In vitro efficacy of artemisinin-based treatments against SARS-CoV-2

Abstract

Effective and affordable treatments for patients suffering from coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are needed. We report in vitro efficacy of Artemisia annua extracts as well as artemisinin, artesunate, and artemether against SARS-CoV-2. The latter two are approved active pharmaceutical ingredients of anti-malarial drugs. Concentration-response antiviral treatment assays, based on immunostaining of SARS-CoV-2 spike glycoprotein, revealed that treatment with all studied extracts and compounds inhibited SARS-CoV-2 infection of VeroE6 cells, human hepatoma Huh7.5 cells and human lung cancer A549-hACE2 cells, without obvious influence of the cell type on antiviral efficacy. In treatment assays, artesunate proved most potent (range of 50% effective concentrations (EC₅₀) in different cell types: 7-12 µg/mL), followed by artemether (53-98 µg/mL), A. annua extracts (83-260 µg/mL) and artemisinin (151 to at least 208 µg/mL). The selectivity indices (SI), calculated based on treatment and cell viability assays, were mostly below 10 (range 2 to 54), suggesting a small therapeutic window. Time-of-addition experiments in A549-hACE2 cells revealed that artesunate targeted SARS-CoV-2 at the post-entry level. Peak plasma concentrations of artesunate exceeding EC₅₀ values can be achieved. Clinical studies are required to further evaluate the utility of these compounds as COVID-19 treatment.

Figure 1

Artemisinin and related API derivatives artesunate and artemether.

Figure 2

Pretreatment efficacy of extracts and compounds against SARS-CoV-2 in a concentration–response antiviral treatment assay in VeroE6 cells. VeroE6 cells seeded the previous day in 96-well plates were treated with the specified concentrations of extracts (A) A. annua ethanolic extract, and (B) A. annua aqueous extract, or compounds artemisinin (C), artesunate (D), and artemether (E) for 1.5 h prior to infection with SARS-CoV-2. After a 2-day incubation, infected cells were visualized by immunostaining for SARS-CoV-2 spike glycoprotein and counted automatically as described in “Materials and methods”. % residual infectivity for individual wells was calculated by relating counts of infected treated wells to the mean count of 14 infected nontreated control wells. Datapoints (red dots) are means of seven replicates with standard errors of the means (SEM) obtained in one representative experiment. Sigmoidal dose response curves (red lines) were fitted and EC₅₀ values were calculated in GraphPad Prism as described in “Materials and methods”. % Cell viability and CC₅₀ values were determined in replicate assays without infection with SARS-CoV-2 as described in “Materials and methods”. Datapoints (blue triangles) are means of 3 replicates with SEM obtained in one representative experiment. The dotted red/blue lines indicate the concentrations at which an antiviral effect (< 70% residual infectivity)/cytotoxic effect (< 90% cell viability) due to DMSO is expected according to Figure S6.

Figure 3

Treatment efficacy of extracts and compounds against SARS-CoV-2 in a concentration–response antiviral treatment assay in VeroE6 cells. VeroE6 cells seeded the previous day in 96-well plates were infected with SARS-CoV-2 and after 1 h incubation treated with the specified concentrations of extracts (A) A. annua ethanolic extract, and (B) A. annua aqueous extract or compounds artemisinin (C), artesunate (D), and artemether (E). After a 2-day incubation, infected cells were visualized by immunostaining for SARS-CoV-2 spike glycoprotein and counted automatically as described in “Materials and methods”. % residual infectivity for individual wells was calculated by relating counts of infected treated wells to the mean count of 14 infected nontreated control wells. Datapoints (red dots) are means of seven replicates with SEM obtained in one representative experiment. Sigmoidal dose response curves (red lines) were fitted and EC₅₀ values were calculated in GraphPad Prism as described in “Materials and methods”. % Cell viability and CC₅₀ values were determined in replicate assays without infection with SARS-CoV-2 as described in “Materials and methods”. Datapoints (blue triangles) are means of three replicates with SEM obtained in one representative experiment. The dotted red/blue lines indicate the concentrations at which an antiviral effect (< 70% residual infectivity)/cytotoxic effect (< 90% cell viability) due to DMSO is expected according to Figure S6.

Figure 4

Treatment efficacy of A. annua extract and compounds against SARS-CoV-2 in a concentration–response antiviral treatment assay in Huh7.5 cells. Huh7.5 cells seeded the previous day in 96-well plates were infected with SARS-CoV-2 and directly treated with the specified concentrations of A. annua ethanolic extract (A) or compounds artemisinin (B), artesunate (C), and artemether (D). After a 3-day incubation, infected cells were visualized by immunostaining for SARS-CoV-2 spike glycoprotein and counted automatically as described in “Materials and methods”. % residual infectivity for individual wells was calculated by relating counts of infected treated wells to the mean count of 14 infected nontreated control wells. Datapoints (red dots) are means of seven replicates with SEM obtained in one representative experiment. Sigmoidal dose response curves (red lines) were fitted and EC₅₀ values were calculated in GraphPad Prism as described in “Materials and methods”. % Cell viability and CC₅₀ values were determined in replicate assays without infection with SARS-CoV-2 as described in “Materials and methods”. Datapoints (blue triangles) are means of 3 replicates with SEM obtained in one representative experiment. The dotted red/blue lines indicate the concentrations at which an antiviral effect (< 70% residual infectivity)/cytotoxic effect (< 90% cell viability) due to DMSO is expected according to Figure S8.

Figure 5

Treatment efficacy of A. annua extract and compounds against SARS-CoV-2 in a concentration–response antiviral treatment assay in A549-hACE2 cells. A549-hACE2 cells seeded the previous day in 96-well plates were infected with SARS-CoV-2 and directly treated with the specified concentrations of A. annua ethanolic extract (A) or compounds artemisinin (B), artesunate (C), and artemether (D). After a 2-day incubation, infected cells were visualized by immunostaining for SARS-CoV-2 spike glycoprotein and counted automatically as described in “Materials and methods”. % residual infectivity for individual wells was calculated by relating counts of infected treated wells to the mean count of 14 infected nontreated control wells. Datapoints (red dots) are means of seven replicates with SEM obtained in one representative experiment. Sigmoidal dose response curves (red lines) were fitted and EC₅₀ values were calculated in GraphPad Prism as described in “Materials and methods”. % Cell viability and CC₅₀ values were determined in replicate assays without infection with SARS-CoV-2 as described in “Materials and methods”. Datapoints (blue triangles) are means of 3 replicates with SEM obtained in one representative experiment. The dotted red/blue lines indicate the concentrations at which an antiviral effect (< 70% residual infectivity)/cytotoxic effect (< 90% cell viability) due to DMSO is expected according to Figure S10.

Figure 6

Time-of-artesunate-addition assay. A549-hACE2 cells were infected with SARS-CoV-2 for 2 h. Artesunate at 14 µg/mL was added at different time points after viral inoculation: 0 h, addition at the time of viral inoculation with presence of the drug during the 2 h viral infection phase; 2 h, addition 2 h post inoculation, immediately after the 2 h viral infection phase; 4 h and 6 h, addition 4 h and 6 h post inoculation, respectively. After a 2-day incubation, infected cells were visualized by immunostaining for SARS-CoV-2 spike glycoprotein and counted automatically as described in “Materials and methods”. % inhibition was calculated as (100%—% residual infectivity). % residual infectivity for individual wells was calculated by relating counts of infected treated wells to the mean count of 12 infected nontreated control wells. Datapoints (columns) are means of six replicates with SEM.