key: cord-0981983-i94vca32
authors: Bae, Joon-Yong; Lee, Gee Eun; Park, Heedo; Cho, Juyoung; Kim, Yung-Eui; Lee, Joo-Yeon; Ju, Chung; Kim, Won-Ki; Kim, Jin Il; Park, Man-Seong
title: Pyronaridine and artesunate are potential antiviral drugs against COVID-19 and influenza
date: 2020-07-28
journal: bioRxiv
DOI: 10.1101/2020.07.28.225102
sha: 681b3c44ce2d1af55baedab833fe6e93e46cdd1e
doc_id: 981983
cord_uid: i94vca32

Since the first human case was reported in Wuhan Province, China in December 2019, SARS-CoV-2 has caused millions of human infections in more than 200 countries worldwide with an approximately 4.01% case-fatality rate (as of 27 July, 2020; based on a WHO situation report), and COVID-19 pandemic has paralyzed our global community. Even though a few candidate drugs, such as remdesivir (a broad antiviral prodrug) and hydroxychloroquine, have been investigated in human clinical trials, their therapeutic efficacy needs to be clarified further to be used to treat COVID-19 patients. Here we show that pyronaridine and artesunate, which are the chemical components of anti-malarial drug Pyramax®, exhibit antiviral activity against SARS-CoV-2 and influenza viruses. In human lung epithelial (Calu-3) cells, pyronaridine and artesunate were highly effective against SARS-CoV-2 while hydroxychloroquine did not show any effect at concentrations of less than 100 μM. In viral growth kinetics, both pyronaridine and artesunate inhibited the growth of SARS-CoV-2 and seasonal influenza A virus in Calu-3 cells. Taken together, we suggest that artesunate and pyronaridine might be effective drug candidates for use in human patients with COVID-19 and/or influenza, which may co-circulate during this coming winter season.

Since the first human case was reported in Wuhan Province, China in December 2019, SARS-CoV-2 has caused millions of human infections in more than 200 countries worldwide with an approximately 4.01% case-fatality rate (as of 27 July, 2020; based on a WHO situation report), and COVID-19 pandemic has paralyzed our global community. Even though a few candidate drugs, such as remdesivir (a broad antiviral prodrug) and hydroxychloroquine, have been investigated in human clinical trials, their therapeutic efficacy needs to be clarified further to be used to treat COVID-19 patients. Here we show that pyronaridine and artesunate, which are the chemical components of anti-malarial drug Pyramax®, exhibit antiviral activity against SARS-CoV-2 and influenza viruses. In human lung epithelial (Calu-3) cells, pyronaridine and artesunate were highly effective against SARS-CoV-2 while hydroxychloroquine did not show any effect at concentrations of less than 100 μM. In viral growth kinetics, both pyronaridine and artesunate inhibited the growth of SARS-CoV-2 and seasonal influenza A virus in Calu-3 cells. Taken together, we suggest that artesunate and pyronaridine might be effective drug candidates for use in human patients with COVID-19 and/or influenza, which may co-circulate during this coming winter season.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic, and more than 16 million cases of coronavirus disease 2019 (COVID-19) have been reported across at least 200 countries and territories worldwide, resulting in approximately 646,000 deaths (as of 27 July 2020) (1). To treat COVID-19 patients and to prepare for a possible second wave in advance, effective antiviral drugs against SARS-CoV-2 are urgently needed.

The antimalarial drugs chloroquine and hydroxychloroquine have been testified against infections caused by many DNA and RNA viruses, including human coronaviruses (2) . However, the results of clinical studies of those drugs for the treatment of COVID-19 have not been favorable (3) . Here, we are the first to report the potential of another antimalarial drug, Pyramax ® , a fixed-dose combination of pyronaridine and artesunate that is currently under a phase II clinical trial in Republic of Korea (4), for COVID-19 treatment. The combination of pyronaridine and artesunate has previously been reported to exhibit broad-spectrum antiviral activity (5, 6) . In particular, pyronaridine showed more effective protection and a greater reduction in viral load than other lysosomotropic antimalarial drugs in Ebola virus-challenged mice and guinea pigs (7, 8) . Additionally, artesunate reduced the viral load by 20-fold in cytomegalovirus-infected rats (9) .

In this study, we show that pyronaridine and artesunate also exhibit antiviral activity against SARS-CoV-2 and influenza viruses. better than those of artesunate in Vero cells and were also better than those of pyronaridine in Calu-3 cells (at 24 hpi: IC50 6.413 µM, CC50 43.08 µM, and SI 6.718; and at 48 hpi: IC50 8.577 µM, CC50 > 100 µM, and SI > 11.66) (Fig. 1B) . By determining viral growth kinetics in Calu-3 cells, we confirmed these two drugs could reduce viral replication in a dose-dependent manner whereas hydroxychloroquine showed no antiviral effects at 1.56-50 mM concentrations (Fig. 1C) , and the results of the time-of-addition assay might indicate that artesunate functions at a stage after viral entry (Fig. 1D ). As demonstrated in the monotherapy approaches ( Fig. 1A-C) , combinations of various concentrations of pyronaridine and artesunate also exhibited antiviral efficacy against SARS-CoV-2, and of these drugs, artesunate demonstrated much better effects against the growth of seasonal influenza A(H1N1) virus in Calu-3 cells (Fig. 1E) , which highlights the possible broad-spectrum use of artesunate against COVID-19 and influenza .

The antiviral effects of both artesunate and its active metabolite dihydroartemisinin (DHA) against DNA viruses have been well described and appear to be mediated through NF-kB and Sp1, which are involved in the type-1 interferon (IFN) pathway (7) . In addition to having antiviral effects, artesunate has been reported to have direct protective effects against sepsis-induced lung injury by inhibiting the Toll-like receptor 4 (TLR4) inflammatory signaling pathway in mice (10) . The mode of action described above is consistent with our data, which indicate that the antiviral efficacy of artesunate against SARS-CoV-2 in Calu-3 cells is far superior to that in Vero cells, which are well known for their type-1 IFN pathway deficiency. Notably, the inhibitory effects of artesunate against SARS-CoV-2 infection were worse than those of hydroxychloroquine in Vero cells but better than those of hydroxychloroquine in Calu-3 cells, which are derived from human airway epithelial cells and thus may be more representative of the susceptible cells in actual human airway infection than Vero cells.

Our data demonstrate a new possibility of repurposing antimalarial drugs against both SARS-CoV-2 and influenza A viruses. Fortunately, artesunate is an FDA-approved drug for malaria with no safety issues identified to date. Our data suggest that artesunate has the potential to be used as a primary selective drug if COVID-19 occurs during the upcoming influenza season in winter. In addition, an in vivo study needs to be conducted to evaluate the efficacy of artesunate against SARS-CoV-2 and influenza viruses. If its antiviral efficacy is proven effective in clinical trials, the combination of artesunate and pyronaridine could be an effective drug for use in human patients with COVID-19 and/or influenza, which may circulate mainly during the winter season. The data, associated protocols, code, and materials described in the manuscript are available upon request. 

WHO (2020) World Health Organization

New insights on the antiviral effects of chloroquine against coronavirus: what to expect for COVID-19?

Risk of QT Interval Prolongation Associated With Use of Hydroxychloroquine With or Without Concomitant Azithromycin Among Hospitalized Patients Testing Positive for Coronavirus Disease 2019 (COVID-19)

Approval of a phase II clinical trial: A Multi-center, Randomized, Double-blind, Parallel, Placebo-Controlled, Phase Ⅱ Clinical Trial to Evaluate Efficacy and Safety of Pyramax in Mild to Moderate COVID-19 Patients

In vivo/ex vivo efficacy of artemether-lumefantrine and artesunateamodiaquine as first-line treatment for uncomplicated falciparum malaria in children: an open label randomized controlled trial in Burkina Faso

The antiviral activities of artemisinin and artesunate

Repurposing the antimalarial pyronaridine tetraphosphate to protect against Ebola virus infection

Repurposing Pyramax for the treatment of ebola virus disease: additivity of the lysosomotropic pyronaridine and non-lysosomotropic artesunate (preprint

The anti-malaria drug artesunate inhibits replication of cytomegalovirus in vitro and in vivo

Artesunate Protects Against Sepsis-Induced Lung Injury Via Heme Oxygenase-1 Modulation

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

hydroxychloroquine on SARS-CoV-2 replication were evaluated in Vero or Calu-3 cells. The virus-only group was treated with cell culture medium. (D) The antiviral effects of artesunate was measured by a time-to-addition assay

Calu-3 cells. The virus-only group was treated with cell culture medium. Combination effects of various concentrations of pyronaridine and artesunate on SARS-CoV-2 was evaluated by qRT-PCR using the Vero cell supernatants. The virus-only group was treated with cell culture medium

The antiviral effects of pyronaridine and artesunate against human seasonal influenza A(H1N1) virus were evaluated in Calu-3 cells. Oseltamivir was used as a control drug. The virusonly group was treated with cell culture medium