cancer cells and are considered as targets for therapeutic intervention in oncology. Results from phase I and II have shown a clinical benefit of CQ when used in combination with other therapeutic molecules [4].

Anti-inflammatory and immuno-suppressive properties

CQ/HCQ immunosuppressive and anti-inflammatory properties, known since the 1950s, are at the origin of the pharmaceutical indication of these molecules in long-term treatment of rheumatoid arthritis or lupus erythematosus. However, cellular mechanisms supporting their efficacy have only emerged in the last decade. Until recently the most accepted hypothesis was the inhibition of the activation of Pattern Recognition Receptors (PRR) from the Toll-Like Receptors (TLR) family., following endosomal pH alkalization. This family of receptors recognizes CpG motifs present in nucleic acid sequences and plays a key role in the induction of the inflammatory response [11,12].

However, a large study conducted in 2011 on HCQ pre-treated plasmacytoid dendritic cells (pDC) or peripheral blood monocytic cells incubated with sera from patients with rheumatoid arthritis or systemic lupus erythematosus, showed only a minor effect on the acidic pH of endosomal vesicles. Instead, this study reported a direct interference of HCQ with the fixation of CpG ligands to PRR, resulting in signaling blockade [13, 14]. This inhibitory effect of HCQ on TLR signaling was also observed in an earlier study in rheumatoid arthritis patients [15]. Moreover, a very recent study reports that HCQ can also inhibit endosomal TLR dimerization, another critical signaling step [16].

In vitro antiviral properties and effects on cellular immune responses

Low pH fusion with cellular endosomal membranes is a mechanism used by several viruses, mostly RNA viruses, to release their genome into the cell. It is a key step of the initiation of viral infection cycle. This step is one of the potential targets for type 4 aminoquinolines like CQ and its derivatives, all positively charged [17]. Inhibition of endosomal fusion is however not the unique mechanism by which of CQ/HCQ can block the infectious cycle, as demonstrated by several studies on viruses which entry is not dependent on acidic fusion, like herpes simplex virus, rotavirus, simian virus 40, or poxviruses [18-21].

Alkalization of vesicles from the trans-or late Golgi network interacts with the activity and/or synthesis of several cellular enzymes, including acid hydrolases, pro-hormone convertases and glycosyltransferases. These enzymes are involved directly or indirectly at several steps of the viral cycle, and in some functions of the host cell that are exploited by the virus, like autophagy activation. Enzymatic dysfunction also impact the correct execution of post-translational modifications important for virus/receptor interaction, like glycosylation, or for proteolytic cleavages needed for virus assembly and maturation (for example, in the final structural shift from non-infectious to infectious particles in flaviviruses) [22]. In vitro, depending on the nature of the virus and on experimental conditions, CQ/HCQ were shown to act on one or several of these steps [23-27].

We presented above the anti-inflammatory effect of CQ in some non-infectious pathologies, through direct interaction with TLR family members. A major function of TLR is to alert the organism upon recognition of a microbial infection and to trigger innate and adaptative immune responses. Signaling is initiated after direct interaction between TLR and pathogen-associated molecular pattern (PAMP) present in viral genomes. Chloroquine was shown to inhibit autophagy and TLR activation after infection, and to reduce secretion of pro-inflammatory cytokines like tumor necrosis factor, interleukin 6 and gamma interferon [28,29].

By contrast, a co-stimulatory effect of CQ on TLR7/8 activation was observed in presence of an imidazoquinoline, R848. In human, TLR7/8 is involved in recognition of single stranded RNA viruses (that includes coronaviruses) [30]. This synergistic effect on TLR7/8 is reminiscent of the one observed between R848 and CpG-containing oligonucleotides [31]. These data suggest that CQ/HCQ could have, under conditions to be determined, a positive effect in vivo on the activation of the immune response to infection.

Clinical and pre-clinical data

Clinical studies on infectious diseases that include CQ or HCQ in therapeutic or prophylactic protocols are largely heterogenous. Great variations are observed regarding the dose, the frequency of administration and the duration of the treatment, with, most of the time, no explicit rational. These studies are summarized in Table 1, which also includes as comparators the curative protocol of Plasmodium infection and the most current treatment for rheumatoid arthritis. In all presented studies, the administration route was per os.

Human acquired immunodeficiency virus, type I, HIV-1 (retrovirus)

Despite their optimal capacities to suppress viremia, current anti-HIV-I therapies have difficulties to restore the CD4 balance, and to reduce CD8 levels in infected patients. In vitro studies indicate that CQ, in addition to its antiviral activity against HIV-1, can inhibit immune activation in pDCs. This inhibitory effect on pDC activation was also demonstrated in vivo in rhesus macaques infected with the simian immunodeficiency virus (SIV), but had no effect on blood monocytic cells composition [32].

Nine clinical studies, at least, have been conducted, all with a low number of patients, and either as monotherapy or in combination with (or compared to) other treatments (Table 1) [33-42]. Six of these studies report a clinical benefit, minor in most of them, at best equivalent to current therapies. Two studies reported no benefit, and two others described an exacerbation of the disease [41]. Protocols were however so different that side-by-side comparisons were not possible , and so it is difficult to learn from these trials.

Materno-fetal transmission is a concern in HIV-I chronically infected, pregnant women. A lower transmission rate was observed in women under antimalarial treatment and was correlated with detection of CQ in the umbilical cord. Children born from these mothers still had detectable plasma CQ levels at 15 and 18 months [35]. Inversely, CQ was shown to increase virus load in breast milk, and breast feeding is thus strongly discouraged in these instances [43].

Type A influenza virus A (paramyxovirus)

Chloroquine was evaluated in only one study, as a potential prophylactic treatment to face an H1N1 influenza pandemic (Table 1) [44]. As of today, this study remains the largest ever conducted with CQ in the context of viral infection (> 700 subjects). No clinical benefit was reported either on infection incidence, or on incidence of severe cases. Preclinical studies in ferret, the only animal to develop flu clinical symptoms after infection with human influenza virus, did not show any significant effect either [45]. A more recent study, using a mouse infection model based on lethality for progeny, confirmed that oral absorption of CQ was unable to prevent influenza H1N1 strain infection, but revealed a drastic inhibitory effect on progeny death when CQ was instilled intranasally at 50mg/kg (70% survivors in the treated group, none in the mock-infected group) [46].

Chikungunya virus (alphavirus)

Three post-infection studies were conducted, aiming at improvement of the clinical status of the infected subjects, and more specifically at reduction of muscular-skeletal pain and arthritis [47-49]. No clinical benefit was observed, in any of these studies, but two of them reported an exacerbating effect consisting of an increase of pro-inflammatory cytokines level [48,49].

The 2018 publication of Roques and coll. also reports the results of a prophylactic study conducted in Macaca fascicularis [49]. Monkeys inoculated intravenously on day(D)0 with 14 mg/kg for 5 days, then infected 1h after the 6th treatment on D5, showed significant increase of viremia together with lower viral clearance, compared to non-treated, infected animals. Moreover, these animals exhibited an exacerbated IFN type I response correlated to plasma viremia, severe lymphopenia, and delayed induction of IgM and cellular responses. The CQ doses in this study were chosen to approximate a plasmatic concentration close to the IC50 preliminarily established in in vitro inhibition studies.

Dengue virus (flavivirus)

Two studies were conducted in subjects with fever and at least two other disease symptoms (retro-orbital or muscular pain, vomiting, skin rash) for minimum 72 hours [50,51]. In the first study, conducted in 2010 on Brazilian patients, CQ treatment had no effect on viremia duration or NS1 antigenemia, a marker of active viral replication [50]. In the second study, conducted in 2013 on Vietnamese patients, treated subjects reported an improvement of their quality of life, progressively reverting after treatment was stopped [51]. No biological parameter was evaluated in this last study.

Hepatitis C virus (hepacivirus)

Only one randomized, triple-blind, placebo-controlled study was conducted on a low number of patients (n=10) with a negative prognostic for IFN/ribavirin treatment. Significant reduction of viremia was reported in treated patients, compared to placebo group [52]. Normalization of some anthropomorphic and biochemical parameters, and of ferritin and vitamin C levels was also observed in these patients [53].

Zika virus (flavivirus)

One of the main challenges of Zika epidemy is prevention of the congenital infection and of the associated risk of microcephaly for the new-born. The presumption of a positive effect of CQ in pregnant women is strong, and its rational was presented in a 2018 publication urging for its introduction in clinical trials [54]. This rational is based on multiple in vitro studies, and one in vivo study demonstrating fetal protection from Zika-induced microcephaly of pregnant BALB/c mice [55]. This study was reproduced in another murine model, SCID-Beige mice [56]. Here again, the supposed action mode is the inhibition of viral genome release from endocytic vesicles, and inhibition of autophagy, usually activated by the virus to increase its replication.

Coronaviruses associated with severe acute respiratory syndrome, SARS-CoV-1 et -2 (coronavirus)

Considering the amplitude of the COVID-19 pandemic due to SARS-CoV-2 virus, several pilot studies including a low number of infected patients were launched in a hurry and lacked appropriate controls (Table 1) [57-63]. These studies have been followed by larger observational and historical studies presenting several biases, also published in a hurry, and extensively debated in scientific forums and in public medias. In addition, the CQ/HCQ arm has been recently removed from the two large American and European trials Solidarity and Discovery, respectively, designed to compare several COVID=19 therapeutic options, but the data are not yet published.

We summarized here the few preliminary, contradictory studies that started the still ongoing controversy. The first study included French volunteers (n=20) at low stage of infection, i.e. as soon as infection was confirmed, treated with a combination of HCQ and azithromycin (AZT, an antibiotic with antiviral properties). Reduction of nasopharyngeal viral load was reported in 75% treated patients, compare to non-treated patients [57] A larger, retrospective study (n=1061) conducted by the same team confirmed the safety of the AZT/HCQ combination, associated to a low fatality rate in patients treated before COVID-19 complications arrived [58]. Shortening of the median time to achieve an undetectable viral RNA was also observed in a large Chinese cohort (n=197) treated with CQ, with a higher side-effects rate at low dose than at high dose [59]

Another study based on HCQ/AZT combination patients presenting co-morbidity factors at an advanced stage of the disease (10 out of 11 under oxygen support) were enrolled. No clinical benefit, and no reduction of viral load were noted in these patients [60]. Clinical improvement was reported in a small randomized study including patients with overt pneumonia, with faster recovery in those treated with HCQ only than in the placebo group [61]. Decrease mortality was also reported in a larger controlled study and associated with attenuation of inflammatory cytokine storm [62]. These data were not in agreement with those obtained in a larger randomised, controlled study in which no clinical benefit of HCQ treatment was reported in patients with pneumonia requiring oxygen [63].

No clinical trial was conducted on SARS-CoV-1 patients, but CQ was evaluated in pregnant C57BL/6 mice for its capacity to protect 5-day litter against an intracranial, lethal challenge with SARS-CoV. Results showed a survival rate of 78 to 98% of 5 days old suckling mice when CQ was administered to mothers 2 days before parturition [64]. CQ was transmitted to the new-borns through the transplacental route or acquired from maternal milk.

Analyses and recommendations

Including all infection models, thirteen studies had shown a clinical benefit, usually modest, of CQ/HCQ treatment, seven did not report any benefit, and the last four report exacerbating effects (Table I, see column “impact”). Protocols heterogeneity, controls definition, targeted viruses, scarcity of studies for some models, makes it difficult to learn from these studies, let alone making predictions for other viral infections. Most of these trials were launched from results obtained in vitro. Results tend to show that clinical data are better correlated with animal data when they are available, which is pleading for more efforts in developments of preclinical models before entering clinical trials.

Chloroquine and its derivatives have been used for more than 100 years to fight the parasite responsible for malaria. However, our knowledge of their potential mode of action against other infections models has not evolved at the same pace in the last 20 years, despite the fact that their potential in the anti-microbial combat has been proposed for a long time [65]. The basis of our understanding of these molecules lies mainly on information acquired from the two approved pharmaceutical indications (antimalarial and drug treatment in inflammatory diseases with non-infectious ethology).

Comparison of the different trials presented here raised several unsolved questions, that will be important to address in future studies:

In vitro studies

We need to better understand, for each studied infection model, the role of the different forms of CQ/HCQ, enantiomers and metabolites, including their relative proportions and their specific pharmacodynamics, and to anticipate the potential impact of lot to lot variations during production. As mentioned above, stereoselectivity has been observed in some infection models and might impact the balance between efficacy and toxicity, depending on the dose.

It would also be useful and important to develop other CQ derivatives with in vitro antiviral activity, and to select the best candidate against each type of virus. This recommendation is supported by three publications. The first one reports that some derivatives mimicking HCQ are far better inhibitors of Plasmodium growth than CQ [66]. The second one, more recent, reports similar inhibition efficacy of Plasmodium by CQ and HCQ, but higher inhibitory effect of HCQ against SARS-CoV-2 [67]. The third one, just published, reports the screening of several commercial anti-infectious molecules, including CQ derivatives, and highlights other potential candidates that could inhibit SARS-CoV-2 replication, based on endosomes alkalization [68].

Preclinical/clinical studies

Administration routes, other than the oral one, should be investigated. Intranasal administration might be an option to explore.

Treatment kinetic is a well-known study parameter in acute infections. Beyond the usual criteria, such as dose effects on viral load and immunological parameters, it would also be useful, especially for multiple administrations, to follow in parallel plasma retention (including metabolites, which potential effects are still unknown) and urinary excretion of the drug.

Availability of a preclinical model should be a priority. In absence of relevant animal model, new technologies, like biomimetic models based on in vitro reconstruction of human organ-like structures should be considered.

Particular attention must be given to efficacy criteria. Absence of viral detection used as a binary criterion (YES/NO) could be misleading. A quantitative assay enables to compare natural and CQ-induced viral decay and is thus more suitable. A complete study should include virus quantitation in all patients, together with therapeutic criteria and immunological dosages, like, for example, interleukins dosage.

Mathematical and statistical tools need to be considered in order to limit studies size and number, while collecting a maximum of data. They include experimental design [69], which enables calculation of the minimum number of patients to enroll according to the number of study parameters, and multi-parametric analysis [70], which allows evaluation of synergistic effects between several parameters from the same study.

Conclusion: To be continued

Chloroquine, as well as its derivatives, exhibit potent, pleiomorphic in vitro antiviral properties, and some clinical data provide a glimpse of a therapeutic potential that we do not master yet. Several studies have been launched around the world in the context of COVID-19 pandemic and we probably will learn more from these studies than from data gathered in the last 130 years since the discovery of chloroquine. These studies should clarify in the coming months the potential therapeutic indication of CQ for treatment of SARS-CoV-2 infection.

Table 1. List of clinical studies.

Abbreviations: CQ : chloroquine. HCQ : hydroxychloroquine. ART: antiretroviral therapy. AZT: azithromycin. d.: day. w.: week. m.: month. pos.: positive. neg.: negative. Nr: not reported. Na: non applicable.

↑: increase ; ↓: decrease ; *: asymptomatic patients ; **: pregnant women.

Liens d’intérêts

Les auteurs déclarent ne pas avoir de lien d’intérêt en rapport avec cet article.

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