2. Short Communication
Evaluation of a dry powder delivery system for laninamivir in a ferret
model of influenza infection
Jacqueline Panozzo a,b,1
, Ding Yuan Oh a,c,1
, Kenneth Margo d
, David A. Morton d
, David Piedrafita c
,
Jennifer Mosse c
, Aeron C. Hurt a,e,⇑
a
WHO Collaborating Centre for Reference and Research on Influenza, VIDRL, The Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000, Australia
b
School of Applied Sciences and Engineering, Monash University, Churchill, Victoria 3842, Australia
c
School of Applied and Biomedical Sciences, Federation University, Churchill, Victoria 3842, Australia
d
Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia
e
Melbourne School of Population and Global Health, University of Melbourne, Parkville, Victoria 3010, Australia
a r t i c l e i n f o
Article history:
Received 26 March 2015
Revised 13 May 2015
Accepted 20 May 2015
Available online 26 May 2015
Keywords:
Laninamivir
Influenza
Powder delivery
Ferrets
Antiviral
Neuraminidase inhibitor
a b s t r a c t
Laninamivir is a long-acting antiviral requiring only a single dose for the treatment of influenza infection,
making it an attractive alternative to existing neuraminidase inhibitors that require multiple doses over
many days. Like zanamivir, laninamivir is administered to patients by inhalation of dry powder. To date,
studies investigating the effectiveness of laninamivir or zanamivir in a ferret model of influenza infection
have administered the drug in a solubilised form. To better mimic the delivery action of laninamivir in
humans, we assessed the applicability of a Dry Powder Insufflator™ (DPI) as a delivery method for lan-
inamivir octanoate (LO) in ferrets to determine the effectiveness of this drug in reducing influenza A and
B virus infections. In vitro characterisation of the DPI showed that both the small particle sized LO (0.7–
6.0 lm diameter) and the large particle sized lactose carrier (20–100 lm diameter) were effectively dis-
charged. However, LO delivered to ferrets via the DPI prior to infection with either A(H1N1)pdm09 or B
viruses had a limited effect on nasal inflammation, clinical symptoms and viral shedding compared to
placebo. Our preliminary findings indicate the feasibility of administering powder drugs into ferrets,
but a better understanding of the pharmacokinetics and pharmacodynamics of LO in ferrets following
delivery by the DPI is warranted prior to further studies.
Ó 2015 Elsevier B.V. All rights reserved.
Infection with influenza A and influenza B viruses causes signif-
icant human morbidity and mortality annually (WHO, 2014).
Currently the leading class of influenza antiviral drugs are the neu-
raminidase inhibitors (NAIs), namely zanamivir, oseltamivir, pera-
mivir and laninamivir (Chairat et al., 2013). Laninamivir,
administered as the pro-drug laninamivir octanoate (LO), is a
long-acting drug requiring only a single dose for treatment of influ-
enza infection. LO is currently licensed only in Japan, where it is the
most commonly prescribed NAI and is delivered as a dry powder
which is inhaled at a dosage of 40 mg for adults and 20 mg for
children (Ikematsu and Kawai, 2011; IMS Health, 2013). Human
clinical trials in Japan have demonstrated the effectiveness of LO
in reducing the duration of symptoms (Kashiwagi et al., 2013;
Katsumi et al., 2012; Koseki et al., 2014; Shobugawa et al., 2012;
Watanabe, 2013). However a reduction in symptoms was not
observed in a recent phase II clinical trial in the USA, although
LO did significantly reduce both viral shedding and the incidence
of secondary bacterial infections (Biota, 2014).
Ferrets are the preferred animal model to assess influenza virus
infection, virulence and transmission (Govorkova et al., 2007; Itoh
et al., 2009; Maines et al., 2009; Munster et al., 2009; Zhang et al.,
2013), and have been widely used in antiviral studies to assess
drug effectiveness (Govorkova et al., 2007, 2011; Marriott et al.,
2014; Oh et al., 2015), different treatment strategies (Maines
et al., 2009; Oh et al., 2014) and the selection of resistant viruses
(Hurt et al., 2010). Because oseltamivir, the most widely used
NAI globally, is an orally administered drug, delivery to ferrets
(or other animals) is relatively straightforward. However, zanami-
vir and laninamivir are both delivered to humans as a dry powder
http://dx.doi.org/10.1016/j.antiviral.2015.05.007
0166-3542/Ó 2015 Elsevier B.V. All rights reserved.
⇑ Corresponding author at: WHO Collaborating Centre for Reference and Research
on Influenza, VIDRL, The Peter Doherty Institute for Infection and Immunity, 792
Elizabeth St, Melbourne, Victoria 3000, Australia. Tel.: +61 3 93429314.
E-mail addresses: jacquipanozzo@gmail.com (J. Panozzo), DingThomas.Oh@
influenzacentre.org (D.Y. Oh), Kenneth.Margo@monash.edu (K. Margo), David.
Morton@monash.edu (D.A. Morton), david.piedrafita@federation.edu.au
(D. Piedrafita), jennifer.mosse@federation.edu.au (J. Mosse), Aeron.hurt@
influenzacentre.org (A.C. Hurt).
1
These authors contributed equally to this work.
Antiviral Research 120 (2015) 66–71
Contents lists available at ScienceDirect
Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral
3. formulation that requires active inhalation, therefore mimicking
this type of administration in animals is considerably more chal-
lenging. In the small number of zanamivir and laninamivir animal
studies conducted to date, the drugs have been hydrated and deliv-
ered to animals intranasally in liquid form rather than as a dry
powder (Kubo et al., 2010; Pizzorno et al., 2014; Ryan et al.,
1995). It is likely that intranasal liquid delivery will result in a very
different drug deposition compared to inhaled dry powder.
Therefore there is an unmet need to develop a dry powder delivery
method for appropriate assessment of these drugs in animal
models.
The Dry Powder Insufflator™ (DPI; Penn-century, USA) (Fig. 1A)
(Supplementary methods), is a device that aerosolises powders for
intratracheal delivery to animals, and has been used to administer
powder drugs or vaccines into macaques, rats, mice and guinea
pigs (Amidi et al., 2008; Grainger et al., 2004; Nahar et al., 2013;
Sung et al., 2009). In this study we used the DPI to deliver LO to fer-
rets and determined the effectiveness of the drug in reducing influ-
enza A and B virus replication and disease.
In humans, LO is delivered as a blend with lactose, therefore we
first assessed the quality of the LO:lactose powder cloud generated
by the DPI (Fig. 1A). Using a Malvern Mastersizer (Malvern
Instruments, UK) we showed that micronised LO had a fine particle
size distribution of 0.7–6.0 lm compared to the larger particle size
of lactose (approximately 20–100 lm) (Fig. 1B). Analysis of the
cumulative particle size distribution of the LO:lactose blend (pre-
pared at a 20:80 w/w ratio) in the plume delivered by the DPI
and measured by a Spraytec (Malvern Instruments, UK)
(Supplementary methods), showed that approximately 20% of the
particles by volume were less than 6 lm, indicating that LO was
delivered by the DPI at the correct proportion of the overall blend
in a well dispersed aerosol cloud (Fig. 1C). However, additional
experiments showed that approximately 10–30% of the total mass
was not delivered from the DPI after a single discharge of air, with
more powder remaining in the device when a larger mass was
loaded (% of drug being delivered, total mass loaded: 68.5%,
5 mg; 82.8%, 3 mg; 90.6%, 2 mg). A second delivery of air was typ-
ically successful in discharging the remaining mass of the powder,
Fig. 1. Characterisation of the delivery of laninamivir octanoate (LO) and lactose through a Penn-Century Dry Powder Insufflator™ (DPI). (A) Picture of the DPI used in this
study discharging LO/lactose powder (note that the picture does not include the 14 cm custom length nylon delivery tube). (B) The particle size distribution of LO and lactose.
(C) The cumulative particle sizes of the LO/lactose blend (prepared as 20% LO:80% lactose) from laser diffraction.
J. Panozzo et al. / Antiviral Research 120 (2015) 66–71 67
4. but because multiple deliveries can cause pulmonary damage to
animals so only a single delivery was used in the ferret studies
(Guillon et al., 2012).
LO (20:80 LO:lactose blend) or placebo (lactose) were adminis-
tered to the ferrets at 2.5 mg/kg 2 h prior to intranasal virus inoc-
ulation with 105
tissue culture infectious doses (TCID50) of
0 1 2 3 4 5 6 7 8 9 10
0
1
2
3
4
5
6
Day pi
Virustitre
(Log10TCID50/mL)
4
5
6
0 1 2 3 4 5 6 7 8 9 10
0
1
2
3
4
5
6
Day pi
Virustitre
(Log10TCID50/mL)
1
2
3
0 1 2 3 4 5 6 7 8 9 10
0
5
10
15
20
Day pi
Cellcount(106
/mL)
1
2
3
0 1 2 3 4 5 6 7 8 9 10
0
5
10
15
20
Day pi
Cellcount(106
/mL)
4
5
6
0 1 2 3 4 5 6 7 8 9 10
37
38
39
40
Day pi
Bodytemperature(o
C)
1
2
3
0 1 2 3 4 5 6 7 8 9 10
37
38
39
40
Day pi
Bodytemperature(o
C)
4
5
6
0 1 2 3 4 5 6 7 8 9 10
85
90
95
100
105
Day pi
Bodyweight(%initial)
1
2
3
0 1 2 3 4 5 6 7 8 9 10
85
90
95
100
105
Day pi
Bodyweight(%initial)
4
5
6
Placebo LO treated
A B
C D
E F
G H
WT flu A
Fig. 2. Effectiveness of laninamivir octanoate treatment on influenza A/Perth/265/2009 infection. Ferrets were treated with either lactose as placebo (n = 3) or laninamivir
octanoate (LO) (n = 3) 2 h prior to intranasal infection with 105
TCID50 (500 lL; 250 lL per nostril) of influenza virus A/Perth/265/2009. (A and B) Viral titre and (C and D) cell
count from nasal washes. (E and F) Body temperature. (G and H) Body weight; dotted line represents baseline. Each data point represents data from a single ferret. Placebo
treated ferrets: 1, 2 and 3. LO treated ferrets: 4, 5 and 6.
68 J. Panozzo et al. / Antiviral Research 120 (2015) 66–71
5. Madin-Darby canine kidney (MDCK) cell grown influenza virus
(see Supplementary methods). 2.5 mg/kg of LO:lactose blend (at
a ratio of 20:80) contained 0.5 mg/kg of pure LO, an equivalent
dose to that given in humans. Ferrets were infected with either
an influenza A(H1N1)pdm09 virus (A/Perth/265/2009) or an influ-
enza B virus (B/Yamanashi/166/1998). The laninamivir IC50 values
of the A(H1N1)pdm09 and B viruses, generated using a
fluorescence-based NA inhibition assay (see Supplementary meth-
ods), were 0.2 ± 0.01 nM and 1.6 ± 0.03 nM respectively, similar to
those of recently circulating laninamivir-sensitive viruses (Leang
et al., 2014).
Compared with placebo treated ferrets, treatment with LO did
not alter A(H1N1)pdm09 virus shedding, with animals displaying
similar peak viral load (mean log10TCID50/mL; placebo:
0 1 2 3 4 5 6 7 8 9 10
0
1
2
3
4
5
6
7
8
Day pi
Virustitre
(Log10TCID50/mL)
7
8
9
0 1 2 3 4 5 6 7 8 9 10
0
1
2
3
4
5
6
7
8
Day pi
Virustitre
(Log10TCID50/mL)
10
11
12
0 1 2 3 4 5 6 7 8 9 10
0
5
10
15
Day pi
Cellcount(106
/mL)
7
8
9
0 1 2 3 4 5 6 7 8 9 10
0
5
10
15
Day pi
Cellcount(106
/mL)
10
11
12
0 1 2 3 4 5 6 7 8 9 10
36
38
40
42
Day pi
Bodytemperature(o
C)
7
8
9
0 1 2 3 4 5 6 7 8 9 10
36
38
40
42
Day pi
Bodytemperature(o
C)
10
11
12
0 1 2 3 4 5 6 7 8 9 10
98
100
102
104
106
110
120
Day pi
Bodyweight(%initial)
7
8
9
0 1 2 3 4 5 6 7 8 9 10
98
100
102
104
Day pi
Bodyweight(%initial)
10
11
12
Placebo LO treated
A B
C D
E F
G H
WT flu B
Fig. 3. Effectiveness of laninamivir octanoate treatment on influenza B/Yamanashi/166/1998 infection. Ferrets were treated with either lactose as placebo (n = 3) or
laninamivir octanoate (LO) (n = 3) 2 h prior to intranasal infection with 105
TCID50 (500 lL; 250 lL per nostril) of influenza virus B/Yamanashi/166/1998. (A and B) Viral titre
and (C and D) cell count from nasal washes. (E and F) Body temperature. (G and H) Body weight; dotted line represents baseline. Each data point represents data from a single
ferret. Placebo treated ferrets: 7, 8 and 9. LO treated ferrets: 10, 11 and 12.
J. Panozzo et al. / Antiviral Research 120 (2015) 66–71 69
6. 4.61 ± 0.59; LO treated: 4.83 ± 0; P > 0.99), area under curve (AUC)
(placebo: 16.61 ± 2.91; LO treated: 19.39 ± 2.64; P = 0.4) and shed-
ding duration (Figs. 2A and B, and S1A). Inflammation, as assessed
by nasal cell concentration, was similar between placebo
and LO treated ferrets (peak cell concentration:
13.83 ± 3.77 Â 106
cells/mL and 10.50 ± 1.04 Â 106
cells/mL
respectively), although 2 of 3 placebo treated ferrets had a partic-
ularly high peak cell count (ferret No. 2: 19 Â 106
cells/mL on day
7; ferret No. 1: 16 Â 106
cells/mL on day 9) (Fig. 2C and D). All fer-
rets in the placebo group had a rise in body temperature on day 2
post-infection (pi) (compared with day 0), resulting in a significant
difference in mean temperature (P = 0.0091) (Figs. 2E and S1C). In
comparison, only 1/3 LO treated ferrets (ferret No. 5) had a sub-
stantially higher body temperature compared with day 0
(Fig. 2F). A weight loss of >10% was not seen in any of the placebo
treated ferrets, but observed in 1/3 LO treated ferrets (Fig. 2G and
H). In additional, both placebo and LO treated group displayed sim-
ilar levels of influenza-specific antibodies (Fig. S1E).
Treatment of influenza B infected ferrets with LO also resulted
in no significant difference in peak viral load (mean
log10TCID50/mL: placebo: 5.39 ± 0.44; LO treated: 5.05 ± 0.11;
P = 0.60), AUC (placebo: 20.95 ± 1.31; LO treated: 20.89 ± 0.90;
P > 0.99) or shedding duration (Figs. 3A and B, and S2A).
Inflammatory responses in the nasal cavity of both placebo and
LO treated groups were similar with cell concentrations peaking
on either day 8 (placebo) or 7 pi (LO treated) (Fig. 3C and D). The
body temperature of 2/3 placebo treated ferrets (ferret Nos. 7
and 9) showed an increase at day 1 and day 2 pi respectively
(Fig. 3E), with temperatures remaining elevated in ferret No. 7
(38.9–40.3 °C) throughout the experiment (Fig. 3E). Similarly, 2/3
LO treated ferrets showed a considerable increase in body temper-
ature at days 1 and 2 pi (Fig. 3F). None of the placebo or LO treated
ferrets experienced >10% weight loss (Fig. 3G and H). In additional,
both placebo and LO treated group displayed similar levels of
influenza-specific antibodies (Fig. S2E).
The laninamivir IC50 of all viruses from ferrets post-LO treat-
ment (day 6 pi) had similar IC50 values to that of the inoculated
virus (A(H1N1)pdm09, 0.2 ± 0.01 nM; influenza B, 1.6 ± 0.03 nM),
indicating that they remained sensitive to the drug. Sequence anal-
ysis of the viruses found no mutations in the HA and NA genes
(data not shown).
To date, only one previous study has investigated the effect of
LO in a ferret model of influenza infection (Kubo et al., 2010). In
that study, LO (delivered in a solubilised, not a dry powder form)
significantly lowered influenza B virus titres on day 2 pi compared
to a saline control (Kubo et al., 2010). While we saw a trend
towards lower influenza B viral titres in the LO-treated ferrets
(mean log10TCID50/mL: placebo: 5.27 ± 0.40; LO treated:
4.72 ± 0.29; P = 0.40) it was not statistically significant. A closer
examination of the reduction in viral titre and its association with
the administration route of LO in ferrets and/or alternative animal
models, such as guinea pigs, may be warranted. This study is the
first to report the effect of laninamivir on influenza A virus infec-
tions in ferrets.
Although the DPI has been widely used for delivering powdered
drugs to animals (Nahar et al., 2013), we identified several limita-
tions. These include the total dose not being discharged in a single
air delivery and the loss of some powder from ‘backflow’ during
drug administration. While the backflow of the powder cloud
was able to be reduced with a more gentle air shot, this resulted
in a larger mass of powder being retained in the device (data not
shown).
The dosage of LO used in this study was based on the human
dose of approximately 0.5 mg/kg (40 mg of LO for an 80 kg average
male adult), which is associated with a significant reduction
(P < 0.001) in viral shedding in humans (Biota, 2014), and was
higher than those used in the previous ferret study (0.24 mg/kg
solubilised LO) (Kubo et al., 2010). However, given the lack of effec-
tiveness seen here, a pharmacokinetic/dynamic (PK/PD) study in
ferrets to determine the concentrations of LO and laninamivir
(the active metabolite) following DPI administration will be impor-
tant prior to further studies. In addition, a better understanding of
the distribution of the powder within the respiratory tract and the
effect of LO treatment on cytokine levels is also important. Finally,
future studies should also investigate whether a ‘natural’ infection
(i.e., transmission from an infected ferret), rather than an intrana-
sal ‘artificial’ infection, may more closely mimic infection in
humans and serve as a better method for assessing NAI effective-
ness in ferrets.
In summary, the feasibility of delivering LO into ferrets by DPI
has been evaluated, but further model development for this
method of delivery, including PK/PD analysis, is warranted before
an accurate assessment of the effect of LO treatment on different
influenza virus infections can be studied.
Acknowledgements
This work was supported by NHMRC/A*STAR grant (1055793).
The Melbourne WHO Collaborating Centre for Reference and
Research on Influenza is supported by the Australian
Government Department of Health.
We thank Lauren Redman, Nikki Hearne, Rachael Murphy,
Crispin Agpasa and Miranda Spiteri and John Moody at Animal
Services bioCSL for providing assistance in animal handling.
Laninamivir was kindly provided free of charge by
Daiichi-Sankyo, Japan to the WHO Collaborating Centre for
Reference and Research on Influenza, Melbourne.
Laninamivir octanoate was kindly provided free of charge by
Biota Pharmaceuticals, Australia to Monash Institute of
Pharmaceutical Sciences.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.antiviral.2015.05.
007.
References
Amidi, M., Krudys, K.M., Snel, C.J., Crommelin, D.J., Della Pasqua, O.E., Hennink, W.E.,
Jiskoot, W., 2008. Efficacy of pulmonary insulin delivery in diabetic rats: use of a
model-based approach in the evaluation of insulin powder formulations. J.
Controlled Release 127, 257–266.
Biota, 2014. Biota Reports Top-Line Data From Its Phase 2 ‘‘IGLOO’’ Trial of
Laninamivir Octanoate Atlanta, USA.
Chairat, K., Tarning, J., White, N.J., Lindegardh, N., 2013. Pharmacokinetic properties
of anti-influenza neuraminidase inhibitors. J. Clin. Pharmacol. 53, 119–139.
Govorkova, E.A., Ilyushina, N.A., Boltz, D.A., Douglas, A., Yilmaz, N., Webster, R.G.,
2007. Efficacy of oseltamivir therapy in ferrets inoculated with different clades
of H5N1 influenza virus. Antimicrob. Agents Chemother. 51, 1414–1424.
Govorkova, E.A., Marathe, B.M., Prevost, A., Rehg, J.E., Webster, R.G., 2011.
Assessment of the efficacy of the neuraminidase inhibitor oseltamivir against
2009 pandemic H1N1 influenza virus in ferrets. Antiviral Res. 91, 81–88.
Grainger, C.I., Alcock, R., Gard, T.G., Quirk, A.V., van Amerongen, G., de Swart, R.L.,
Hardy, J.G., 2004. Administration of an insulin powder to the lungs of
cynomolgus monkeys using a Penn Century insufflator. Int. J. Pharm. 269,
523–527.
Guillon, A., Montharu, J., Vecellio, L., Schubnel, V., Roseau, G., Guillemain, J., Diot, P.,
de Monte, M., 2012. Pulmonary delivery of dry powders to rats: tolerability
limits of an intra-tracheal administration model. Int. J. Pharm. 434, 481–487.
Hurt, A.C., Lowther, S., Middleton, D., Barr, I.G., 2010. Assessing the development of
oseltamivir and zanamivir resistance in A(H5N1) influenza viruses using a ferret
model. Antiviral Res. 87, 361–366.
Ikematsu, H., Kawai, N., 2011. Laninamivir octanoate: a new long-acting
neuraminidase inhibitor for the treatment of influenza. Expert Rev. Anti
Infect. Ther. 9, 851–857.
IMS Health, 2013. http://www.imshealth.com/ (accessed 23.06.13).
Itoh, Y., Shinya, K., Kiso, M., Watanabe, T., Sakoda, Y., Hatta, M., Muramoto, Y.,
Tamura, D., Sakai-Tagawa, Y., Noda, T., Sakabe, S., Imai, M., Hatta, Y., Watanabe,
70 J. Panozzo et al. / Antiviral Research 120 (2015) 66–71
7. S., Li, C., Yamada, S., Fujii, K., Murakami, S., Imai, H., Kakugawa, S., Ito, M.,
Takano, R., Iwatsuki-Horimoto, K., Shimojima, M., Horimoto, T., Goto, H.,
Takahashi, K., Makino, A., Ishigaki, H., Nakayama, M., Okamatsu, M., Warshauer,
D., Shult, P.A., Saito, R., Suzuki, H., Furuta, Y., Yamashita, M., Mitamura, K.,
Nakano, K., Nakamura, M., Brockman-Schneider, R., Mitamura, H., Yamazaki, M.,
Sugaya, N., Suresh, M., Ozawa, M., Neumann, G., Gern, J., Kida, H., Ogasawara, K.,
Kawaoka, Y., 2013. In vitro and in vivo characterization of new swine-origin
H1N1 influenza viruses. Nature 460, 1021–1025.
Kashiwagi, S., Watanabe, A., Ikematsu, H., Awamura, S., Okamoto, T., Uemori, M.,
Ishida, K., 2013. Laninamivir octanoate for post-exposure prophylaxis of
influenza in household contacts: a randomized double blind placebo
controlled trial. J. Infect. Chemother.: Off. J. Jpn. Soc. Chemother. 19, 740–749.
Katsumi, Y., Otabe, O., Matsui, F., Kidowaki, S., Mibayashi, A., Tsuma, Y., Ito, H., 2012.
Effect of a single inhalation of laninamivir octanoate in children with influenza.
Pediatrics 129, E1431–E1436.
Koseki, N., Kaiho, M., Kikuta, H., Oba, K., Togashi, T., Ariga, T., Ishiguro, N., 2014.
Comparison of the clinical effectiveness of zanamivir and laninamivir octanoate
for children with influenza A (H3N2) and B in the 2011–2012 season. Influenza
Other Respir. Viruses 8, 151–158.
Kubo, S., Tomozawa, T., Kakuta, M., Tokumitsu, A., Yamashita, M., 2010. Laninamivir
prodrug CS-8958, a long-acting neuraminidase inhibitor, shows superior anti-
influenza virus activity after a single administration. Antimicrob. Agents
Chemother. 54, 1256–1264.
Leang, S.K., Kwok, S., Sullivan, S.G., Maurer-Stroh, S., Kelso, A., Barr, I.G., Hurt, A.C.,
2014. Peramivir and laninamivir susceptibility of circulating influenza A and B
viruses. Influenza Other Respir. Viruses 8, 135–139.
Maines, T.R., Jayaraman, A., Belser, J.A., Wadford, D.A., Pappas, C., Zeng, H., Gustin,
K.M., Pearce, M.B., Viswanathan, K., Shriver, Z.H., Raman, R., Cox, N.J.,
Sasisekharan, R., Katz, J.M., Tumpey, T.M., 2009. Transmission and
pathogenesis of swine-origin 2009 A(H1N1) influenza viruses in ferrets and
mice. Science (New York, N.Y.) 325, 484–487.
Marriott, A.C., Dove, B.K., Whittaker, C.J., Bruce, C., Ryan, K.A., Bean, T.J., Rayner, E.,
Pearson, G., Taylor, I., Dowall, S., Plank, J., Newman, E., Barclay, W.S., Dimmock,
N.J., Easton, A.J., Hallis, B., Silman, N.J., Carroll, M.W., 2014. Low dose influenza
virus challenge in the ferret leads to increased virus shedding and greater
sensitivity to oseltamivir. PLoS One 9, e94090.
Munster, V.J., de Wit, E., van den Brand, J.M., Herfst, S., Schrauwen, E.J., Bestebroer,
T.M., van de Vijver, D., Boucher, C.A., Koopmans, M., Rimmelzwaan, G.F., Kuiken,
T., Osterhaus, A.D., Fouchier, R.A., 2009. Pathogenesis and transmission of
swine-origin 2009 A(H1N1) influenza virus in ferrets. Science (New York, N.Y.)
325, 481–483.
Nahar, K., Gupta, N., Gauvin, R., Absar, S., Patel, B., Gupta, V., Khademhosseini, A.,
Ahsan, F., 2013. In vitro, in vivo and ex vivo models for studying particle
deposition and drug absorption of inhaled pharmaceuticals. Eur. J. Pharm. Sci.:
Off. J. Eur. Fed. Pharm. Sci. 49, 805–818.
Oh, D.Y., Lowther, S., McCaw, J.M., Sullivan, S.G., Leang, S.K., Haining, J., Arkinstall, R.,
Kelso, A., McVernon, J., Barr, I.G., Middleton, D., Hurt, A.C., 2014. Evaluation of
oseltamivir prophylaxis regimens for reducing influenza virus infection,
transmission and disease severity in a ferret model of household contact. J.
Antimicrob. Chemother. 69, 2458–2469.
Oh, D.Y., Barr, I.G., Hurt, A.C., 2015. A novel video tracking method to evaluate the
effect of influenza infection and antiviral treatment on ferret activity. PLoS One
10, e0118780.
Pizzorno, A., Abed, Y., Rheaume, C., Boivin, G., 2014. Oseltamivir–zanamivir
combination therapy is not superior to zanamivir monotherapy in mice
infected with influenza A(H3N2) and A(H1N1)pdm09 viruses. Antiviral Res.
105, 54–58.
Ryan, D.M., Ticehurst, J., Dempsey, M.H., 1995. GG167 (4-guanidino-2,4-dideoxy-
2,3-dehydro-N-acetylneuraminic acid) is a potent inhibitor of influenza virus in
ferrets. Antimicrob. Agents Chemother. 39, 2583–2584.
Shobugawa, Y., Saito, R., Sato, I., Kawashima, T., Dapat, C., Dapat, I.C., Kondo, H.,
Suzuki, Y., Saito, K., Suzuki, H., 2012. Clinical effectiveness of neuraminidase
inhibitors – oseltamivir, zanamivir, laninamivir, and peramivir – for treatment
of influenza A(H3N2) and A(H1N1)pdm09 infection: an observational study in
the 2010–2011 influenza season in Japan. J. Infect. Chemother. 18, 858–864.
Sung, J.C., Garcia-Contreras, L., Verberkmoes, J.L., Peloquin, C.A., Elbert, K.J., Hickey,
A.J., Edwards, D.A., 2009. Dry powder nitroimidazopyran antibiotic PA-824
aerosol for inhalation. Antimicrob. Agents Chemother. 53, 1338–1343.
Watanabe, A., 2013. A randomized double-blind controlled study of laninamivir
compared with oseltamivir for the treatment of influenza in patients with
chronic respiratory diseases. J. Infect. Chemother. 19, 89–97.
WHO, 2014. Influenza (Seasonal) Fact Sheet No. 211. World Health Organisation.
Zhang, Q., Shi, J., Deng, G., Guo, J., Zeng, X., He, X., Kong, H., Gu, C., Li, X., Liu, J., Wang,
G., Chen, Y., Liu, L., Liang, L., Li, Y., Fan, J., Wang, J., Li, W., Guan, L., Li, Q., Yang, H.,
Chen, P., Jiang, L., Guan, Y., Xin, X., Jiang, Y., Tian, G., Wang, X., Qiao, C., Li, C., Bu,
Z., Chen, H., 2013. H7N9 influenza viruses are transmissible in ferrets by
respiratory droplet. Science (New York, N.Y.) 341, 410–414.
J. Panozzo et al. / Antiviral Research 120 (2015) 66–71 71
