Synergistic Effect of Nitazoxanide with Neuraminidase Inhibitors against Influenza A Viruses In Vitro

The emergence of drug-resistant influenza A virus (IAV) strains represents a serious threat to global human health and underscores the need for novel approaches to anti-influenza chemotherapy. Combination therapy with drugs affecting different IAV targets represents an attractive option for influenza treatment. We have previously shown that the thiazolide anti-infective nitazoxanide (NTZ) inhibits H1N1-IAV replication, by selectively blocking viral hemagglutinin maturation. Herein we investigated the anti-influenza activity of NTZ against a wide range of human and avian IAVs (H1N1, H3N2, H5N9, H7N1), including amantadine-resistant and oseltamivir-resistant strains, in vitro. We also investigated whether combination therapy of NTZ with neuraminidase inhibitors oseltamivir and zanamivir exerts synergistic, additive or antagonistic antiviral effects against influenza viruses. NTZ was effective against all IAV tested with IC50 ranging from 0.9 to 3.2 μM, and SI ranging from >50 to >160, depending on the strain and the multiplicity of infection (m.o.i.). Combination therapy studies were performed in cell culture-based assays using A/PuertoRico/8/1934(H1N1), A/WSN/1933(H1N1) or avian A/Chicken/Italy/9097/1997(H5N9) IAVs; dose-effect analysis and synergism/antagonism quantification were performed using isobologram analysis according to the Chou-Talalay method. Combination indices (CI) analysis indicated that combined NTZ/oseltamivir treatment was synergistic against A/PuertoRico/8/1934(H1N1) and A/WSN/1933(H1N1) IAVs with CI values ranging between 0.39 and 0.63, independently of the m.o.i. used. Similar results were obtained when NTZ was administered in combination with zanamivir (CI=0.3-0.48). Combined NTZ/oseltamivir treatment was synergistic also against the avian A/Chicken/Italy/9097/1997(H5N9) IAV (CI=0.18-0.31). Taken together, the results suggest that regimens that combine neuraminidase inhibitors and nitazoxanide exert synergistic anti-influenza effects.