Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) – the causative pathogen of coronavirus disease 2019 (COVID-19) – continues to spread across many parts of the world. The COVID-19 pandemic is one of the largest public health problems in recent memory. Over 54.7 million have been infected with SARS-CoV-2, and over 1.32 million have died. As of now, no effective and safe specific antiviral therapies are available against this infection. While the hunt for a vaccine that immunizes against SARS-CoV-2 is ongoing, researchers around the world are also on the lookout for repurposing other antimicrobial agents to see their potential against this novel virus in therapeutic treatments.
Itraconazole is a broad-spectrum antifungal agent that has been shown to have some in vitro activity against SARS-CoV-2 by a team of Belgian and German researchers. When tried on patients with COVID-19, it was however found to be ineffective. The study, titled "In Vitro Activity of Itraconazole Against SARS-CoV-2," has been released on bioRxiv* the preprint server, while the article undergoes peer-review.
At present, no effective and safe medicines or vaccines are available against SARS CoV-2. Global repurposing of drugs is underway, wrote the researchers. Identification of these agents could help clinical benefit to patients with COVID-19, they write. Itraconazole is an antifungal agent that has shown efficacy against animal coronaviruses. It is a member of the triazole group of broad-spectrum antifungals – acting against a wide range of fungi.
Itraconazole against coronaviruses
SARS-CoV-2 is a betacoronavirus with a single-strand, positive-sense RNA and an envelope. Itraconazole, among others, has been identified as a potential candidate. It has crossed an initial screening in an in vitro using a luciferase reporter-expressing recombinant murine betacoronavirus as well as against a feline alphacoronavirus that could lead to feline infectious peritonitis (FIP) in cats.
Itraconazole is also active against other respiratory viruses, such as influenza A and human rhinovirus, the team wrote. Against fungi, it acts by inhibiting cholesterol trafficking within the fungal cells and thus prevents replication. Similarly, it can act against viruses, the team wrote. They added that interferon priming could be an additional mechanism that allows its antiviral action.
For this study, the team studied the in vitro activity of 17-OH itraconazole in Caco-2 and VeroE6-eGFP cells infected with SARS-CoV-2. The virus samples were obtained from COVID-19 patients.
For this study, cell assays were used to check the in vitro antiviral activity of itraconazole as well as 17-OH itraconazole on clinical isolates from COVID-19 positive patients from Germany and Belgium.
Cell cultures used were:
- Caco-2 cells (human colon carcinoma cell line). They were cultured in "Minimal Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) with penicillin (100 IU/mL) and streptomycin (100 μg/mL) at 37°C in a 5% CO2 atmosphere."
- VeroE6-eGFP (African green monkey kidney cell line) "cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS, 0.075% sodium bicarbonate, penicillin and streptomycin (100 μg/mL) at 37°C in a 5% CO2 atmosphere."
Assessment of antiviral activity
Itraconazole and its metabolite, 17-OH Itraconazole, and remdesivir were checked for antiviral activity. The test for antiviral activity was inhibition of virus-induced cytopathogenic effect (CPE).
Viral RNA Yield Reduction:
- Antiviral activity was assessed by using viral yield in Caco-2 cells
- Itraconazole diluted in MEM was added to the cell culture in 4-fold dilutions ranging between 0.01 μM to 141 50 μM
- 17-OH itraconazole was diluted in MEM in 4-fold dilutions between 0.02 μM to 100 μM
- Remdesivir was diluted between 0.02 μM to 100 μM.
Overall, antiviral activity was seen with Itraconazole in human Caco-2 cells (Half maximal effective dose was 2.3 μM on MTT assay). 17-OH itraconazole – the primary metabolite of Itraconazole also showed inhibition of SARS-CoV-2 activity at a half-maximal dose of 3.6 μM. Positive control Remdesivir showed inhibition at a half-maximal dose of 0.4 μM.
Authors wrote, "Itraconazole and 17-OH itraconazole resulted in a viral yield reduction in vitro of approximately 2-log10 and approximately 1-log10, as measured in both Caco-2 cells and VeroE6-eGFP cells, respectively." Positive control remdesivir or GS-441524 showed an estimated three log10 drop and more than four log10 drop in Caco-2 cells and VeroE6-eGFP cells, respectively, they wrote.
Toxicity was minimal with Itraconazole. The authors wrote, "Minimal cytotoxicity was seen in Caco-2 cells with both itraconazole (CC50 >50 μM) and 17-OH itraconazole (CC50 >100 μM). For remdesivir CC50 values >100 μM were observed in Caco-2 cells."
Conclusions and implications
Authors write that both Itraconazole and 17-OH itraconazole show "in vitro low micromolar activity against SARS-CoV-2" but was weaker than remdesivir. They did not, however, find any beneficial effect of Itraconazole on hospitalized COVID-19 patients in a clinical study (EudraCT Number: 2020-001243-15). They concluded that more studies on non-hospitalized patients is needed to check for efficacy of Itraconazole against SARS-CoV-2.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
- Ellen Van Damme, Sandra De Meyer, Denisa Bojkova, Sandra Ciesek, Jindrich Cinatl, Steven De Jonghe, Dirk Jochmans, Pieter Leyssen, Christophe Buyck, Johan Neyts, Marnix Van Loock. In Vitro Activity of Itraconazole Against SARS-CoV-2. bioRxiv. doi: https://doi.org/10.1101/2020.11.13.381194, https://www.biorxiv.org/content/10.1101/2020.11.13.381194v1