Moxidectin and Ivermectin Inhibit SARS-CoV-2 Replication in Vero E6 Cells but Not in Human Primary Bronchial Epithelial Cells
Nilima Dinesh Kumar, Bram M Ter Ellen, Ellen M Bouma, Berit Troost, Denise P I Van De Pol, Heidi H Van Der Ende-Metselaar, Djoke Van Gosliga, Leonie Apperloo, Orestes A Carpaij, Maarten Van Den Berge, Martijn C Nawijn, Ymkje Stienstra, Izabela A Rodenhuis-Zybert, Nilima Dinesh Kumar, Bram M Ter, Jolanda M Smit
Antiviral therapies are urgently needed to treat and limit the development of severe COVID-19 disease. Ivermectin, a broad-spectrum anti-parasitic agent, has been shown to have anti-SARS-CoV-2 activity in Vero cells at a concentration of 5 mM. These limited in vitro results triggered the investigation of ivermectin as a treatment option to alleviate COVID-19 disease. However, in April 2021, the World Health Organization stated the following: "The current evidence on the use of ivermectin to treat COVID-19 patients is inconclusive." It is speculated that the in vivo concentration of ivermectin is too low to exert a strong antiviral effect. Here, we performed a head-to-head comparison of the antiviral activity of ivermectin and the structurally related, but metabolically more stable moxidectin in multiple in vitro models of SARS-CoV-2 infection, including physiologically relevant human respiratory epithelial cells. Both moxidectin and ivermectin exhibited antiviral activity in Vero E6 cells. Subsequent experiments revealed that these compounds predominantly act on the steps following virus cell entry. Surprisingly, however, in human-airway-derived cell models, both moxidectin and ivermectin failed to inhibit SARS-CoV-2 infection, even at concentrations of 10 mM. These disappointing results call for a word of caution in the interpretation of anti-SARS-CoV-2 activity of drugs solely based on their activity in Vero cells. Altogether, these findings suggest that even using a high-dose regimen of ivermectin, or switching to another drug in the same class, is unlikely to be useful for treatment of SARS-CoV-2 in humans. KEYWORDS moxidectin, ivermectin, antiviral, SARS-CoV-2, ALI, in vitro W ithin less than 1.5 years, the pandemic SARS coronavirus 2 (SARS-CoV-2) has infected over 153 million individuals and resulted in over 3.2 million deaths worldwide (1-3). The social and economic burden of this still-ongoing pandemic is staggering, and, besides vaccine development, it is of utmost importance to develop therapeutic interventions to reduce disease symptoms. To date, multiple compounds have been shown to exert SARS-CoV-2 antiviral activity in vitro and several compounds have reached clinical trials (4, 5). Remdesivir and hydroxychloroquine were thought to be effective early in the pandemic, but after a careful evaluation in an interim solidarity trial, the WHO released a conditional yet strong recommendation against the usage of
% cytotoxicity ¼ ðcompound-treated LDH activity 2 spontaneous LDH activityÞ ðmaximum LDH activity 2 spontaneous LDH activityÞ Â 100 Live/dead staining and flow cytometry. PBECs cultured under ALI conditions were treated with 10 mM moxidectin, ivermectin, or an equivalent volume of EtOH at the basolateral side for 48 h at 37°C. Subsequently, cells were harvested by trypsinization and stained with fixable viability dye eFluor780 (Thermo Fisher Scientific) for 20 min at 4°C. Next, cells were washed with fluorescence-activated cell sorter (FACS) buffer (1X phosphate-buffered saline, 2% FBS, 1% EDTA), centrifuged, and fixed with 4% paraformaldehyde for 10 min at 4°C. After fixation, cells were washed, centrifuged, and resuspended in FACS buffer. Flow cytometry analyses were performed using the LSR-2 flow cytometer (BD Biosciences, San Jose, CA, USA) and data was further analyzed using Kaluza analysis software, version 2.1 (Beckman Coulter, Fullerton, CA, USA). Antiviral assays in Vero E6 and Calu-3. Vero E6 cells were seeded at a density of 1.3 Â 10 5 cells/well in 12-well plates. The next day, the medium was replaced with 0.25 ml of DMEM (2% FBS) containing the virus inoculum (MOI 1), in the presence of either increasing concentrations of compounds or the equivalent volume of EtOH. Following 2 h adsorption at 37°C, the virus inoculum was removed, after which the cells were washed twice and fresh DMEM (10% FBS) containing either the compounds or EtOH was added. At 8 hpi, cell..
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