Pre-clinical evaluation of antiviral activity of nitazoxanide against SARS-CoV-2

Abstract

Background: To address the emergence of SARS-CoV-2, multiple clinical trials in humans were rapidly started, including those involving an oral treatment by nitazoxanide, despite no or limited pre-clinical evidence of antiviral efficacy.

Methods: In this work, we present a complete pre-clinical evaluation of the antiviral activity of nitazoxanide against SARS-CoV-2.

Findings: First, we confirmed the in vitro efficacy of nitazoxanide and tizoxanide (its active metabolite) against SARS-CoV-2. Then, we demonstrated nitazoxanide activity in a reconstructed bronchial human airway epithelium model. In a SARS-CoV-2 virus challenge model in hamsters, oral and intranasal treatment with nitazoxanide failed to impair viral replication in commonly affected organs. We hypothesized that this could be due to insufficient diffusion of the drug into organs of interest. Indeed, our pharmacokinetic study confirmed that concentrations of tizoxanide in organs of interest were always below the in vitro EC₅₀.

Interpretation: These preclinical results suggest, if directly applicable to humans, that the standard formulation and dosage of nitazoxanide is not effective in providing antiviral therapy for Covid-19.

Funding: This work was supported by the Fondation de France "call FLASH COVID-19", project TAMAC, by "Institut national de la santé et de la recherche médicale" through the REACTing (REsearch and ACTion targeting emerging infectious diseases), by REACTING/ANRS MIE under the agreement No. 21180 ('Activité des molécules antivirales dans le modèle hamster'), by European Virus Archive Global (EVA 213 GLOBAL) funded by the European Union's Horizon 2020 research and innovation program under grant agreement No. 871029 and DNDi under support by the Wellcome Trust Grant ref: 222489/Z/21/Z through the COVID-19 Therapeutics Accelerator".

Keywords: Animal model; Antiviral therapy; COVID-19; Nitazoxanide; Pre-clinical research; SARS-CoV-2.

Figure 1

Antiviral activity of NTZ and TIZ in Vero E6 and Caco-2 cells. Dose response curve and cell viability for: NTZ in Vero E6 (a) and Caco-2 (b) cells and for TIZ in Vero E6 cells (c). D: Table of EC₅₀, EC₉₀, CC₅₀. Results presented in the table for NTZ in Vero E6 are the mean ± SD from three independent experiments. Graphical representation is from one representative experiment.

Figure 2

Antiviral activity of NTZ in a bronchial human airway epithelium. Kinetics of virus excretion at the apical side of the epithelium measured using an RT-qPCR assay (A) and a TCID₅₀ assay (B). Data represent mean ± SD. Statistical significance was calculated by Kruskal-Wallis test versus untreated group for (A) and 1-way ANOVA versus untreated group for (B). Remdesivir at 10µM was used as a positive drug control. *, **, *** and **** indicate and average significant value lower than that of the untreated group, with a p-value ranging between 0.01-0.05, 0.001-0.01, 0.0001-0.001 and <0.0001, respectively. Result are the mean ± SD of two independent experiment with in each experiment two independent inserts (Details in Supplementary Data 1).

Figure 3

Antiviral activity of oral treatment of NTZ in a hamster model. Groups of 6 hamsters were intranasally infected with 10⁴ TCID₅₀ of virus. a Experimental timeline. b, f, j Viral replication in lung based on infectious titers (measured using a TCID₅₀ assay) expressed in TCID₅₀/g of lung (n=6 animals/group). c, g, k Viral replication in lung based on viral RNA yields (measured using an RT-qPCR assay) expressed in viral genome copies/g of lung (n=6 animals/group). d, h, l Plasma viral loads (measured using an RT-qPCR assay) are expressed in viral genome copies/mL of plasma (the dotted line indicates the detection threshold of the assay) (n=6 animals/group). e, i, m Clinical course of the disease (n=6 animals/group). Normalized weight at day n was calculated as follows: % of initial weight of the animal at day n. Data represent mean ± SD (Details in Supplementary Data 2). Two-sided statistical analysis were performed using Shapiro–Wilk normality test, Fisher's exact test, Student t-test, Mann–Whitney test and two-way ANOVA with Post-hoc Dunnett's multiple comparisons test. *** and ** symbols indicate that the average value for the group is significantly lower than that of the untreated group with a p-value ranging between 0.0001-0.001 and 0.001-0.01 respectively (Details in Supplementary Data 2 and 3).

Figure 4

Lung histopathological changes. Groups of 4 animals were intranasally infected with 10⁴ TCID₅₀ of virus and sacrificed at 5 dpi. Based on severity of inflammation, alveolar hemorrhagic necrosis and vessel lesions, a cumulative score from 0 to 10 was calculated and assigned to a grade of severity (I, II, III and IV). a Experimental timeline. b Scoring of pathological changes (Details in Supplementary Data 4). Two-sided statistical analysis was performed using Shapiro–Wilk normality test, Fisher's exact test, and Student t-test. c Representative images of bronchial inflammation (scale bar: 100µ): severe peribronchiolar inflammation and bronchiole filled with numerous neutrophilic, marked peribronchiolar inflammation and normal bronchi. d Representative images of alveolar inflammation (scale bar: 100µ): severe infiltration of alveolar walls, alveoli filled with neutrophils/macrophages, marked infiltration of alveolar walls, some alveoli filled with neutrophils/macrophages and normal alveoli. e Representative images of vessel inflammation (scale bar: 100µ): moderate accumulation of inflammatory cells in arteriolar walls and normal arteriole.

Figure 5

Antiviral activity of intranasal treatment of NTZ in a hamster model. Groups of 6 hamsters were intranasally infected with 10⁴ TCID₅₀ of virus. a Experimental timeline. b Viral replication in lung based on infectious titers (measured using a TCID₅₀ assay) expressed in TCID₅₀/g of lung (n=6 animals/group). c Viral replication in lung based on viral RNA yields (measured using an RT-qPCR assay) expressed in viral genome copies/g of lung (n=6 animals/group). d Plasma viral loads (measured using an RT-qPCR assay) are expressed in viral genome copies/mL of plasma (the dotted line indicates the detection threshold of the assay) (n=6 animals/group). e Viral replication in nasal turbinates based on infectious titers (measured using a TCID₅₀ assay) expressed in TCID₅₀/copy of ɣ-actine gene (n=6 animals/group). f Viral replication in nasal turbinates based on viral RNA yields (measured using an RT-qPCR assay) expressed in viral genome copies/copy of ɣ-actine gene (n=6 animals/group). g Clinical course of the disease (n=6 animals/group). Normalized weight at day n was calculated as follows: % of initial weight of the animal at day n. Data represent mean ± SD (Details in Supplementary Data 2). Two-sided statistical analysis were performed using Shapiro–Wilk normality test, Fisher's exact test, Student t-test and two-way ANOVA with Post-hoc Dunnett's multiple comparisons test. ** symbols indicate that the average value for the group is significantly lower than that of the untreated group with a p-value ranging between 0.001-0.01 (Details in Supplementary Data 2 and 3).

Figure 6

Simulated pharmacokinetic parameters of TIZ in human and hamster at steady state. Predicted steady-state pharmacokinetic parameters of TIZ, i.e. C_max (A), AUC (B) and C_min (C), in human associated with receiving 1000mg/day BID of NTZ (grey box) were compared with pharmacokinetic parameters of hamster receiving 50, 100, 200, 500 and 1000mg/kg/day BID of NTZ (colored boxes). Boxes and whiskers represent the median with inter-quantile range and the 95% prediction intervals, respectively.