Characterization of orally efficacious influenza drug with high resistance barrier in ferrets and human airway epithelia
Science Translational Medicine • 23 Oct 2019 • Vol 11, Issue 515 • DOI: 10.1126/scitranslmed.aax5866
Treating flu, escaping resistance
Influenza viruses can cause severe illness and death. Although vaccines are continuously developed, their efficacy is moderate, particularly in the elderly. Current therapies based on neuraminidase inhibitors are only partially effective and subjected to resistance. Now, Toots et al. developed a drug called EIDD-2801 with anti-influenza virus activity. The authors demonstrated that EIDD-2801 had therapeutic effects in infected ferrets, reducing symptoms and virus load. The treatment was effective against multiple influenza strains in human airway epithelial cultures, and the viruses did not develop pharmacoresistance. The results suggest that EIDD-2801 is a promising candidate for treating seasonal and pandemic influenza.
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.
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Supplementary Material
Summary
Fig. S1. PK of NHC and EIDD-2801 in mice.
Fig. S2. Single-dose PK of EIDD-2801 in ferrets.
Fig. S3. Multidose PK of EIDD-2801 in ferrets.
Fig. S4. Ferret efficacy study timeline.
Fig. S5. Histopathology scores of Ca09-infected ferret lungs.
Fig. S6. Escalating-dose adaptation of IAV to NHC.
Fig. S7. Fixed-dose serial passaging of IAV in the presence of NHC.
Fig. S8. Genetic changes in IAV-WSN RNA during fixed-dose passaging.
Fig. S9. Immunofluorescence of influenza-infected 3D airway epithelium cultures.
Fig. S10. Therapeutic efficacy of NHC in the 3D airway epithelium culture.
Fig. S11. Cytotoxicity of NHC in the 3D airway epithelium culture.
Fig. S12. NHC effect on nuclear and mitochondrial gene expressions.
Fig. S13. Recapitulation of NHC PK profiles in 3D human airway epithelium culture.
Fig. S14. Immunohistochemistry of nasal turbinates extracted from vehicle- and EIDD-2801–treated animals.
Fig. S15. Immunohistochemistry of lungs extracted from vehicle- and EIDD-2801–treated animals.
Fig. S16. Immunofluorescence of 3D airway epithelium cultures.
Fig. S17. Immunofluorescence of 3D airway epithelium cultures after NHC exposure.
Table S1. PK parameters for NHC in cynomolgus macaques.
Table S2. Single-dose PK parameters for NHC in ferrets.
Table S3. Lung concentrations of NHC and NHC-TP.
Table S4. Multidose PK parameters for NHC in ferrets.
Table S5. Antibodies used in this study.
Table S6. Primers used in this study.
Data file S1. Amino acid changes during baloxavir adaptation.
Data file S2. Summary of amino acid changes during baloxavir adaptation.
Data file S3. Amino acid changes during NHC adaptation.
Data file S4. Summary of amino acid changes during NHC adaptation.
Data file S5. Primary data.
Resources
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File (aax5866_data_file_s3.zip)
File (aax5866_data_file_s4.csv)
File (aax5866_data_file_s5.xlsx)
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Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
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History
Received: 4 April 2019
Revision received: 19 June 2019
Accepted: 20 September 2019
Acknowledgments
We thank V. von Messling for lending the telemetric ferret body temperature measurement system and sharing ferret quantitative PCR primer sequences; V. Edpuganti, L. Moellering, and J. A. Marlow for assistance with the pharmacological analyses; M. T. Saindaine and M. A. Lockwood for chemical syntheses; K. Ganti and A. C. Lowen for counseling on immunohistochemistry procedures; and A. L. Hammond for the critical reading of the manuscript. Funding: This work was supported, in part, by contracts HDTRA1-15-C-0075 (to G.R.P.) and HHSN272201500008C (to G.R.P.) from the DTRA and the NIH/NIAID, respectively; by Public Health Service grants AI071002 (to R.K.P.), AI119196 (to R.K.P.), and HD079327 (to R.K.P.) from the NIH/NIAID and NIH/NICHD, respectively; and by a pilot grant from the Georgia Research Alliance (to G.R.P. and R.K.P.). The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication. Author contributions: Conceptualization: M.T., G.R.P., and R.K.P. Data curation: M.T., A.A.K., A.L.G., M.G.N., and R.K.P. Formal analysis: M.T., R.P., A.A.K., A.L.G., and R.K.P. Funding acquisition: G.R.P. and R.K.P. Chemical discovery: G.R.B., M.G.N., and G.R.P. Investigation: M.T., J.-J.Y., R.M.C., M.H., Z.M.S., N.M., A.H.B., P.G.R., D.G.M., R.C.S., A.L.G., and R.K.P. Project administration: M.G.N. and R.K.P. Resources: A.L.G., G.R.B., M.G.N., and R.K.P. Supervision: A.L.G., G.R.P., and R.K.P. Validation: A.A.K., A.L.G., M.G.N., and R.K.P. Visualization: M.T., A.L.G., and R.K.P. Preparation of original draft: M.T. and R.K.P. Review and editing: R.P., A.A.K., A.L.G., M.G.N., and G.R.P. Competing interests: G.R.B., M.G.N., and G.R.P. hold patent 20190022116, “N4-hydroxycytidine and derivatives and anti-viral uses related thereto,” covering composition of matter and method of use of EIDD-2801 for influenza therapy. This study could affect their personal financial status. All the other authors declare that they have no competing interests. Data and materials availability: The distribution of influenza virus strains and noncommercial chemical material described in this study for research use is regulated by material transfer agreements from Georgia State University and Emory University. All data from next-generation sequencing reads are deposited and accessible in the NCBI SRA under BioProject PRJNA528811. All other data associated with this study are present in the paper or the Supplementary Materials.
Authors
Funding Information
Defense Threat Reduction Agency: HDTRA1-15-C-0075
Defense Threat Reduction Agency: HHSN272201500008C
RE: Characterization of orally efficacious influenza drug with high resistance barrier in ferrets and human airway epithelia
To the Editor,
I read with very keen interest the article by Toots et al, (1) and titled "Characterization of orally efficacious influenza drug with high resistance barrier in ferrets and human airway epithelia".
Influenza virus infections remain of global public health concern. The current development, distribution, availability and delivery of seasonal vaccines and therapeutics are all critical measures to limit the impact of Influenza virus outbreaks.
Despite significant work and attention, current Influenza vaccination strategies are typically comprised of seasonal influenza virus vaccines inducing immunity against a lone subtype of Influenza virus where annual re-design of these commonly available seasonal Influenza vaccines are required to manage and adapt accordingly to the ongoing viral mutations that occur.
In the current context of Influenza infections, we still await the development of very much needed "Universal" Influenza vaccine and efficacious therapeutics.
Influenza therapeutics involving Neuraminidase inhibitors have demonstrated limited effectiveness and are also prone to resistance.
Toots et al, (1) showed that the EIDD-2801 drug was able to generate anti-influenza viral activity in cultured cells and mice. EIDD-2801, is an isopropylester prodrug of the ribonucleoside analog N4-hydroxycytidine (NHC, EIDD-1931), demonstrating orally bioavailable in ferrets and nonhuman primates.
The underlying mechanism of activity that was reported was lethal viral mutagenesis, with the development of viral resistance conditions prevented, promoting the avoiding pharmacoresistance.
These findings together with the associated pharmacokinetic characteristics described, highlight EIDD-2801 as a candidate for further development within the context of seasonal and pandemic Influenza virus conditions.
A timely set of results with immediate clinical relevance.
Respectfully yours,
Dr Dianne Sika-Paotonu CQS MRSNZ
Associate Dean (Pacific)
Senior Lecturer, Pathology & Molecular Medicine
Head, University of Otago Wellington Pacific Office
Wellington School of Medicine and Health Sciences, University of Otago, New Zealand
Honorary Research Associate, Telethon Kids Institute, Australia
Honorary Research Associate, Victoria University of Wellington, New Zealand
Associate Investigator Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, New Zealand
References:
1. Toots, M., Yoon, J. J., Cox, R. M., Hart, M., Sticher, Z. M., Makhsous, N., ... & Shean, R. C. (2019). Characterization of orally efficacious influenza drug with high resistance barrier in ferrets and human airway epithelia. Science translational medicine, 11(515).