In vitro activity of itraconazole against SARS-CoV-2

Abstract

Although vaccination campaigns are currently being rolled out to prevent coronavirus disease (COVID-19), antivirals will remain an important adjunct to vaccination. Antivirals against coronaviruses do not exist, hence global drug repurposing efforts have been carried out to identify agents that may provide clinical benefit to patients with COVID-19. Itraconazole, an antifungal agent, has been reported to have activity against animal coronaviruses. Using cell-based phenotypic assays, the in vitro antiviral activity of itraconazole and 17-OH itraconazole was assessed against clinical isolates from a German and Belgian patient infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Itraconazole demonstrated antiviral activity in human Caco-2 cells (EC₅₀ = 2.3 µM; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay). Similarly, its primary metabolite, 17-OH itraconazole, showed inhibition of SARS-CoV-2 activity (EC₅₀ = 3.6 µM). Remdesivir inhibited viral replication with an EC₅₀ = 0.4 µM. 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. The viral yield reduction brought about by remdesivir or GS-441524 (parent nucleoside of the antiviral prodrug remdesivir; positive control) was more pronounced, with an approximately 3-log₁₀ drop and >4-log₁₀ drop in Caco-2 cells and VeroE6-eGFP cells, respectively. Itraconazole and 17-OH itraconazole exert in vitro low micromolar activity against SARS-CoV-2. Despite the in vitro antiviral activity, itraconazole did not result in a beneficial effect in hospitalized COVID-19 patients in a clinical study (EudraCT Number: 2020-001243-15).

Keywords: 17-OH itraconazole; Caco-2 cells; SARS-CoV-2; VeroE6-eGFP cells; in vitro; itraconazole.

Figure 1

Effect of either (A) itraconazole, (B) 17‐OH itraconazole, or (C) remdesivir on SARS‐CoV‐2 replication in CPE assays and on viability of Caco‐2 cells. Mean percent inhibition for each readout across two series of three independent experiments (itraconazole, remdesivir) or three independent experiments (17‐OH itraconazole) with triplicate measurements are plotted. The error bars represent the standard deviation. Orange represents CPE visual read‐out; purple represents MTT assay; and green represents cytotoxicity. (A) EC₅₀ by visual scoring of inhibition of CPE = 1.5 μM; EC₅₀ by MTT assay = 2.3 μM (B) EC₅₀ by visual scoring of inhibition of CPE = 1.2 μM; EC₅₀ by MTT assay = 3.6 μM (C) EC₅₀ by visual scoring of inhibition of CPE = 0.3 μM; EC₅₀ by MTT assay = 0.4 μM. CPE, cytopathogenic effect; EC₅₀, concentration of the compound that inhibited 50% of the infection; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)−2,5‐diphenyltetrazolium bromide; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2

Figure 2

Effect of itraconazole, 17‐OH itraconazole or remdesivir on SARS‐CoV‐2 vRNA yield and viability in Caco‐2 cells. (A) Mean differences in vRNA in the supernatant between untreated cultures and treated cultures at 48 h postinfection with SARS‐CoV‐2‐FFM1 of three independent experiments each containing two replicates is shown. Error bars represent the standard deviation. (B) Mean viability of the cells, based on three independent experiments each containing three replicates is shown. Error bars represent the standard deviation. SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2; vRNA, viral RNA

Figure 3

Effect of itraconazole, 17‐OH itraconazole, or GS‐441524 on SARS‐CoV‐2 vRNA yield and viability in VeroE6‐eGFP cells (A) and (B) Mean differences in vRNA in the supernatant between untreated cultures and treated cultures at 48 h postinfection with SARS‐CoV‐2‐Belgium of one independent experiment containing two replicates (A) or three replicates (B) is shown. Error bars represent the standard deviation. (C, D) Mean viability of the cells, based on MTT readout of uninfected cells. Error bars represent the standard deviation. SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2; vRNA, viral RNA