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Evaluation of a dry powder delivery system for laninamivir in a ferret model of influenza infection
Antiviral Research 120 (2015) 66–71
Contents lists available at ScienceDirect
Antiviral Research journal homepage: www.elsevier.com/locate/antiviral
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 School of Applied Sciences and Engineering, Monash University, Churchill, Victoria 3842, Australia 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 b c
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 laninamivir 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 discharged. 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 significant human morbidity and mortality annually (WHO, 2014). Currently the leading class of influenza antiviral drugs are the neuraminidase inhibitors (NAIs), namely zanamivir, oseltamivir, peramivir 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 influenza 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 ⇑ 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: [email protected] (J. Panozzo), DingThomas.Oh@ influenzacentre.org (D.Y. Oh), [email protected] (K. Margo), David. [email protected] (D.A. Morton), david.piedrafi[email protected] (D. Piedrafita), [email protected] (J. Mosse), Aeron.hurt@ influenzacentre.org (A.C. Hurt). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.antiviral.2015.05.007 0166-3542/Ó 2015 Elsevier B.V. All rights reserved.
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, zanamivir and laninamivir are both delivered to humans as a dry powder
J. Panozzo et al. / Antiviral Research 120 (2015) 66–71
formulation that requires active inhalation, therefore mimicking this type of administration in animals is considerably more challenging. In the small number of zanamivir and laninamivir animal studies conducted to date, the drugs have been hydrated and delivered 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 ferrets and determined the effectiveness of the drug in reducing influenza A and B virus replication and disease.
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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 (prepared 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 typically 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.
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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 administered to the ferrets at 2.5 mg/kg 2 h prior to intranasal virus inoculation with 105 tissue culture infectious doses (TCID50) of
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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.
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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 influenza 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 methods), 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:
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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.
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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 shedding 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 ± 3.77 106 cells/mL and 10.50 ± 1.04 106 cells/mL respectively), although 2 of 3 placebo treated ferrets had a particularly high peak cell count (ferret No. 2: 19 106 cells/mL on day 7; ferret No. 1: 16 106 cells/mL on day 9) (Fig. 2C and D). All ferrets 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 substantially 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 similar 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 temperature 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 treatment (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 analysis 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 infections in ferrets. Although the DPI has been widely used for delivering powdered drugs to animals (Nahar et al., 2013), we identified several limitations. 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 effectiveness 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 important 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 intranasal ‘artificial’ infection, may more closely mimic infection in humans and serve as a better method for assessing NAI effectiveness 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,
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Antiviral Research journal homepage: www.elsevier.com/locate/antiviral
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 School of Applied Sciences and Engineering, Monash University, Churchill, Victoria 3842, Australia 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 b c
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 laninamivir 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 discharged. 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 significant human morbidity and mortality annually (WHO, 2014). Currently the leading class of influenza antiviral drugs are the neuraminidase inhibitors (NAIs), namely zanamivir, oseltamivir, peramivir 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 influenza 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 ⇑ 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: [email protected] (J. Panozzo), DingThomas.Oh@ influenzacentre.org (D.Y. Oh), [email protected] (K. Margo), David. [email protected] (D.A. Morton), david.piedrafi[email protected] (D. Piedrafita), [email protected] (J. Mosse), Aeron.hurt@ influenzacentre.org (A.C. Hurt). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.antiviral.2015.05.007 0166-3542/Ó 2015 Elsevier B.V. All rights reserved.
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, zanamivir and laninamivir are both delivered to humans as a dry powder
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formulation that requires active inhalation, therefore mimicking this type of administration in animals is considerably more challenging. In the small number of zanamivir and laninamivir animal studies conducted to date, the drugs have been hydrated and delivered 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 ferrets and determined the effectiveness of the drug in reducing influenza A and B virus replication and disease.
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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 (prepared 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 typically 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.
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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 administered to the ferrets at 2.5 mg/kg 2 h prior to intranasal virus inoculation with 105 tissue culture infectious doses (TCID50) of
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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.
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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 influenza 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 methods), 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:
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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.
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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 shedding 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 ± 3.77 106 cells/mL and 10.50 ± 1.04 106 cells/mL respectively), although 2 of 3 placebo treated ferrets had a particularly high peak cell count (ferret No. 2: 19 106 cells/mL on day 7; ferret No. 1: 16 106 cells/mL on day 9) (Fig. 2C and D). All ferrets 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 substantially 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 similar 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 temperature 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 treatment (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 analysis 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 infections in ferrets. Although the DPI has been widely used for delivering powdered drugs to animals (Nahar et al., 2013), we identified several limitations. 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 effectiveness 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 important 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 intranasal ‘artificial’ infection, may more closely mimic infection in humans and serve as a better method for assessing NAI effectiveness 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,
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