Characterization of orally efficacious influenza drug with high resistance barrier in ferrets and human airway epithelia

Abstract

Influenza viruses constitute a major health threat and economic burden globally, frequently exacerbated by preexisting or rapidly emerging resistance to antiviral therapeutics. To address the unmet need of improved influenza therapy, we have created EIDD-2801, an isopropylester prodrug of the ribonucleoside analog N4-hydroxycytidine (NHC, EIDD-1931) that has shown broad anti-influenza virus activity in cultured cells and mice. Pharmacokinetic profiling demonstrated that EIDD-2801 was orally bioavailable in ferrets and nonhuman primates. Therapeutic oral dosing of influenza virus–infected ferrets reduced group pandemic 1 and group 2 seasonal influenza A shed virus load by multiple orders of magnitude and alleviated fever, airway epithelium histopathology, and inflammation, whereas postexposure prophylactic dosing was sterilizing. Deep sequencing highlighted lethal viral mutagenesis as the underlying mechanism of activity and revealed a prohibitive barrier to the development of viral resistance. Inhibitory concentrations were low nanomolar against influenza A and B viruses in disease-relevant well-differentiated human air-liquid interface airway epithelia. Correlating antiviral efficacy and cytotoxicity thresholds with pharmacokinetic profiles in human airway epithelium models revealed a therapeutic window >1713 and established dosing parameters required for efficacious human therapy. These data recommend EIDD-2801 as a clinical candidate with high potential for monotherapy of seasonal and pandemic influenza virus infections. Our results inform EIDD-2801 clinical trial design and drug exposure targets.

Analysis of the Potential for N 4 -Hydroxycytidine To Inhibit Mitochondrial Replication and Function

“…at 200 mg/kg/day, despite the fact that plasma levels of NHC were expected to be above 10 M at any time throughout the treatment course (26). It has been shown that NHC was efficiently anabolized to the triphosphate form in primary hepatocytes and in animal organs in vivo (16,26,31). The decrease in quantity of mitochondrial protein or the potential decrease of functional activity of mitochondrial proteins may result in less production of ATP by the mitochondria even if the mitochondrial number has not changed.…”

“…While NHC-5=-triphosphate does not inhibit DNA-polymerases ␣, ␤, and ␥ at 1,000 M (Table S3), incorporation into cellular RNA could be a cause of observed cytotoxicity. Incorporation of NHC residues into Pol II RNA could be suggested from an elevated level of mutations found in Pol II-transcribed mRNA in the presence of NHC (26). Cellular RNA Pol II possesses a safeguard activity from misincorporation and is able to excise misincorporated nucleotides (27).…”

“…In fact, mutations in nuclear and mitochondrial mRNA were observed in tissue culture cells that were treated with NHC at 10 M for 3 days; however, there was no increase in the frequency of mitochondrial or nuclear mRNA mutations in ferrets that were given seven doses of NHC twice daily (b.i.d.) at 200 mg/kg/day, despite the fact that plasma levels of NHC were expected to be above 10 M at any time throughout the treatment course (26). It has been shown that NHC was efficiently anabolized to the triphosphate form in primary hepatocytes and in animal organs in vivo (16,26,31).…”

The epidemiology and pathogenesis of coronavirus disease (COVID-19) outbreak

“…Furthermore, there are a number of other compounds that are in development. These include the clinical candidate EIDD-2801 compound that has shown high therapeutic potential aganist seasonal and pandemic influenza virus infections and this represents another potential drug to be considered for the treatment of COVID-19 infection [28]. Along those lines, until more specific therapeutics become available, it is reasonable to consider more broad-spectrum antivirals that provide drug treatment options for COVID-19 infection include Lopinavir/Ritonavir, Neuraminidase inhibitors, peptide (EK1), RNA synthesis inhibitors.…”

Next-generation direct-acting influenza therapeutics

“…However, the transition mutation frequency in ferret lung tissue was unchanged from that in controls after extended treatment of animals with 200 mg/kg daily dose and thus over 14-times the efficacious dose for influenza therapy. 106 Potential adverse effects of EIDD-2801 on embryo development are untested at present, and formal 2-species multi-dose toxicity studies will be required to establish initial human doses examined in phase 1 clinical trials. In the absence of drug, random mutation frequency in the virus population is low (green).…”