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.

Proof-of-concept for prophylactic efficacy of the extracts was obtained using a plaque-reduction assay in VeroE6 cells. Subsequent concentration-response studies using a high-throughput antiviral assay, based on immunostaining of SARS-CoV-2 spike glycoprotein, revealed that pretreatment and treatment with extracts, artemisinin, and artesunate inhibited SARS-CoV-2 infection of VeroE6 cells. In treatment assays, artesunate (50% effective concentration (EC50): 7 μg/mL) was more potent than the tested plant extracts (128-260 μg/mL) or artemisinin (151 μg/mL) and artemether (>179 μg/mL), while generally EC50 in pretreatment assays were slightly higher. The selectivity index (SI), calculated based on treatment and cell viability assays, was highest for artemisinin (54), and roughly equal for the extracts (5-10), artesunate (6) and artemether (<7). Similar results were obtained in human hepatoma Huh7.5 cells. 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.

SciScore for 10.1101/2020.10.05.326637: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Cells were incubated with the anti-N monoclonal antibodies (1:25 dilution in PBS-T) for 45 min, followed by incubation with secondary antibody (Alexa 488-labeled goat anti-mouse at a 1:500 dilution; Thermo Fisher).	anti-N suggested: None anti-mouse suggested: None
Following two washes with PBS containing 0.1% Tween-20, plates were incubated with secondary antibody F(ab’)2-Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, HRP (Invitrogen #A24476,	anti-Hum…

SciScore for 10.1101/2020.10.05.326637: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Cells were incubated with the anti-N monoclonal antibodies (1:25 dilution in PBS-T) for 45 min, followed by incubation with secondary antibody (Alexa 488-labeled goat anti-mouse at a 1:500 dilution; Thermo Fisher).	anti-N suggested: None anti-mouse suggested: None
Following two washes with PBS containing 0.1% Tween-20, plates were incubated with secondary antibody F(ab’)2-Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, HRP (Invitrogen #A24476,	anti-Human IgG suggested: (Thermo Fisher Scientific Cat# A24476, RRID:AB_2535945)
Experimental Models: Cell Lines
Sentences	Resources
30, 31 VeroE6 cells were plated at 10,000 cells per well of poly-D-lysine-coated 96-well plates (Thermo Fisher Scientific, Rochester, NY, USA).	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
The MOI for infection was chosen aiming at on average 3000-4000 counts per well for VeroE6 cells and on average 300-600 counts per well for the less permissive Huh7.5 cells in infected nontreated control wells upon termination of the respective assays.	Huh7.5 suggested: RRID:CVCL_7927)
Software and Algorithms
Sentences	Resources
Sigmoidal dose response curves were fitted and median cytotoxic concentration (CC50) values were calculated with GraphPad Prism 8.0.0 using a bottom constraint of 0 and the formula Y= Top/(1+10^((LogCC50-X)*HillSlope)) as further specified in Figures S5 and S8.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

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

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