Enisamium Reduces Influenza Virus Shedding and Improves Patient Recovery by Inhibiting Viral RNA Polymerase Activity

Abstract

Infections with respiratory viruses constitute a huge burden on our health and economy. Antivirals against some respiratory viruses are available, but further options are urgently needed. Enisamium iodide (laboratory code FAV00A, trade name Amizon) is an antiviral, marketed in countries of the Commonwealth of Independent States for the treatment of viral respiratory infections, but its clinical efficacy and mode of action are not well understood. In this study, we investigated the efficacy of enisamium in patients aged between 18 and 60 years with confirmed influenza virus and other viral respiratory infections. Enisamium treatment resulted in reduced influenza virus shedding (at day 3, 71.2% in the enisamium group tested negative versus 25.0% in placebo group [P < 0.0001]), faster patient recovery (at day 14, 93.9% in the enisamium group had recovered versus 32.5% in placebo group [P < 0.0001]), and reduced disease symptoms (from 9.6 ± 0.7 to 4.6 ± 0.9 score points in enisamium group versus 9.7 ± 1.1 to 5.6 ± 1.1 score points in placebo group [P < 0.0001]) compared to those in the placebo group. Using mass spectrometry, and cell-based and cell-free viral RNA synthesis assays, we identified a hydroxylated metabolite of enisamium, VR17-04. VR17-04 is capable of inhibiting influenza virus RNA synthesis and is present in plasma of patients treated with enisamium. VR17-04 inhibits the activity of the influenza virus RNA polymerase more potently than its parent compound. Overall, these results suggest that enisamium is metabolized in humans to an inhibitor of the influenza virus RNA polymerase that reduces viral shedding and improves patient recovery in influenza patients. (This study has been registered at ClinicalTrials.gov under identifier NCT04682444.).

Keywords: FAV00A; RNA polymerase; RNA synthesis; adenovirus; adenoviruses; enisamium iodide; influenza A and B; influenza A and B viruses; respiratory syncytial virus; respiratory viruses; treatment.

Copyright © 2021 te Velthuis et al.

Figures

FIG 1

Patient enrollment, demographics, and antigen status. (A) Schematic of patient recruitment, randomization, and treatment. (B) Demographics of enisamium- and placebo-treated patient groups. (C) Frequency of virus antigen detection in nasal swabs of patients treated with enisamium or placebo by immunofluorescence staining.

FIG 2

Enisamium treatment reduces viral antigen levels and improves patient activity. (A) Patients in whom virus antigens were not detected by immunofluorescence staining of nasal swabs. (B) Patients without routine activities. P values were determined by Fisher’s exact test.

FIG 3

Enisamium treatment reduces objective symptoms in patients with viral respiratory disease. (A) Patients without objective symptoms at different visit days. (B) Patients without abnormal breath sounds at different visit days. (C) Patients without pharyngeal hyperemia at different visit days. (D) Patients without fever at different visit days. P values were determined by Fisher’s exact test.

FIG 4

Enisamium treatment reduces subjective symptoms in patients with viral respiratory disease. (A) Patients without subjective symptoms at different visit days. (B) Patients without headache at different visit days. (C) Patients without weakness at different visit days. (D) Patients without sore throat at different visit days. (E) Patients without cough at different visit days. (F) Patients without elevated body temperature at different visit days. P values were determined by Fisher’s exact test.

FIG 5

Inhibition of IAV infection and RNA synthesis by enisamium in cell culture. (A) Effect of enisamium on influenza A/WSN/1933 (H1N1) virus titers in A549 cells as determined by plaque assay (black line). Cytotoxicity (blue line) was determined in uninfected cells after 48 h of incubation with FAV00A. (B to D) Effect of enisamium on influenza A/WSN/1933 (H1N1) virus titers in RD cells (B), Caco-2 cells (C), and HepG2 cells (D) as determined by microplaque assay (black line). Cytotoxicity (percentage of live cells, blue line) was determined in uninfected cells after 48 h of incubation with enisamium. Data points represent means and standard deviations of three independent enisamium titrations and matching virus plaque experiments. (E) Overview of IC50, IC90, 50% cytotoxic concentration (CC50), and selectivity index (SI) values in different cell lines.

FIG 6

Enisamium is metabolized in humans and metabolite VR17-04 inhibits the viral RNA polymerase in vitro. (A) Effect of enisamium on the steady-state IAV vRNA, 5S rRNA, and GFP levels, with quantification shown in the graph. Levels of 5S rRNA and IAV vRNA were analyzed by primer extension (middle panel). PA and tubulin expression were analyzed by Western blotting (bottom panel). A mutant IAV RNA polymerase containing two amino acid substitutions in the PB1 active site (PB1a) was used as a negative control. Data points represent means and standard deviations of three independent enisamium titrations and matching GFP measurements or primer extensions. M, marker. (B) Effect of extracts from A549 cells treated with enisamium on IAV RNA polymerase activity in vitro. Five hundred or 2,500 μg of enisamium was added to A549 cells for 24 h. Next, cells were lysed and 1/10 of the lysate was added to in vitro polymerase assays. RNA polymerase products were analyzed by 20% denaturing PAGE. (C) Quantification of the activity of the IAV RNA polymerase in vitro in the presence of enisamium, VR17-04, or T-705 triphosphate (T-705-TP). Data points represent means and standard deviations of three independent titrations in RNA polymerase assays. (D) Phase I metabolites identified in human plasma samples. (E) Activity of the IAV RNA polymerase in the presence of different VR17-04 concentrations.