Supplementary Materials for · Fig. S10. Therapeutic efficacy of NHC in the 3D airway epithelium...
Transcript of Supplementary Materials for · Fig. S10. Therapeutic efficacy of NHC in the 3D airway epithelium...
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: [email protected]
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.