Background The antifungal drug itraconazole exerts in vitro activity against SARS-CoV-2 in Vero and human Caco-2 cells. Preclinical and clinical studies are required to investigate if itraconazole is effective for the treatment and/or prevention of COVID-19. Methods Due to the initial absence of preclinical models, the effect of itraconazole was explored in a clinical, proof-of-concept, open-label, single-center study, in which hospitalized COVID-19 patients were randomly assigned to standard of care with or without itraconazole. Primary outcome was the cumulative score of the clinical status until day 15 based on the 7-point ordinal scale of the World Health Organization. In parallel, itraconazole was evaluated in a newly established hamster model of acute SARS-CoV-2 infection and transmission, as soon as the model was validated. Findings In the hamster acute infection model, itraconazole did not reduce viral load in lungs, stools or ileum, despite adequate plasma and lung drug concentrations. In the transmission model, itraconazole failed to prevent viral transmission. The clinical trial was prematurely discontinued after evaluation of the preclinical studies and because an interim analysis showed no signal for a more favorable outcome with itraconazole: mean cumulative score of the clinical status 49 vs 47, ratio of geometric means 1.01 (95% CI 0.85 to 1.19) for itraconazole vs standard of care. Interpretation Despite in vitro activity, itraconazole was not effective in a preclinical COVID-19 hamster model. This prompted the premature termination of the proof-of-concept clinical study. Funding: KU Leuven, Research Foundation - Flanders (FWO), Horizon 2020, Bill and Melinda Gates Foundation
Figure 1In vitro antiviral activity. (a) Antiviral activity of itraconazole and hydroxy-itraconazole at different concentrations in the SARS-CoV-2 / VeroE6-eGFP assay system. Green fluorescence indicates cells surviving SARS-CoV-2 infection. Itraconazole was tested in two independent experiments while hydroxy-itraconazole (17-OH-itraconazole) was tested in one experiment. (b) Antiviral activity of azithromycine, hydroxychloroquine and GS-441524 in two independent experiments. (c) Antiviral activity of itraconazole and other azoles different concentrations in the SARS-CoV-2 / VeroE6-eGFP assay system. VC indicates “Virus Control” i.e. infected untreated cultures; CC indicates “Cell Control” i.e. uninfected untreated cultures; GS-441524 is the parent nucleoside of remdesivir.
2.3 Clinical study
2.1.1 Study design
The Direct Antivirals Working against new Corona virus (DAWn)-Itraconazole study was an open-label, randomized, single-center pilot trial in hospitalized COVID-19 patients. The trial was conducted at the University Hospitals Leuven, Belgium, between March and June 2020. The study compared standard of care with or without itraconazole in a 1:1 randomization. Standard of care was based on guidelines of national and international organizations and outlined in an institutional policy.
The study was conducted in accordance with the International Conference on Harmonization Guidelines for Good Clinical Practice and the Declaration of Helsinki. The protocol was approved by the institutional Ethics Committee and by the Belgian Federal Agency for Medicines and Health Products (EudraCT 2020-001243-15). The trial was part of the DAWn clinical studies [11 ]. An independent Data Safety Monitoring Board (DSMB) reviewed trial safety outcomes. The full protocol and statistical analysis plan are available in Supplement.
Hospitalized patients aged 18 years or older with COVID-19, confirmed by PCR or typical chest CT-scan, were eligible if they displayed at least one of the following features: radiographic infiltrates, Sp02 ≤ 94% on room air, or requiring supplemental oxygen. Subjects were excluded in case of elevated liver tests (ALT/AST > 5 times the upper limit of normal), pregnancy or breast feeding, heart failure with severely reduced ejection fraction (≤ 30%), or concomitant treatment with lopinavir/ritonavir or potent CYP450 inducers.
Because of safety concerns due to the risk of transmission of SARS-CoV-2, verbal informed consent in the presence of an independent witness was obtained during hospitalization of all patients. Written informed consent was obtained after discharge from quarantine.
The primary outcome was defined as cumulative clinical status on day 15. This endpoint consists of the sum of daily clinical status scores on the 7-point WHO ordinal scale from day 1 to 15 included. The WHO ordinal scale consists of the following 7 categories: 1) not hospitalized, no limitations on activities; 2) not hospitalized, limitations on activities; 3) hospitalized, not requiring supplemental oxygen; 4) hospitalized, requiring supplemental oxygen; 5) hospitalized, on non-invasive ventilation or high flow oxygen devices; 6) hospitalized, requiring extracorporeal membrane oxygenation (ECMO) or invasive mechanical ventilation; and 7) death.
The secondary outcome was defined as time to sustained clinical improvement or live discharge, whichever comes first, whereby a sustained clinical improvement is defined as an improvement of more than 2 points on the 7-point ordinal scale versus the highest value of day 0 and 1 and sustained for at least 3 days. Other secondary outcomes included: time to events (admission to ICU, death, discharge); mortality on day 29, duration of supplemental oxygen, need for and duration of mechanical ventilation, duration of hospitalization, duration of intensive care stay, daily National Early Warning Score (NEWS). Safety outcomes included adverse events (AE) graded as grade 4 or 5 or serious adverse events (SAE) and ECG monitoring. Quantitative PCR for SARS-CoV-2 in (nasopharyngeal) swab on day 1 and day 6 were exploratory outcomes.
For pharmacokinetic evolution, itraconazole and its main metabolite, hydroxy-itraconazole, trough concentrations were measured at different time points with liquid chromatography tandem mass spectrometry (Waters Acquity TDQ system with Recipe ClinMass® antimycotics kit). The trough concentrations measured before and after 72 hours after initiation of treatment were defined as early and late exposure, respectively.
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