Research ArticleInfluenza

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

See allHide authors and affiliations

Science Translational Medicine  23 Oct 2019:
Vol. 11, Issue 515, eaax5866
DOI: 10.1126/scitranslmed.aax5866
  • Fig. 1 Anti-influenza efficacy of EIDD-2801 in ferrets.

    (A) Plasma PK profiles of NHC and therapeutic candidate EIDD-2801 in cynomolgus macaques (N = 8) after 100 mg/kg oral dose. F indicates oral bioavailability. p.o., per os. (B) 2D structures of EIDD-2801 and its hydrolysis product, NHC. (C to I) Ferrets infected intranasally with Ca/09 (C to E and I; n = 3 to 6) or Wi/05 (F to H; n = 3 to 7) were treated orally with EIDD-2801 (100 or 20 mg/kg) twice daily for 3.5 days post-infection (pI). Shed (C and F) and nasal turbinate (E and H) virus titers are shown. (D and G) Body temperature was continuously monitored telemetrically. Lowes analysis of data obtained from all animals per group. Prophylactic treatment of Ca/09-infected animals with oseltamivir is shown for comparison. Symbols in (A) to (H) represent biological repeats, and graphs indicate medians ± SD. Two-way ANOVA (shed titers) or one-way ANOVA (nasal turbinate titers) with Dunnett’s post hoc test. Differences in resolve of fever were assessed through time-to-event Mantel-Cox test. LoD, limit of detection; MTFR, median time to fever resolve; N/A, not applicable. (I) Representative images of lung sections.

  • Fig. 2 Resistance profiling.

    (A) Virus titers after passages shown in fig. S7 (n = 3). (B) Adaptation of influenza virus to baloxavir marboxil and NHC. Adaptation profile and amino acid (AA) frequencies of resistance mutations after deep sequencing of baloxavir marboxil–experienced (n = 2) viruses (top). Deep sequencing to identify mutation frequency (5% cutoff) in influenza virus polymerase components after 10 passages at 1 or 2 μM NHC or vehicle (bottom panels). (C) C-to-U and G-to-A transition events after 5 and 10 passages at 1 or 2 μM NHC relative to vehicle. Symbols show biological repeats, columns are means, and error bars represent SD. Statistical significance was explored by two-way ANOVA and Dunnett’s post hoc test. (D) Repassaging of NHC-experienced virus populations from (A) at 4 μM NHC. Symbols represent biological repeats, and lines connect means. n = 4 for all NHC- and vehicle-experienced virus populations in (B) to (D). RLU, relative luciferase unit.

  • Fig. 3 Anti-IAV and IBV efficacy in well-differentiated human airway epithelium cultures.

    (A) Confocal microscopy 21 days after ALI induction, showing hallmarks of airway epithelia: tight junctions (anti–ZO-1), adherens junctions (anti–E-cadherin), goblet cells (anti-Muc5AC), and ciliated cells (anti–β-tubulin). Nuclei stained with DAPI. (B) TEER measurements throughout the 21-day differentiation at ALI. Symbols represent individual Transwells (n = 10), and line shows mean; two-way ANOVA with Dunnett’s post hoc test. (C) Ca/09- or B/Brisbane/60/2008-infected airway epithelia cultures treated basolaterally with 1.8 μM NHC or DMSO volume equivalents. Stained are viral antigens (Ca/09, anti-NS1; B/Brisbane/60/2008, anti-IBV), tight junctions, and nuclei. (D) NHC dose-response curves against IAV and IBV in 3D epithelia cultures. Oseltamivir tested against Ca/09 only (n = 3 to 6 per concentration point). Apically shed virus was harvested 3 days after infection. EC50 calculations through four-parameter variable-slope regression modeling. Symbols show biological repeats, and lines connect means. (E) TEER after 3-day exposure to 50 μM NHC or vehicle (n = 3). Lines connect means. (F) Confocal microscopy of samples from (E) showing tight junctions and nuclei. Enlarged immunofluorescence images in fig. S9 and figs. S16 and S17.

  • Fig. 4 Simulation of influenza therapy in well-differentiated human airway epithelium models.

    (A) Transitions in HBTEC nuclear (SDH-A) and mitochondrial (COX-1) mRNAs after 3-day NHC treatment; ≥5 clones and ≥ 5000 nucleotides (nt) per concentration examined. Fisher’s exact test was applied. ns, not significant. (B) Transitions in ferret lung nuclear (TNFα) and mitochondrial (COX-15) mRNAs after seven oral twice-daily (b.i.d.) EIDD-2801 doses at 100 mg/kg; 11 clones and ≥7000 nucleotides were examined. (C) Recapitulation of EIDD-2801 (128 mg/kg) oral ferret NHC plasma PK profile in the basolateral chamber of well-differentiated human airway epithelium cultures. NHC concentrations applied are shown in gray columns, and corresponding NHC-TP concentrations were measured by LC-MS/MS after 4, 12, and 24 hours. Symbols show biological repeats (n = 3 per time point). (D) NHC-TP concentrations in human airway epithelia recapitulating oral EIDD-2801 twice-daily treatment at 128, 20, or 7 mg/kg in ferrets. Solid lines connect measured NHC-TP from (C) and fig. S13, dashed lines extrapolate twice-daily treatment, and dotted lines mark robust efficacy (EC99), sterilizing antiviral activity, and cytotoxicity thresholds. (E to G) Ca/09-infected ferrets treated therapeutically with oral EIDD-2801 (7 mg/kg) twice daily after a single 20 mg/kg loading dose (LD). (E) Shed viral load. Symbols (n = 3) show biological repeats, and lines connect medians; two-way ANOVA with Sidak’s post hoc test. (F) Lowes analysis of continuously monitored body temperature. Differences in resolve of fever were assessed through time-to-event Mantel-Cox test. (G) Select cytokine and chemokine mRNA inductions in nasal turbinates 2.5 days after infection. Values are relative to uninfected animals, symbols show biological repeats (n = 3), and columns are means ± SD. Welch’s unequal variances t test was applied. IL-6, interleukin-6; IFN-β, interferon-β.

  • Fig. 5 Experimental validation of simulation results.

    Ca/09-infected ferrets treated therapeutically with EIDD-2801 (7 mg/kg) twice daily for 3.5 days. (A) Shed viral load. Biological repeats (n = 5) are shown, and lines connect medians; two-way ANOVA with Sidak’s post hoc test. (B) Lowes analysis of body temperature. Differences in resolve of fever were assessed through time-to-event Mantel-Cox test. (C) Total number of cells in nasal lavages. (D) Total white blood cell counts. Symbols in (C) and (D) show biological repeats (n = 5), and lines connect means; two-way ANOVA with Sidak’s post hoc test. (E) Virus titers in upper and lower respiratory tracts 3.5 days after infection. Symbols show biological repeats (n = 3), and graphs indicate medians ± SD. Individual statistical assessments of titers in the distinct respiratory tract compartments with unpaired t tests. NT, nasal turbinates; BALF, bronchioalveolar lavage fluid. (F) Virus distribution in individual lung lobes. Symbols show titers in caudal and cranial lung lobes for individual animals, and graphs indicate medians ± SD. (G) Immunohistochemistry of nasal turbinates from vehicle- and EIDD-2801–treated animals. Specific detection with anti-IAV HA antiserum and DAB staining with hematoxylin counterstain. Red arrows mark isolated DAB-positive cells detected in treated animals. Scale bars, 100 μm (overview microphotographs) and 25 μm (inserts). Representative fields of view are shown. (H) Immunohistochemistry of lung sections from vehicle- and EIDD-2801–treated animals. Staining and size bars as in (G).

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/515/eaax5866/DC1

    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.

  • The PDF file includes:

    • 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.
    • Legends for data files S1 to S5

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (.html format). Amino acid changes during baloxavir adaptation.
    • Data file S2 (Microsoft Excel format). Summary of amino acid changes during baloxavir adaptation.
    • Data file S3 (.html format). Amino acid changes during NHC adaptation.
    • Data file S4 (Microsoft Excel format). Summary of amino acid changes during NHC adaptation.
    • Data file S5 (Microsoft Excel format). Primary data.

Stay Connected to Science Translational Medicine

Navigate This Article