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Evaluationofadrypowderdeliverysystemforlaninamivirinaferretmodelofinfluenzainfection
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Antiviral Research 120 (2015) 66–71
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Antiviral Research
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Short Communication
Evaluation of a dry powder delivery system for laninamivir in a ferretmodel of influenza infection
http://dx.doi.org/10.1016/j.antiviral.2015.05.0070166-3542/� 2015 Elsevier B.V. All rights reserved.
⇑ Corresponding author at: WHO Collaborating Centre for Reference and Researchon Influenza, VIDRL, The Peter Doherty Institute for Infection and Immunity, 792Elizabeth St, Melbourne, Victoria 3000, Australia. Tel.: +61 3 93429314.
E-mail addresses: [email protected] (J. Panozzo), [email protected] (D.Y. Oh), [email protected] (K. Margo), [email protected] (D.A. Morton), [email protected](D. Piedrafita), [email protected] (J. Mosse), [email protected] (A.C. Hurt).
1 These authors contributed equally to this work.
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, Australiab School of Applied Sciences and Engineering, Monash University, Churchill, Victoria 3842, Australiac School of Applied and Biomedical Sciences, Federation University, Churchill, Victoria 3842, Australiad Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australiae 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 2015Revised 13 May 2015Accepted 20 May 2015Available online 26 May 2015
Keywords:LaninamivirInfluenzaPowder deliveryFerretsAntiviralNeuraminidase 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 overmany 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 infectionhave administered the drug in a solubilised form. To better mimic the delivery action of laninamivir inhumans, 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 andB 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 Bviruses had a limited effect on nasal inflammation, clinical symptoms and viral shedding compared toplacebo. 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 followingdelivery by the DPI is warranted prior to further studies.
� 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 along-acting drug requiring only a single dose for treatment of influ-enza infection. LO is currently licensed only in Japan, where it is themost commonly prescribed NAI and is delivered as a dry powderwhich is inhaled at a dosage of 40 mg for adults and 20 mg for
children (Ikematsu and Kawai, 2011; IMS Health, 2013). Humanclinical trials in Japan have demonstrated the effectiveness of LOin 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 notobserved in a recent phase II clinical trial in the USA, althoughLO did significantly reduce both viral shedding and the incidenceof secondary bacterial infections (Biota, 2014).
Ferrets are the preferred animal model to assess influenza virusinfection, virulence and transmission (Govorkova et al., 2007; Itohet al., 2009; Maines et al., 2009; Munster et al., 2009; Zhang et al.,2013), and have been widely used in antiviral studies to assessdrug effectiveness (Govorkova et al., 2007, 2011; Marriott et al.,2014; Oh et al., 2015), different treatment strategies (Maineset al., 2009; Oh et al., 2014) and the selection of resistant viruses(Hurt et al., 2010). Because oseltamivir, the most widely usedNAI 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
J. Panozzo et al. / Antiviral Research 120 (2015) 66–71 67
formulation that requires active inhalation, therefore mimickingthis type of administration in animals is considerably more chal-lenging. In the small number of zanamivir and laninamivir animalstudies conducted to date, the drugs have been hydrated and deliv-ered to animals intranasally in liquid form rather than as a drypowder (Kubo et al., 2010; Pizzorno et al., 2014; Ryan et al.,1995). It is likely that intranasal liquid delivery will result in a verydifferent drug deposition compared to inhaled dry powder.Therefore there is an unmet need to develop a dry powder deliverymethod for appropriate assessment of these drugs in animalmodels.
The Dry Powder Insufflator™ (DPI; Penn-century, USA) (Fig. 1A)(Supplementary methods), is a device that aerosolises powders forintratracheal delivery to animals, and has been used to administerpowder drugs or vaccines into macaques, rats, mice and guineapigs (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.
Fig. 1. Characterisation of the delivery of laninamivir octanoate (LO) and lactose througstudy discharging LO/lactose powder (note that the picture does not include the 14 cm cu(C) The cumulative particle sizes of the LO/lactose blend (prepared as 20% LO:80% lacto
In humans, LO is delivered as a blend with lactose, therefore wefirst assessed the quality of the LO:lactose powder cloud generatedby the DPI (Fig. 1A). Using a Malvern Mastersizer (MalvernInstruments, UK) we showed that micronised LO had a fine particlesize distribution of 0.7–6.0 lm compared to the larger particle sizeof lactose (approximately 20–100 lm) (Fig. 1B). Analysis of thecumulative particle size distribution of the LO:lactose blend (pre-pared at a 20:80 w/w ratio) in the plume delivered by the DPIand measured by a Spraytec (Malvern Instruments, UK)(Supplementary methods), showed that approximately 20% of theparticles by volume were less than 6 lm, indicating that LO wasdelivered by the DPI at the correct proportion of the overall blendin a well dispersed aerosol cloud (Fig. 1C). However, additionalexperiments showed that approximately 10–30% of the total masswas not delivered from the DPI after a single discharge of air, withmore powder remaining in the device when a larger mass wasloaded (% 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,
h a Penn-Century Dry Powder Insufflator™ (DPI). (A) Picture of the DPI used in thisstom length nylon delivery tube). (B) The particle size distribution of LO and lactose.se) from laser diffraction.
68 J. Panozzo et al. / Antiviral Research 120 (2015) 66–71
but because multiple deliveries can cause pulmonary damage toanimals so only a single delivery was used in the ferret studies(Guillon et al., 2012).
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Fig. 2. Effectiveness of laninamivir octanoate treatment on influenza A/Perth/265/2009octanoate (LO) (n = 3) 2 h prior to intranasal infection with 105 TCID50 (500 lL; 250 lL pecount from nasal washes. (E and F) Body temperature. (G and H) Body weight; dotted lintreated ferrets: 1, 2 and 3. LO treated ferrets: 4, 5 and 6.
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
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infection. Ferrets were treated with either lactose as placebo (n = 3) or laninamivirr nostril) of influenza virus A/Perth/265/2009. (A and B) Viral titre and (C and D) celle represents baseline. Each data point represents data from a single ferret. Placebo
J. Panozzo et al. / Antiviral Research 120 (2015) 66–71 69
Madin-Darby canine kidney (MDCK) cell grown influenza virus(see Supplementary methods). 2.5 mg/kg of LO:lactose blend (ata ratio of 20:80) contained 0.5 mg/kg of pure LO, an equivalentdose to that given in humans. Ferrets were infected with eitheran influenza A(H1N1)pdm09 virus (A/Perth/265/2009) or an influ-enza B virus (B/Yamanashi/166/1998). The laninamivir IC50 valuesof the A(H1N1)pdm09 and B viruses, generated using a
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Fig. 3. Effectiveness of laninamivir octanoate treatment on influenza B/Yamanashi/16laninamivir octanoate (LO) (n = 3) 2 h prior to intranasal infection with 105 TCID50 (500 land (C and D) cell count from nasal washes. (E and F) Body temperature. (G and H) Body wferret. Placebo treated ferrets: 7, 8 and 9. LO treated ferrets: 10, 11 and 12.
fluorescence-based NA inhibition assay (see Supplementary meth-ods), were 0.2 ± 0.01 nM and 1.6 ± 0.03 nM respectively, similar tothose of recently circulating laninamivir-sensitive viruses (Leanget al., 2014).
Compared with placebo treated ferrets, treatment with LO didnot alter A(H1N1)pdm09 virus shedding, with animals displayingsimilar peak viral load (mean log10TCID50/mL; placebo:
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6/1998 infection. Ferrets were treated with either lactose as placebo (n = 3) orL; 250 lL per nostril) of influenza virus B/Yamanashi/166/1998. (A and B) Viral titreeight; dotted line represents baseline. Each data point represents data from a single
70 J. Panozzo et al. / Antiviral Research 120 (2015) 66–71
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 assessedby nasal cell concentration, was similar between placeboand LO treated ferrets (peak cell concentration:13.83 ± 3.77 � 106 cells/mL and 10.50 ± 1.04 � 106 cells/mLrespectively), although 2 of 3 placebo treated ferrets had a partic-ularly high peak cell count (ferret No. 2: 19 � 106 cells/mL on day7; ferret No. 1: 16 � 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 2post-infection (pi) (compared with day 0), resulting in a significantdifference in mean temperature (P = 0.0091) (Figs. 2E and S1C). Incomparison, 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 placebotreated ferrets, but observed in 1/3 LO treated ferrets (Fig. 2G andH). 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 resultedin no significant difference in peak viral load (meanlog10TCID50/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 andLO treated groups were similar with cell concentrations peakingon either day 8 (placebo) or 7 pi (LO treated) (Fig. 3C and D). Thebody temperature of 2/3 placebo treated ferrets (ferret Nos. 7and 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, 2/3LO treated ferrets showed a considerable increase in body temper-ature at days 1 and 2 pi (Fig. 3F). None of the placebo or LO treatedferrets experienced >10% weight loss (Fig. 3G and H). In additional,both placebo and LO treated group displayed similar levels ofinfluenza-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 inoculatedvirus (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 ofLO in a ferret model of influenza infection (Kubo et al., 2010). Inthat study, LO (delivered in a solubilised, not a dry powder form)significantly lowered influenza B virus titres on day 2 pi comparedto a saline control (Kubo et al., 2010). While we saw a trendtowards 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 closerexamination of the reduction in viral titre and its association withthe administration route of LO in ferrets and/or alternative animalmodels, such as guinea pigs, may be warranted. This study is thefirst to report the effect of laninamivir on influenza A virus infec-tions in ferrets.
Although the DPI has been widely used for delivering powdereddrugs to animals (Nahar et al., 2013), we identified several limita-tions. These include the total dose not being discharged in a singleair delivery and the loss of some powder from ‘backflow’ duringdrug administration. While the backflow of the powder cloudwas able to be reduced with a more gentle air shot, this resultedin a larger mass of powder being retained in the device (data notshown).
The dosage of LO used in this study was based on the humandose of approximately 0.5 mg/kg (40 mg of LO for an 80 kg averagemale 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/kgsolubilised LO) (Kubo et al., 2010). However, given the lack of effec-tiveness seen here, a pharmacokinetic/dynamic (PK/PD) study inferrets 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 ofthe distribution of the powder within the respiratory tract and theeffect 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 inhumans and serve as a better method for assessing NAI effective-ness in ferrets.
In summary, the feasibility of delivering LO into ferrets by DPIhas been evaluated, but further model development for thismethod of delivery, including PK/PD analysis, is warranted beforean accurate assessment of the effect of LO treatment on differentinfluenza virus infections can be studied.
Acknowledgements
This work was supported by NHMRC/A*STAR grant (1055793).The Melbourne WHO Collaborating Centre for Reference andResearch on Influenza is supported by the AustralianGovernment Department of Health.
We thank Lauren Redman, Nikki Hearne, Rachael Murphy,Crispin Agpasa and Miranda Spiteri and John Moody at AnimalServices bioCSL for providing assistance in animal handling.
Laninamivir was kindly provided free of charge byDaiichi-Sankyo, Japan to the WHO Collaborating Centre forReference and Research on Influenza, Melbourne.
Laninamivir octanoate was kindly provided free of charge byBiota Pharmaceuticals, Australia to Monash Institute ofPharmaceutical Sciences.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.antiviral.2015.05.007.
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