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

Supplementary Materials for

Characterization of orally efficacious influenza drug with high resistance

barrier in ferrets and human airway epithelia

Mart Toots, Jeong-Joong Yoon, Robert M. Cox, Michael Hart, Zachary M. Sticher, Negar Makhsous, Roland Plesker, Alec H. Barrena, Prabhakar G. Reddy, Deborah G. Mitchell, Ryan C. Shean, Gregory R. Bluemling,

Alexander A. Kolykhalov, Alexander L. Greninger, Michael G. Natchus, George R. Painter, Richard K. Plemper*

*Corresponding author. Email: rplemper@gsu.edu

Published 23 October 2019, Sci. Transl. Med. 11, eaax5866 (2019)

DOI: 10.1126/scitranslmed.aax5866

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

Other Supplementary Material for this manuscript includes the following: (available at stm.sciencemag.org/cgi/content/full/11/515/eaax5866/DC1)

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.

Fig. S1. PK of NHC and EIDD-2801 in mice. Plasma concentrations of NHC over time after a

single oral dose of EIDD-2801 or NHC administered to mice at the specified dose

concentrations. Three animals per group, plasma sample analysis by LC-MS/MS. Symbols

represent biological repeats (N = 3); lines show mean values. Only NHC could be detected in

mouse plasma after EIDD-2801 dosing. Mice were chosen for this experiment, since NHC is not

orally bioavailable in macaques (Fig 1A).

Fig. S2. Single-dose PK of EIDD-2801 in ferrets. Plasma concentrations of NHC over time

after a single oral dose of EIDD-2801 administered to ferrets at the specified dose

concentrations. Plasma sample analysis by LC-MS/MS. Symbols represent biological repeats;

lines show mean values; 128 mg/kg and 512 mg/kg groups: N = 4; 4 mg/kg and 20 mg/kg

groups: N = 3

Fig. S3. Multidose PK of EIDD-2801 in ferrets. Plasma concentrations of NHC over time after

seven oral doses of EIDD-2801 in ferrets at the specified dose concentrations. 20 mg/kg single

dose concentration from Fig S2 are shown for comparison. Plasma sample analysis by LC-

MS/MS. Symbols represent biological repeats (N = 3); lines show mean values. No significant

accumulation of the drug was observed. Statistical analysis with 2-way ANOVA and Sidak’s

post hoc test.

Fig. S4. Ferret efficacy study timeline. Schematic of the EIDD-2801 efficacy testing protocol

used for the ferret studies, showing treatment regimen, dosing cycles, and analysis of clinical

signs.

Fig. S5. Histopathology scores of Ca09-infected ferret lungs. Histopathology analyses of

ferret lungs extracted 3.5 days after intranasal infection with Ca/09. Lungs were perfused with

10% neutral-buffered formalin and 4 lung lobes (terminal and middle parts; two technical repeats

for each sample) from each animal analyzed. Pathology scoring was performed by a licensed

pathologist. Clinical scores from vehicle, EIDD-2801 dosed at 100 and 20 mg/kg and SOC

oseltamivir dosed at 20 mg/kg, in all cases following a b.i.d. protocol, are shown. Oseltamivir

dosing was initiated prophylactically only. Columns represent all technical and biological repeats

(N = 31-43) showing means SD. Symbols represent scores assigned to individual tissue slides.

Statistical analyses with 1-way ANOVA followed by Dunnett’s post hoc test.

Fig. S6. Escalating-dose adaptation of IAV to NHC. Adaptation schematic (left) of the

escalating-dose adaptation of IAV A/WSN/1933 (H1N1) harboring a nanoLuc reporter to NHC.

For adaptation to NHC MDCK cells were infected with 0.005 MOI of IAV-WSN and treated

with 0.25 M NHC. Luciferase readouts were measured after 2 days, supernatant was collected

and used to infect new cells; NHC concentration was doubled after every passage. IAV-WSN

was undetectable by nanoLuc after the fifth passage. Viral titers after each passage of the

escalating-dose adaptation protocol of IAV-WSN to NHC (right). Symbols represent biological

repeats (N = 3); lines show mean values; LoD, limit of detection.

Fig. S7. Fixed-dose serial passaging of IAV in the presence of NHC. MDCK cells were

infected with 0.005 MOI of IAV-WSN expressing nanoLuc reporter gene and treated with 10, 4,

2 or 1 M NHC. Luciferase readouts were measured in every 2 days, supernatant was collected

and used to infect new cells. Luciferase readouts were used to normalize the infection volume.

No influenza-specific RT-PCR product could be detected after 3 or 4 passages in case of 10 M

NHC and after 5 passages in case of 4 M NHC. Fixed-dose passaging at NHC 1 or 2 M

resulted in no loss of infectious virus after 10 passages (viral titers throughout the passaging are

shown in Fig 2A).

Fig. S8. Genetic changes in IAV-WSN RNA during fixed-dose passaging.

Transition/transversion mutation frequency in IAV-WSN RNA during fixed-dose passaging with

1 or 2 M NHC. Total RNA was extracted from IAV-WSN infected cells after passage #5 (left)

and #10 (right) and subjected to whole genome next-generation deep sequencing. Symbols

represent biological repeats (N = 4), columns represent mean values SDs. Frequency of

specific transitions or transversions is expressed relative to vehicle-adapted virus populations.

None of the transitions or transversions were significantly different between differentially treated

virus populations. Statistical analyses with 2-way ANOVA followed by Dunnett’s post hoc test.

Fig. S9. Immunofluorescence of influenza-infected 3D airway epithelium cultures. Confocal

immunofluorescence microscopy describing airway integrity in DMSO and NHC treated 3D

airway epithelium cultures; virus (NS1 for IAV; whole virus for IBV) was stained in red, tight

junctions (ZO-1) in green, and nuclei in blue (DAPI). Corresponding graphs show total numbers

of intact and fragmented nuclei detected in randomly selected areas (approximately 13,800 m2

each) of IAV (top panel) or IBV (bottom panel)-infected epithelium cultures treated with vehicle

(DMSO) or 1.8 M NHC in basolateral chamber. Uninfected and untreated cultures served as

comparison. Symbols show counts of individual areas, columns indicate means SD. 1-way

ANOVA with Sidak’s post hoc test.

Fig. S10. Therapeutic efficacy of NHC in the 3D airway epithelium culture. Therapeutic

efficacy of NHC, administered basolaterally to 3D human airway epithelium models. 3D cultures

were infected with Ca/09 and shed viral load measured daily over a 6-day time period by TCID50

titration. Treatment with 1.8 M NHC (sterilizing concentration) was initiated at the specified

timepoints. Symbols represent biological repeats (N = 3) and lines show mean values. Statistical

analyses with 2-way ANOVA followed by Dunnett’s post hoc test. LoD – limit of detection.

Fig. S11. Cytotoxicity of NHC in the 3D airway epithelium culture. NHC effect on TEER in

the 3D airway epithelium cultures, serving as cytotoxicity biomarker. Symbols represent

biological repeats (N = 3), line shows four-parameter variable-slope regression model, and

numbers specify calculated CC50 concentration, 95% confidence interval in parentheses.

Fig. S12. NHC effect on nuclear and mitochondrial gene expressions. In-cell ELISA (Abcam;

ab110217) in HBTECs was performed after 3-day exposure of cells to NHC. Symbols show

biological repeats (N = 3); lines show mean values; 2-way ANOVA with Sidak’s multiple

comparison post hoc test.

Fig. S13. Recapitulation of NHC PK profiles in 3D human airway epithelium culture.

Recapitulation of oral NHC plasma PK profiles in ferrets corresponding to 20 mg/kg (A, red) and

7 mg/kg (B, magenta) in the basolateral chamber of 3D human airway epithelium cultures.

Shaded blocks indicate basolateral NHC concentrations applied, based on the in vivo data shown

in Fig. S2. Corresponding NHC-TP concentrations in the epithelium tissue were determined at 4

and 12 hours after experiment start by LC-MS/MS. Values are expressed per 106 cells, symbols

show biological repeats (N = 3); lines show mean values.

Fig. S14. Immunohistochemistry of nasal turbinates extracted from vehicle- and EIDD-

2801–treated animals. Specific detection with anti-IAV HA antiserum and DAB staining,

hematoxylin counterstain. Isotype-matched non-specific antibody was used for specificity

control (negative control IgG). Scale bars are 100 m for the main images and 25 m for the

inserts, red arrows highlight isolated DAB-positive cells in treated animals.

Fig. S15. Immunohistochemistry of lungs extracted from vehicle- and EIDD-2801–treated

animals. Specific detection with anti-IAV HA antiserum and DAB staining, hematoxylin

counterstain. Separate HE staining of sections equivalent to the insert are shown in addition.

Scale bars are 100 m for the main images and 25 m for the inserts. Red arrows highlight

DAB-positive cells in vehicle-treated animals.

Fig. S16. Immunofluorescence of 3D airway epithelium cultures. Confocal

immunofluorescence microscopy to assess characteristic features of well-differentiated 3D

airway epithelium cultures: tight junctions (ZO-1), adherens junctions (E-Cadherin), goblet cells

(Muc5AC) and ciliated cells (-tubulin). Nuclei were stained with DAPI.

Fig. S17. Immunofluorescence of 3D airway epithelium cultures after NHC exposure. 3D

airway epithelium tissue integrity after 3-day basolateral exposure to NHC. Evaluation by

visualizing tight junctions (ZO-1 confocal immunofluorescence microscopy) and nuclei (DAPI).

Table S1. PK parameters for NHC in cynomolgus macaques. PK parameters for NHC in

cynomolgus macaque plasma after a single dose of NHC or EIDD-2801 at specified route and

dose concentrations. Parameters of NHC were calculated using the WinNonlin (Pharsight)

software package. N = 6 (3 males and 3 females) for the intravenously (IV) dosed group, N = 8

(4 males and 4 females) for each of the orally (PO) dosed groups; N/A: not applicable.

Compound; Route;

Dose concentration

tmax

[hours]

Cmax

[nmol/ml] AUC 024 h

[hrnmol/ml]

AUC/Dose

[hnmol kg/mmolml] t1/2

[hours]

F

[%]

NHC; IV; 10 mg/kg 0.8 0 36.5 4.8 11.6 3.2 300.1 82.7 0.18 0.04 N/A

NHC; PO; 100 mg/kg 0.81 0.53 3.3 1.8 6.6 3.1 17.1 8.0 2.08 1.54 5.7 2.7

EIDD-2801; PO; 130 mg/kg 1.62 0.74 10.2 2.9 38.5 14.2 97.6 35.9 1.77 0.86 32.5 12.0

Table S2. Single-dose PK parameters for NHC in ferrets. Calculation of PK parameters for

NHC after a single p.o. dose of EIDD-2801 in ferrets at specified dose concentrations (N = 3 per

group). Parameters of NHC were calculated using the WinNonlin (Pharsight) software package.

Dose concentration

[mg/kg]

Tmax

[hours]

Cmax

[nmol/ml] AUC 024 h

[hrnmol/ml]

AUCinf

[hrnmol/ml]

Cmax/Dose

[h*kg*nmol/ml/mmol]

AUCinf/Dose

[hours]

T(1/2)

[hours]

4 1.7 0.6 3.5 1.5 12.7 4.8 13.2 4.8 290.5 125.8 1087.7 392.4 8.2 1.7

20 1.8 1.9 15.4 1.9 71.8 32.1 72.5 32.1 251.9 31.6 1189.1 526.2 4.7 1.3

128 1.7 0 100.1 22.3 317.9 42 322.4 42.6 257.3 57.2 828.5 109.4 5.1 0.8

512 2.5 1 209.2 106 786.4 388.4 791.4 390.7 134.1 68 507.3 250.4 4.2 0.6

Table S3. Lung concentrations of NHC and NHC-TP. NHC and NHC-TP concentrations in

ferret lungs after a single p.o. dose of EIDD-2801 at 128 mg/kg [nmol/g]. 3-hour values are

means (N = 3) SD, 12-hour values are means (N = 2) range.

Drug Dose concentration

[mg/kg]

Time post-dose

[hours]

Lung

[nmol/g]

NHC 128 3 89 27

NHC 128 12 10.7 1.2

NHC-TP 128 3 8.8 2.2

NHC-TP 128 12 3.2 1.5

Table S4. Multidose PK parameters for NHC in ferrets. Calculation of PK parameters for

NHC after multiple p.o. doses of ferrets at the specified dose concentrations. Parameters of NHC

were calculated using the WinNonlin (Pharsight) software package; means (N = 3) SD are

shown.

Dose concentration

[mg/kg]

Tmax

[hours]

Cmax

[nmol/ml] AUC 024 h

[hrnmol/ml]

AUCinf

[hrnmol/ml]

T(1/2)

[hours]

7 20 mg/kg 1.5 0.9 25.1 15.2 58.8 8.6 63.8 8.3 6.7 0.1

1 20, 6 7 mg/kg 1.5 0.9 11 4 25.8 4.7 29.2 4 9.2 4.5

Table S5. Antibodies used in this study.

Target Dilution Manufacturer

Muc5AC 1:200 Thermo Scientific; MA5-12175

ZO-1 1:50 BD Biosciences; 610966

E-Cadherin 1:200 BD Biosciences; 610181

-tubulin-647 1:100 Novus Biologicals; NBP237830AF647

influenza A NS1 1:100 Thermo Scientific; PA5-23365

influenza B 1:100 Thermo Scientific; PA5-34975

mouse-FITC 1:1000 SantaCruz; sc-2080

goat-Alexa 568 1:1000 Thermo Scientific; A-11057

rabbit-Alexa 568 1:1000 Thermo Scientific; A-11011

influenza A HA 1:100 GeneTex; GTX127357

rabbit-biotin 1:1000 Calbiochem; OS03B

Table S6. Primers used in this study.

Primer name Primer sequence

CloneJet_F 5’-CGACTCACTATAGGGAGAGCGGC-3’

CloneJet_R 5’-AAGAACATCGATTTTCCATGGCAG-3’

Ferret TNF_F 5’-ATGAGCACTGAAAGCATGATC-3’

Ferret TNF_R 5’-TCACAGGGCAATGATTCCAAAG-3’

Ferret COX15_F 5’-ATGCAGCGATTGCTCTTTC-3’

Ferret COX15_R 5’-TTGGGACTCTTCGGAGTTC-3’

Human COX1_F 5’-CGCCGACCGTTGACTATTC-3’

Human COX1_R 5’-GATTATGGTAGCGGAGGTG-3’

Human SDH-A_F 5’-GGGAACAAGAGGGCATCTG-3’

Human SDH-A_R 5’-CTCTCCACGACATCCTTCC-3’

Ferret GAPDH_F 5’-AACATCATCCCTGCTTCCACTGGT-3’

Ferret GAPDH_R 5’-TGTTGAAGTCGCAGGAGACAACCT-3’

Ferret IL-6_F 5’-AGTGGCTGAAACACGTAACAATTC-3’

Ferret IL-6_R 5’-ATGGCCCTCAGGCTGAACT-3’

Ferret IFN-_F 5’-GGTGTATCCTCCAAACTGCTCTCC-3’

Ferret IFN-_R 5’-CACTCCACACTGCTGCTGCTTAG-3’

Ferret IFN-_F 5’-TCAAAGTGATGAATGATCTCTCACC-3’

Ferret IFN-_R 5’-GCCGGGAAACACACTGTGAC-3’

Ferret CXCL10_F 5’-CTTTGAACCAAAGTGCTGTTCTTATC-3’

Ferret CXCL10_R 5’-AGCGTGTAGTTCTAGAGAGAGGTACTC-3’

Data file S1. Amino acid changes during baloxavir adaptation. Interactive HTML file

Datafile_S1.html showing amino acid changes (frequency 5%) detected through whole genome

deep-sequencing after virus adaptation to baloxavir marboxil.

Data file S2. Summary of amino acid changes during baloxavir adaptation. Summary table

Datafile_S2.csv showing all changes with frequency 5% detected through whole genome next

generation deep sequencing after virus adaptation to baloxavir marboxil.

Data file S3. Amino acid changes during NHC adaptation. Interactive HTML file

Datafile_S3.html showing amino acid changes (frequency 5%) detected through whole genome

deep-sequencing after virus adaptation to NHC.

Data file S4. Summary of amino acid changes during NHC adaptation. Summary table

Datafile_S4.csv showing all changes with frequency 5% detected through whole genome next

generation deep sequencing after virus adaptation to NHC.

Data file S5. Primary data. Provided in Excel format.