key: cord-1007965-q01v8l19
authors: Gendrot, Mathieu; Duflot, Isabelle; Boxberger, Manon; Delandre, Océane; Jardot, Priscilla; Le Bideau, Marion; Andreani, Julien; Fonta, Isabelle; Mosnier, Joel; Rolland, Clara; Hutter, Sébastien; La Scola, Bernard; Pradines, Bruno
title: Antimalarial artemisinin-based combination therapies (ACT) and COVID-19 in Africa: In vitro inhibition of SARS-CoV-2 replication by mefloquine-artesunate
date: 2020-08-14
journal: International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases
DOI: 10.1016/j.ijid.2020.08.032
sha: 39a475bacb1a5c5eb5e2fc9eb6c26aef7a0363b4
doc_id: 1007965
cord_uid: q01v8l19

Abstract Objectives At the end of November 2019, a novel coronavirus responsible for respiratory tract infections (COVID-19) emerged in China. Despite drastic containment measures, this virus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread in Asia and Europe. The pandemic is ongoing with particular hotspot in Southern Europe and America. Many studies predicted a similar epidemic in Africa as that currently seen in Europe and the United States of America. However, reported data do not confirm these predictions. One of the hypotheses that could explain the later emergence and spread of COVID-19 pandemic in African countries is the use of antimalarial drugs to treat malaria, and more particularly artemisinin-based combination therapy (ACT). Methods The antiviral activity of fixed concentrations of ACT at concentrations consistent with those observed in human plasma when ACT are administered at oral doses for uncomplicated malaria treatment was evaluated in vitro against clinically isolated SARS-CoV-2 strain (IHUMI-3) in Vero E6 cells. Results Mefloquine-artesunate exerted the highest antiviral activity with % inhibition of 72.1±18.3% at expected maximum blood concentration (Cmax) for each ACT drug at doses commonly administered in malaria treatment. All the other combinations, artesunate-amodiaquine, artemether-lumefantrine, artesunate-pyronaridine or dihydroartemisinin-piperaquine, showed antiviral inhibition in the same range (27.1 to 34.1%). Conclusions Antimalarial drugs for which concentration data in lungs are available, are concentrated from 10 to 160 fold in lungs than in blood. These in vitro results reinforce the hypothesis that antimalarial drugs could be effective as anti-COVID-19 treatment.

causing coronavirus diseases 2019 emerged in Wuhan, China (Wu F et al., 2020 Gilbert et al. (2020) estimated that the countries at highest importation risk of cases of COVID-19 from China were Egypt, Algeria, South Africa, Nigeria and Ethiopia according to air travel flows from infected provinces of China to Africa and African country's capacity to detect COVID-19. Another study estimated the number of cases of COVID-19 in each African country, and more especially the timing of reporting 10,000 cases for all African countries (Pearson et al., 2020) . For instance, the timing of reporting 10,000 cases was between April 11 and 18 (6,129 cases on June 25, 2020) in Senegal, April 17 and 23 (12,270 cases) in Cameroon, May 7 to 21 (197 cases) in Angola and April 23 and May 3 (5,034 cases) in Ethiopia. The declared data to WHO indicated the spread of COVID-19 in Africa but with lower confirmed cases than expected. Several hypotheses could explain the later emergence and spread of COVID-19 pandemic in Africa, like delay in systematic SARS-CoV-2 detection and appropriate epidemiological surveillance (Kobia and Gitaka, 2020) , quick implementation of lockdown measures, physical distancing and enhanced hygiene, limited international air travel flows (Haider et al., 2020) , climate conditions Wang et al., 2020) , demographic conditions with less people above 65 years old (Diop (angiotensin converting enzyme 2, ACE-2) (Cao et al., 2020) , cross-immunity to SARS-CoV-2 (Grifoni et al., 2020; Katoh et al., 2020) .

Another hypothesis that may explain this later emergence in Africa, and more particularly in malaria endemic areas, would be the use of antimalarial drugs (Izoulet, 2020) . Since 2002, the World Health Organization (WHO) has recommended the use of artemisinin-based combination therapy (ACT) in the treatment of uncomplicated falciparum malaria (artemether-lumefantrine, artesunate-amodiaquine, dihydroartemisinin-piperaquine or artesunate-mefloquine). The combination artesunate-amodiaquine is preferentially used as first-line treatment in Burundi, Cameroon, Democratic Republic of Congo, Gabon, Ivory

Coast for instance, artesunate-mefloquine in Cambodia and Brazil, artemether-lumefantrine in Benin, Central African Republic, Malawi and South Africa and dihydroartemisininpiperaquine in Thailand or Vietnam. Amodiaquine and mefloquine, two quinoline ACT partners, were found to be active in vitro at micromolar concentration against SARS-CoV-1 at 2.5 µM and SARS-CoV-2 at 10 µM, respectively (Barnard et al., 2006; Fan et al., 2020) .

Although in vitro activity is not necessarily linked to clinical efficacy, access of in vitro effectiveness of ACT against SARS-CoV-2 may provide some answers if antimalarial use may have been involved in the later emergence and spread of COVID-19 pandemic in Africa.

The aim of this study was to evaluate the antiviral activity of ACT at concentrations consistent with those observed in human plasma when ACT are administered at oral doses for uncomplicated malaria treatment. 

The clinically isolated SARS-CoV-2 strain (IHUMI-3) (Gautret et al., 2020) was maintained in production in Vero E6 cells (American type culture collection ATCC® CRL-1586™) in MEM with 4% of fetal bovine serum and 1% glutamine (complete medium).

Briefly, 96-well plates were prepared with 5.10 5 cells/mL of Vero E6 (200µL per well), as previously described (Andreani et al., 2020) . ACT concentrations were added 4 h before infection. Vero E Cells were infected with IHUMI-3 strain at an MOI of 0.25. Controls 0%

(Vero E cell infected without drug) and 100% of inhibition (Vero E cell infected with 100 µM of ferroquine, Sigma, Saint Louis, MO, USA) were included. After 48h post-infection, the replication was estimated by RT-PCR using the Superscrit III platinum one step with Rox kit (Invitrogene) after extraction with the BIoExtract SuperBall kit (Biosellal, Dardilly, France).

The primers used were previously described (Amrane et al., 2020) .

The percentage of inhibition of SARS-CoV-2 replication was estimated for each combination as following: (mean CTcombination -mean CTcontrol 0%)/(mean CTcontrol 100% -mean CTcontrol 0%)*100. Data were expressed as mean and standard deviation of 5 different experimentations.

Estimations of the % of inhibition of the SARS-CoV-2 replication by fixed-doses of ACT were summarized in Table 1 . Mefloquine-artesunate exerted the highest antiviral activity with J o u r n a l P r e -p r o o f % inhibition of 72.1 ± 18.3 % at expected maximum blood concentration (Cmax) for each ACT drug at doses commonly administered in malaria treatment. All the other combinations showed antiviral inhibition in the same range (27.1 to 34.1 %).

A patient, who is treated with fixed-doses of ACT at commonly recommended doses for uncomplicated malaria, shows maximum blood concentrations (Cmax) of the two drugs which are able to inhibit 27.1 to 72.1 % of the Vero E Cells infected with the SARS-CoV-2 IHUMI-3 strain. Treatment with artesunate-amodiaquine, artemether-lumefantrine, piperaquinedihydroartemisinin or artesunate-pyronaridine leads to replication inhibition around 30%.

Additionally, some of these antimalarial drugs are concentrated in lungs. A single oral dose of 2 mg (10 mg/kg) of pyronaridine in rats led to a blood Cmax of 223 ng/ml and a lung Cmax of 36.4 µg/g (163 more concentrated in lung than blood) (Park and Pradeep, 2010) . About 0.07% of the administered oral dose (8.6 mg/kg) of amodiaquine was found in rat lung (Winstanley et al., 1988) . Treatment with artesunate-mefloquine (expected blood Cmax at 8.3 and 1 µM) leads to replication inhibition above 70%. This is consistent with the in vitro antiviral activity of mefloquine previously reported at 10 µM (Fan et al., 2020) . A study on postmortem human cases showed that mefloquine levels are 10 times higher in lung than in blood (concentration can go up to 180 mg/kg in the lung) (Jones et al., 1994) . No data is available on drug accumulation in lungs for the other antimalarial drugs, and more particularly dihydroartemsinin. The data on pyronaridine and mefloquine suggest the concentrations expected in lungs allow a 100% replication inhibition of SARS-CoV-2.

Additionally, artesunate exerts anti-inflammatory effects by decreasing the secretion of various pro-inflammatory cytokines including interleukin 6 (IL6), tumor necrosis factor-alpha (TNF), interleukin 1 beta (IL1beta) and interleukin 6 (IL6) through inhibition of nuclear J o u r n a l P r e -p r o o f factor kappa B (NF-kB) (Xu et al., 2007) . The secretions of IL1beta, IL6, interferon gamma (INF) were considerably increased in the cytokine storm due to COVID-19 Qin et al., 2020) . The combination of antiviral activity and anti-inflammatory effects could allow a better clinical efficacy of mefloquine-artesunate.

These in vitro results reinforce the hypothesis that antimalarial drugs could be effective as anti-COVID-19 treatment. Based on our results, we would expect that countries which commonly use ACT report fewer cases and deaths during malaria season. It could be necessary now to evaluate clinically the ACT efficacy to treat COVID-19, and more particularly that of mefloquine-artesunate, and the potential prevention of ACT against SARS-CoV-2 by comparing the antimalarial use and the dynamics of COVID-19 country by country. J o u r n a l P r e -p r o o f

No ethical approval is required.

M Boxberger received a PhD grant supported by L'Occitane Society. All the other authors have no conflict of interest to declare.

Estimation of the % of inhibition of the SARS-CoV-2 replication by fixed-doses of ACT (1x corresponds to expected maximum blood concentration (Cmax) for each ACT drug at doses commonly administered in malaria treatment) Inhibition % at 2x plasma Cmax Inhibition % at 1x plasma Cmax Inhibition % at 0.5x plasma Cmax

Concentrations 

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