Review Laninamivir and Its Prodrug, CS-8958: Long-Acting Neuraminidase Inhibitors for the Treatment of Influenza

Antiviral Chemistry & Chemotherapy 2010; 21:71–84 (doi: 10.3851/IMP1688)

Review Laninamivir and its prodrug, CS-8958: long-acting neuraminidase inhibitors for the treatment of influenza

Makoto Yamashita1*

1Biological Research Laboratories, Daiichi Sankyo Co., Ltd, Tokyo, Japan

*Corresponding author e-mail: [email protected]

Oseltamivir and zanamivir are currently licensed worldwide after an intranasal administration of CS-8958, therapeutic for influenza treatment and chemoprophylaxis. Both drugs administration of a single dose of CS-8958 showed supe- require twice-daily administration for 5 days for treatment. rior efficacy to repeated administrations of zanamivir or A new influenza drug,laninamivir (code name R-125489), oseltamivir in animal infection models for influenza A and and its prodrug form, CS-8958 (laninamivir octanoate or B viruses. These include pandemic (2009) H1N1 virus and laninamivir prodrug), which are long-acting neuramini- HPAI H5N1 virus. Prophylactic single administration of dase inhibitors, are introduced in this review. Laninamivir CS-8958, as early as 7 days prior to infection, also showed potently inhibited the neuraminidase activities of various superior efficacy. Finally, the potential of a single inhala- influenza A and B viruses, including subtypes N1–N9, pan- tion of CS-8958 for influenza patients was demonstrated demic (2009) H1N1 virus, highly pathogenic avian influ- by clinical studies, and CS-8958 has been approved and is enza (HPAI) H5N1 viruses and oseltamivir-resistant viruses. commercially available as Inavir® (Daiichi Sankyo Co., Ltd, Because of the long retention of laninamivir in mouse lungs Tokyo) in Japan.

Introduction

Influenza is a serious respiratory illness, which can be disorder, the pathogenicity of this virus is clearly higher debilitating, and could cause complications that lead to than that of seasonal influenza viruses in animal mod- hospitalization and death, especially in elderly individu- els [4–6] and humans, including those who do not have als. This respiratory disease is caused by influenza A and underlying illnesses [7]. The World Health Organization B viruses, which are pathogens that are highly contagious (WHO) [8] has reported >18,000 deaths worldwide as of among humans. Influenza A viruses are classified into 2 July 2010 and the US Centers for Disease Control and subtypes on the basis of the antigenicities of haemagglu- Prevention [9] estimated a 0.02% mortality rate of mid- tinin (HA) and neuraminidase (NA) molecules. To date, level range depending on age. Another possible concern 16 HA subtypes (H1–H16) and 9 NA subtypes (N1–N9) is a pandemic caused by highly pathogenic avian influ- have been reported. Seasonal influenza or influenza epi- enza (HPAI) H5N1 viruses. In 1997, human infections demics are caused by influenza A viruses H1N1 and with HPAI H5N1 viruses were first documented in Hong H3N2 and influenza B virus [1]. The global burden of Kong [10–12]. Since 2003, these viruses have spread influenza epidemics is believed to be 3.5 million cases of throughout Asia, Europe and Africa with high morbidity severe illness and 300,000–500,000 deaths annually [2]; and mortality among avian species and with occasional these numbers were calculated before the new pandemic transmission to humans, resulting in high mortality. A in 2009. In the past 100 years, humans have experienced total of 507 cases infected with the HPAI H5N1 virus three influenza pandemics: the first in 1918 (H1N1), the and as many as 302 deaths were reported as of 18 Octo- second in 1957 (H2N2) and the third in 1968 (H3N2) ber 2010 [13]. Although human-to-human transmission [1]. Since its emergence in the early spring of 2009, is rare, once the H5N1 viruses acquire this ability, a dev- pandemic (2009) H1N1 influenza A viruses have been astating pandemic might be inevitable. circulating worldwide [3]. Although most infected indi- Two countermeasures, vaccination and treatment viduals have exhibited an uncomplicated mild respiratory with antivirals, are available to control human influenza.

Although vaccination plays a crucial role in influenza virus is already adamantane-resistant [3]. Moreover, the prophylaxis, it is insufficient both for prophylaxis and emergence of adamantane-resistant HPAI H5N1 viruses treatment against a pandemic virus; therefore, antivirals in Vietnam, Cambodia and Thailand [18] has prompted are an important tool that might be used to mitigate the WHO to recommend oseltamivir for the treatment influenza pandemics. Currently, two types of anti-influ- and prophylaxis of human H5N1 influenza virus infec- enza virus drugs are available: M2 ion channel blockers tions [19]. Accordingly, many countries have stockpiled (adamantanes) [14] and NA inhibitors. In Figure 1, a life oseltamivir in anticipation of an H5N1 pandemic. The cycle of influenza virus and action points of the inhibi- second and most recently developed class of drugs with tors are explained; however, in terms of adamantanes, activities against influenza A and B viruses are the NA adamantane-resistant viruses readily emerge and are inhibitors, which bind to the NA of newly formed virus already prevalent worldwide among the seasonal influ- particles and prevent their efficient release from the host enza viruses (both the H1N1 and the H3N2 subtypes) cell [20]. Two NA inhibitors, zanamivir (inhaled drug, [15,16]. The adamantane drugs have not been recom- 10 mg/dose; Relenza) and oseltamivir (oral drug, 75 mg/ mended for use for the treatment or chemoprophylaxis dose; Tamiflu), are currently licensed worldwide. Both of influenza in the US since the 2005 influenza season drugs require twice-daily administration for treatment. [15,17]. The recently emerged pandemic (2009) H1N1 Another NA inhibitor, peramivir (single intravenous

Figure 1. Life cycle of the influenza virus and the inhibition step of neuraminidase inhibitors

A E F

H+ B

vRNA mRNA D

+RNA

A. The haemagglutinin (HA) of influenza virus recognizes neuraminic acid linked to glycoproteins or glycolipids on the cell surface, and viruses thus bind to target cells. B. The viruses bound to cells penetrate into the cell via endocytosis. C. Protons in the endosome enter the virion via the M2 ion channel protein, and the HA structure altered by acidification triggers membrane fusion, followed by the release of viral RNAs (vRNAs) into the cytoplasm. Adamantane drugs block the M2 ion channel. D. From vRNAs, messenger RNAs (mRNAs) for protein synthesis and positive-strand RNA (+RNA) for replication of vRNAs are synthesized in the nucleus. E. vRNAs for progeny viruses are transported close to the cell membrane. F. Viral proteins are synthesized. G. vRNAs and viral proteins are assembled and generate progeny viruses on the cell surface. H. Progeny viruses form aggregates of each other or with cells through the binding of HA to neuraminic acid on viruses and/or cells. I and J. Viral neuraminidase (NA) cleaves the neuraminic acid linkage, releasing progeny viruses from the virus aggregates and expanding infection. NA inhibitors inhibit this step to stop the release of progeny viruses from virus aggregates. The inset shows virus aggregates (arrow) when cultured cells were infected with influenza viruses in the presence of laninamivir.

drip infusion, 300 mg/dose; Rapiacta), was licensed in Figure 2. Chemical structures of laninamivir and CS-8958 2010 in Japan. Oseltamivir is predominant and is used worldwide for the treatment of influenza, and the gen- eration and circulation of oseltamivir-resistant seasonal OH OCH3 influenza viruses have become major concerns [21–25]. H In particular, the worldwide prevalence of oseltamivir- O CO2H resistant mutants of seasonal H1N1 virus with the OH H274Y substitution (histidine to tyrosine at position H3C NH 274, numbering based on the N2 subtype) in NA has HN O been reported. In addition, 95% of H1N1 isolates tested NH from the fourth quarter of 2008 to January 2009 [26] H2N and almost all the H1N1 isolates of the 2008–2009 season in the US [27] were reported to be oseltamivir-resist- Laninamivir (R-125489) ant. Additionally, a number of oseltamivir-resistant pandemic 2009 H1N1 viruses [28] and HPAI H5N1 viruses [25] have already appeared, although their appearance is still sporadic. These epidemics of oseltamivir-resistant influenza viruses therefore necessitate the development O of alternative antiviral agents. H3C O OCH3 Yamashita et al. [29] and Kubo et al. [30] found a new H potent NA inhibitor, laninamivir (code name R-125489), O CO2H and reported that CS-8958 (laninamivir octanoate or laninamivir prodrug) acts as a long-acting NA inhibitor. The OH H3C HN results of in vitro and in vivo experiments of laninamivir HN and CS-8958 [4,29–34] and the clinical studies of CS-8958 O NH [35,36] are introduced in this review. The potential of the H N single administration of CS-8958 for influenza patients 2 was confirmed by the clinical studies conducted for the Laninamivir prodrug (CS-8958) 2008–2009 influenza season [36–38]. CS-8958 has been approved and is commercially available as Inavir® (Dai- ichi Sankyo Co., Ltd, Tokyo) in Japan. Reproduced with permission from the American Society for Microbiology [29].

Compound H1N1 [29], pandemic (2009) H1N1 [4], H3N2 [29], Laninamivir (R-125489) is a potent NA inhibitor HPAI H5N1 viruses [33] and influenza B viruses [29]. of various influenza viruses. Its chemical structure is Laninamivir showed a 50% inhibitory concentra-

(2R,3R,4S)-3-acetamido-2-[(1R,2R)-2,3-dihydroxy- tion (IC50) of 0.41 nM to pandemic (2009) H1N1, A/

1-methoxypropyl]-4-guanidino-3,4-dihydro-2H- California/4/2009, and exhibited a median IC50 of pyran-6-carboxylic acid, as shown in Figure 2 [29]. It 1.55 nM to nine pandemic (2009) H1N1 strains isolated demonstrated a prolonged survival effect in a mouse in Japan. The values were comparable to two other NA infection model with influenza virus. Moreover, the inhibitors, zanamivir and oseltamivir carboxylate, an esterified forms of laninamivir, with acyl chains of active metabolite of oseltamivir. Also, laninamivir inhib- various lengths, significantly improved the prolonged ited NA activity of HPAI H5N1 viruses with an IC50 survival effect in the same model. Among these forms, of 0.28–0.32 nM. This IC50 was comparable to that of 3-(O)-octanoyl laninamivir (CS-8958; Figure 2) is the zanamivir and oseltamivir carboxylate, with the excep- best compound in terms of its life-prolonging effect tion of A/Indonesia/UT3006/05 (H5N1), which exhib- [29]. Both compounds were first discovered by Daiichi ited reduced susceptibility to oseltamivir carboxylate

Sankyo Co., Ltd. (IC50=10 nM). Laninamivir showed less potent inhibitory activities against NA of H3N2 and influenza B viruses In vitro activities compared with zanamivir and oseltamivir carboxylate.

The IC50 of laninamivir to H3N2 was approximately Neuraminidase inhibitory activity 10× lower than that of oseltamivir carboxylate. A summary of NA inhibitory activities is shown in To date, 16 HA subtypes (H1–H16) and 9 NA Table 1. Laninamivir had potent inhibitory activity to subtypes (N1–N9) of influenza A viruses have been NAs of various influenza viruses, such as the seasonal described, but only a limited number of subtypes

Antiviral Chemistry & Chemotherapy 21.2 73 M Yamashita

Table 1. Inhibitory activities of laninamivir to neuraminidases of various influenza viruses

Number of Median IC50, nM (range) Virus type Virus strain strains investigated Laninamivir Zanamivir Oseltamivir carboxylate

Seasonal H1N1 – 11 2.09 (1.29–5.97) 1.38 (0.751–3.62) 1.26 (0.658–2.39) H3N2 – 15 14.2 (7.09–38.8) 7.33 (3.42–14.2) 1.33 (0.706–3.44) B – 23 15.9 (10.4–31.4) 6.41 (3.55–12.0) 9.55 (3.09–15.6) Pandemic (H1N1) 2009 A/California/04/09 – 0.41 0.32 0.96 Japanese isolates 9 1.55 (1.21–1.75) 1.35 (1.19–1.55) 0.679 (0.607–0.831) HPAI H5N1 – 3 0.29 (0.28–0.32) 0.15 (0.07–0.72) 0.35 (0.31–10.00) N1–N9a – 9 (1.81–27.9) (1.40–11.5) (0.925–3.65) H5N1 A/R(duck/Mongolia/54/ – 4.54 2.99 2.17 01–duck/Mongolia/47/01)b H2N2 A/Singapore/1/57 – 11.4 3.66 0.925 H5N3 A/duck/Hokkaido/84/2002 – 13.3 3.65 2.94 H8N4 A/turkey/Ontario/6118/68 – 7.08 5.58 3.65 H12N5 A/duck/Alberta/60/76 – 2.34 1.83 1.43 H11N6 A/duck/England/1/56 – 25.5 8.57 2.81 H7N7 A/seal/Massachusetts/1/80 – 27.9 11.5 2.56 H3N8 A/duck/Ukraine/1/63 – 1.81 1.40 2.12 H11N9 A/duck/Memphis/546/74 – 6.81 3.31 1.70 aData for nine viruses with subtype N1–N9 are described. bReassortant virus generated from A/duck/Mongolia/54/01 (H5N2) and A/duck/Mongolia/47/01 (H7N1).

HPAI, highly pathogenic avian influenza; IC50, 50% inhibitory concentration.

(H1N1, H2N2 and H3N2) have circulated in humans This phenomenon could be partly explained by recep- [1]. In the future, new viruses with other NA subtypes tor specificity of the virus. HA is a receptor recognition will possibly generate and circulate in humans. As protein of human influenza virus, which mainly recog- shown in Table 1, laninamivir inhibited all NA sub- nizes sialic acid with an α-2-6 linkage as a receptor on types and might be effective against emerging viruses the cells [40,41]. Meanwhile, the major sialic acid link- with NA subtypes other than N1 and N2 [29]. age on the Madine–Darby canine kidney (MDCK) cells, As shown in Table 2, laninamivir inhibited NA which was used for the plaque assay, has been reported activity of the oseltamivir-resistant viruses of seasonal to be an α-2-3 linkage [42,43]. This recognition/recep- H1N1, HPAI H5N1 and H3N2, which were isolated tor mismatch induces a weak binding of viruses to the from infected patients [29]. Also, laninamivir main- cells, enabling progeny viruses to be easily released from tained inhibitory activity to oseltamivir-resistant pan- the infected cells without NA action. In this case, influ- demic (2009) H1N1 virus, which possessed the H274Y enza viruses could replicate in the cells in the presence substitution [34]. According to these results, it should of NA inhibitors. be noted that the degrees of resistance of oseltamivir carboxylate and peramivir to H274Y mutants were In vivo efficacy comparable to each other, and were 603-fold and 564- fold, respectively (Table 2). Therapeutic efficacy after a single administration of CS-8958 in animal infection models Virus susceptibility in cell culture In vitro inhibitory activities of laninamivir were The inhibitory effect of laninamivir to viral replication not largely different from those of two other NA in cultured cells was investigated. Also, the 50% effec- inhibitors. However, octanoyl laninamivir, CS-8958, tive concentration (EC50) determined by a plaque assay showed good efficacy by a single intranasal admin- for seasonal H1N1, H3N2 and influenza B viruses, istration in an animal infection model. The licensed and the 90% effective concentration on the basis of the drugs, zanamivir (inhaled drug, 10 mg/dose) and osel- median tissue culture infective dose value of virus titre tamivir (oral drug, 75 mg/dose), require a twice-daily for the pandemic (2009) H1N1 virus are shown in Table administration for 5 days for treatment of influenza. 3. Laninamivir strongly inhibited the replication of these The virus titres in the lungs of mice infected with viruses. The EC50 values have a very large range. It was influenza virus A/PR/8/34 were measured after asin- reported that the susceptibility of influenza viruses to gle intranasal administration of CS-8958, a twice-daily NA inhibitors was variable, indicating that NA inhibi- oral administration of oseltamivir and a twice-daily tory activity is currently the only in vitro assay that is intranasal administration of zanamivir. Groups treated predictive of in vivo susceptibility to NA inhibitors [39]. with CS-8958 at doses of 0.027 and 0.080 mg/kg both

Table 2. Inhibitory activity of laninamivir, zanamivir and oseltamivir carboxylate against oseltamivir-resistant influenza viruses

Oseltamivir Laninamivir Zanamivir carboxylate a b b b Virus type Strain Mutation in NA IC50, nM Ratio IC50, nM Ratio IC50, nM Ratio

Seasonal H1N1 A/Yokohama/67/2006 (clone 1) WT 3.03 – 2.70 – 2.28 – A/Yokohama/67/2006 (clone 11) H274Y 5.62 1.9 3.05 1.1 755 330 H3N2 A/Kawasaki/IMS22A-954/2003 WT 15.4 – 8.29 – 1.25 – A/Kawasaki/IMS22B-955/2003 R292K 10.6 0.69 11.2 1.4 10,400 8,400 A/Yokohama/IMS9A-2029/2003 WT 19.2 – 10.7 – 1.78 – A/Yokohama/IMS9B-2050/2003 E119V 13.2 0.69 7.71 0.72 140 79 A/Kawasaki/MS31A-1030/2002 WT 13.4 – 7.82 – 1.18 – A/Kawasaki/MS31B-1206/2002 N294S 37.3 2.8 13.5 1.7 37.2 32 HPAI H5N1 A/Hanoi/30408/05 WT 0.32 – 0.72 – 0.35 – H274Y 1.1 3.4 0.68 0.94 430 1,200 N294S 1.6 5.0 0.57 0.79 1.6 4.6 A/Vietnam/1203/04 WT 0.28 – 0.15 – 0.31 – H274Y 2.1 7.5 0.22 1.5 1,100 3,500 N294S 1.4 5.0 0.48 3.2 28 90 Pandemic (H1N1) 2009cd Three strains WT 0.74 – 1.20 – 1.90 – Four strains H274Y 1.14 1.5 1.04 0.87 1,146 603 aThe left amino acid mutated to the right one at the indicated residue number, which is based on the N2 neuraminidase (NA) numbering. Amino acids are described using a b c one-letter symbol. The ratio of the 50% inhibitory concentration (IC50) of mutant virus to that of wild-type virus is indicated. Mean IC50 values to three wild-type strains of A/New York/18/2009, A/Washington/29/2009 and A/Singapore/91/2009 and four H274Y mutants of A/Osaka/180/2009, A/Washington/29/2009, A/Hong Kong/2369/2009 d and A/Singapore/57/2009 are shown. Data from [34]. The IC50 values were determined by fluorescent assay using 2-(4-methylumbelliferyl)-α-N-acetylneuraminic acid as a fluorogenic substance. The IC50 values of peramivir to WT and H274Y mutants were 0.22 nM and 124 nM, respectively. HPAI, highly pathogenic avian influenza.

Table 3. Inhibitory activities of laninamivir, zanamivir and oseltamivir carboxylate against replication of various influenza viruses in cell cullture

a Number of EC50, nM Virus type/subtype Virus strain strains investigated Laninamivir Zanamivir Oseltamivir carboxylate

Seasonal H1N1 – 3 0.24–2.4 0.57–11 0.49–13 H3N2 – 4 1.3–230 1.6–360 0.26–80 B – 5 4.1–11 6.4–22 15–120 Pandemic (H1N1) 2009 A/California/04/09 – 4.24 17.67 10.56 N1–N9b – 9 0.26–2.5 0.58–3.6 0.093–0.88 H5N1 A/R(duck/Mongolia/54/01– – 0.34 0.79 0.88 duck/Mongolia/47/01) H2N2 A/Singapore/1/57 – 0.43 0.79 0.11 H5N3 A/duck/Hokkaido/84/2002 – 0.97 1.1 0.40 H8N4 A/turkey/Ontario/6118/68 – 0.26 0.58 0.87 H12N5 A/duck/Alberta/60/76 – 0.75 1.6 0.30 H11N6 A/duck/England/1/56 – 0.83 2.0 0.45 H7N7 A/seal/Massachusetts/1/80 – 2.5 3.6 0.54 H3N8 A/duck/Ukraine/1/63 – 1.1 1.5 0.64 H11N9 A/duck/Memphis/546/74 – 0.66 0.77 0.093 a Data are ranges of the strains investigated or means of three different assays. The 50% effective concentration (EC50) values were determined by a plaque assay, and b are described on the basis of the median tissue culture infective dose. Data for nine viruses with subtype N1–N9 are described. showed statistically significant reductions in virus efficacy of the single administration of CS-8958 was titres compared with the oseltamivir group at a dose of confirmed in a lethal mouse infection model [30]. 10 mg/kg (Figure 3A). The CS-8958 group at a dose of The efficacy of CS-8958 was investigated in ferrets, 0.24 mg/kg (0.5 µmol/kg) showed a similar reduction in which are susceptible to influenza virus and also virus titres compared with the zanamivir group at a dose develop an upper respiratory tract infection as a result of 0.17 mg/kg (0.5 µmol/kg; Figure 3B). The superior of influenza virus infection. At 4 h post-infection, ferrets

Antiviral Chemistry & Chemotherapy 21.2 75 M Yamashita

Figure 3. Therapeutic efficacy of a single administration of infected with B/Malaysia/2506/2004 received a dose of CS-8958 in the mouse infection model saline intranasally, a 0.24 mg/kg (0.5 µmol/kg) dose of CS-8958, a 0.17 mg/kg (0.5 µmol/kg) dose of zanamivir or 25 mg/kg of a twice-daily oral administration A of oseltamivir from 4 hpi. The virus titres in the nasal 8.0 washes are shown in Figure 4 [30]. The single intranasal administration of CS-8959 significantly decreased virus titres in the ferret infection model. 7.0 Therapeutic efficacy after a single administration of a PFU/lungs 6.0 a CS-8958 in HPAI H5N1 virus and in the pandemic 10 (2009) H1N1 infection mouse model

e, log The therapeutic efficacy of CS-8958 against HPAI 5.0 H5N1 influenza viruses was evaluated using a mouse lethal infection model. Mice were infected with three rus titr Vi 4.0 different strains of HPAI H5N1 viruses, and were then given a single dose of CS-8958 intranasally or were 24 48 72 96 treated with oseltamivir administered orally twice-daily for 5 days. Survival of the infected mice was monitored

Time post-infection, h Figure 4. Therapeutic efficacy of a single administration of B CS-8958 in the ferret infection model 9.0

8.0 7.0 NS 7.0 PFU/lungs 10

PFU/ml 6.0 10

6.0 a e, lo g 5.0 b 5.0 irus titr V 4.0 b 4.0 24 48 72 96 e in nasal wash, log 3.0 ral titr Vi Time post-infection, h 2.0 1 2 3 Time post-infection, days Control CS-8958 0.027 mg/kg Oseltamivir 1.0 mg/kg CS-8958 0.08 mg/kg Control Zanamivir 0.5 µmol/kg Oseltamivir 10 mg/kg CS-8958 0.5 µmol/kg Oseltamivir 25 mg/kg CS-8958 0.5 µmol/kg Zanamivir 0.5 µmol/kg

Reproduced with permission from the American Society for Microbiology [30]. Reproduced with permission from the American Society for Microbiology [30]. (A) Virus titres in the mouse lungs after saline administration, repeated oral Ferrets were intranasally infected with B/Malaysia/2506/2004 (1,000 plaque- administrations of oseltamivir (1.0 mg/kg or 10 mg/kg) and a single intranasal forming units [PFU]). Virus titres are shown for the nasal wash after saline administration of CS-8958 (0.027 mg/kg or 0.08 mg/kg). (B) Virus titres in the administration, repeated oral administrations of oseltamivir (25 mg/kg), a single mouse lungs after saline administration, repeated intranasal administrations of intranasal administration of zanamivir (0.5 µmol/kg, corresponding to 0.17 mg/kg) zanamivir (0.5 µmol/kg, corresponding to 0.17 mg/kg) and a single intranasal and a single intranasal administration of CS-8958 (0.5 µmol/kg, corresponding to administration of CS-8958 (0.5 µmol/kg, corresponding to 0.24 mg/kg). Mice 0.24 mg/kg). The administrations were performed once for zanamivir and CS-8958 were infected with A/PR/8/34 (H1N1; 30 plaque-forming units [PFU]) at 0 h. and twice daily for oseltamivir. Virus titres were measured at three time points. The administrations were performed at the indicated time points, once for The virus titres in the nasal washes from the untreated ferrets decreased after 2 CS-8958 (black arrow) and twice daily for other NA inhibitors (gray arrows). days post-infection. This was mainly a result of the limited number of cells in the The virus titres were measured at three time points. Statistically significant noses of the ferrets that were susceptible to virus infection, and was less likely to differences were observed between both groups of CS-8958 and the 10 mg/kg be caused by innate immunity, if any. Therefore, statistical analysis was performed administration group of oseltamivir (A). Statistical analysis was performed by a on the basis of the area under the curve of the viral titres from 1–2 days post- two-way ANOVA. aP<0.001. NS, non-significant. infection by a two-way ANOVA. aP<0.01 and bP<0.001 versus control.

for 21 days and survival data of mice infected with the lungs and brain of HPAI virus A/Hanoi/30408/05 A/Vietnam/1203/04 are shown in Figure 5A. A single clone 7- or A/Indonesia/UT3006/05-infected mice. dose of CS-8958 was efficacious for mice infected with CS-8958 at 0.75 or 1.5 mg/kg dramatically inhibited the HPAI H5N1 viruses [33]. This therapeutic efficacy the replication of both viruses, particularly in the of a single dose of CS-8958 against H5N1 viruses was brain, compared with oseltamivir [33]. These results also confirmed by the determination of virus titres in indicate that CS-8958 confers more potent and long-

Figure 5. Therapeutic efficacy of CS-8958 against HPAI H5N1 virus and pandemic (2009) H1N1 virus in the mouse infection model

A HPAI H5N1 virus 100

a Control CS-8958 0.075 mg/kg 75 a CS-8958 0.75 mg/kg % CS-8958 1.5 mg/kg 50 a Oseltamivir 5 mg/kg Oseltamivir 50 mg/kg Survival rate, rate, Survival 25

0 0246810 12 14 16 18 20 Time post-infection, days B 8 Day 3 8 Day 6 Control Oseltamivir 4 mg/kg Oseltamivir 40 mg/kg 7 a 7 a b Zanamivir 0.8 mg/kg a Zanamivir 8 mg/kg

PFU/g lungs PFU/g b lungs PFU/g CS-8958 0.7 mg/kg

10 6 10 6 b a

5 5 Viral titre, log titre, Viral log titre, Viral

4 4

Control Control Zanamivir CS-8958 Zanamivir CS-8958 Oseltamivir Oseltamivir

(A) Reproduced from [33]. Mice were intranasally infected with A/Vietnam/1203/04 (H5N1; 4×50% mouse lethal dose) and were then given CS-8958 intranasally once (0.075 mg/kg, 0.75 mg/kg or 1.5 mg/kg), oseltamivir orally twice daily for 5 days (5 mg/kg or 50 mg/kg) or distilled water/saline as control. Survival was monitored daily for 21 days. Statistical analysis against the control was done by a log-rank test using a joint ranking method (aP<0.05). (B) Reprinted by permission from Macmillan Publishers, Ltd [4]. Mice were intranasally infected with A/California/04/09 (pandemic [2009] H1N1; 10,000 plaque-forming units [PFU]) and administered oseltamivir (orally, twice daily for 5 days), zanamivir (intranasally, once daily for 5 days), CS-8958 (intranasally, once) or distilled water/phosphate-buffered saline (control). Doses are indicated in the figure. The virus titres in lungs on days 3 and 6 were determined by plaque assays in Madine–Darby canine kidney cells. The statistical significance of differences against control mice was assessed by the Student’s t-test (bP<0.05 and cP<0.01). HPAI, highly pathogenic avian influenza.

Antiviral Chemistry & Chemotherapy 21.2 77 M Yamashita

Figure 6. Therapeutic efficacy of CS-8958 against oseltamivir-resistant viruses with the H274Y substitution in the mouse infection model

ABSeasonal H1N1 with H274Y HPAI H5N1 virus with H274Y 100 b 5.0 b

a 4.0 PFU/lungs

10 b 50 b e, log

3.0 Survival rate, % 25 ral titr Vi

2.0 0 37 61 85 02468101214161820 Time post-infection, h Time post-infection, days

Control CS-8958 0.08 mg/kg Control CS-8958 1.5 mg/kg CS-8958 0.027 mg/kg Oseltamivir 10 mg/kg CS-8958 0.075 mg/kg Oseltamivir 5 mg/kg CS-8958 0.75 mg/kg Oseltamivir 50 mg/kg

(A) Reproduced with permission from the American Society for Microbiology [30]. Mice were infected with oseltamivir-resistant A/Yokohama/67/2006 (H1N1) with the H274Y substitution (1,000 plaque-forming units [PFU]) and were then intranasally administered once with CS-8958 (0.027 mg/kg or 0.08 mg/kg) or orally administered twice daily with oseltamivir (10 mg/kg) or control. Viral titres in the lungs were measured at three time points. Statistically significant differences were observed between CS-8958 and control (aP<0.001). (B) Reproduced from [33]. Mice were intranasally infected with A/Vietnam/1203/04 (H5N1) with the H274Y substitution (4×50% mouse lethal dose) and then were intranasally administered once with CS-8958 (0.075 mg/kg, 0.75 mg/kg or 1.5 mg/kg) or orally administered twice daily for 5 days with either oseltamivir (5 mg/kg or 50 mg/kg) or control. Survival was monitored daily for 21 days. Statistical analysis against the control was done by a log- rank test using a joint ranking method (bP<0.05). lasting protection to mice against H5N1 influenza Yokohama/67/2006 (H1N1) H274Y infection model, viruses than oseltamivir. statistically significant virus titre reduction was observed Next, efficacy against pandemic (2009) H1N1 A/ in a single dose of CS-8958 at 0.08 mg/kg compared California/04/09 virus was investigated in the mouse with a control. No reduction in virus titres was observed infection model. Mice infected with the virus were given in a twice-daily administration of oseltamivir at 10 mg/ a single dose of CS-8958 intranasally, oseltamivir was kg. In the HPAI A/Vietnam/1203/04 (H5N1) H274Y orally administered twice-daily for 5 days or zanamivir virus infection model, only 12.5% and 50% of mice sur- was administered intranasally once-daily for 5 days. The vived at a dose of 5 and 50 mg/kg of oseltamivir (twice virus titres in the mice lungs were measured on days 3 daily for 5 days), respectively. By contrast, one-half of and 6 after infection (Figure 5B) [4]. Again, a single the mice survived with a single dose of 0.075 mg/kg administration of CS-8958 showed significant efficacy of CS-8958. It should be noted that for one-half of the and stronger decrease in viral titres compared with a mice to survive, the dose of CS-8958 was comparable to repeated administration of oseltamivir or zanamivir [4]. a presumed clinical dose of CS-8958, whereas as much as 50 mg/kg (twice daily for 5 days) of oseltamivir (clini- Therapeutic efficacy after a single administration cal dose, 75 mg/dose) was required. Thus, CS-8958 also of CS-8958 in oseltamivir-resistant H1N1 and HPAI showed good efficacy to oseltamivir-resistant virus. H5N1 virus infection mouse model The efficacy after a single dose of CS-8958 was con- Prophylactic efficacy after a single administration of firmed in a mouse model infected with the oseltamivir- CS-8958 in animal models resistant seasonal H1N1 virus monitored by virus titres Once-daily administration of zanamivir (inhaled drug, in lungs (Figure 6A) [30] and with the HPAI H5N1 virus 10 mg/dose) and of oseltamivir (oral drug, 75 mg/dose) monitored by the survival rate (Figure 6B) [33]. Both have been licensed as prophylactic drugs. This suggests viruses have the H274Y substitution. In the seasonal A/ that the half life (t1/2) of each drug in target tissue is

Figure 7. Prophylactic efficacy of CS-8958 in the mouse infection model

AB a 100 100 a a

80 80

60 60 c

b 40 40 Survival, % Survival, % b 20 20

0 0 0 246810 12 14 16 18 20 0 246810 12 14 16 18 20 Time post-infection, days Time post-infection, days

Control 12 h 1 day 4 days 7 days

Reproduced with permission from the American Society for Microbiology [30]. Mice were infected with A/PR/8/34 (H1N1; 100 plaque-forming units) on day 0. (A) Oseltamivir at 110 mg/kg and (B) CS-8958 at 0.17 mg/kg were administered at 12 h, 1 day, 4 days or 7 days before infection. Saline was administered intranasally as a control at 7 days before infection. Survival was monitored daily for 20 days. Statistical analysis against control was done by a log-rank test based on a joint ranking method (aP<0.001, bP<0.05 and cP<0.01). not long; therefore, they require once-daily administra- could be a result of the unique characteristics of the tion. By comparison, the t1/2 of CS-8958 after intranasal compounds described below. administration to mice is quite long. Prophylactic efficacy of a single dose of CS-8958 was investigated in a mouse Long-lasting drug in respiratory organs of animals infection model. A single administration of CS-8958 The lung concentration–time profiles of CS-8958 and prior to infection with A/PR/8/34 showed a life-prolong- laninamivir after a single intranasal administration of ing effect in the mouse infection model. One-half of the CS-8958 at a dose of 0.5 µmol/kg (0.236 mg/kg) to mice administered CS-8958 at 0.17 mg/kg 7 days before mice are shown in Figure 8 [31]. After administra- infection survived. By contrast, only one-quarter of the tion, the lung CS-8958 concentrations decreased with mice treated with oseltamivir at as much as 110 mg/kg a t1/2 of 0.833 h and, at 12 h post-dose or later, the 1 day before infection survived. The dose of oseltamivir CS-8958 concentrations were below the lower limit was much higher than the clinical setting (Figure 7) [30], of quantification. By contrast, the lung laninamivir probably because of a short t1/2 of 3 h and time of maxi- concentrations increased soon after administration, mum concentration of 0.5 h of oseltamivir carboxylate and, subsequently, laninamivir retained a t1/2 of as in mouse plasma after an intravenous administration of long as 41.4 h. Even at 120 h post-dose, laninamivir oseltamivir carboxylate and oral administration of osel- remained in lungs at a concentration of 0.915 nmol/g tamivir, respectively [44]. Prophylactic efficacies against [31], which is considerably higher than the IC50 of H5N1 virus after the single intranasal administration laninamivir against the NA activities of various influ- of CS-8958 were also confirmed in both the lethal mice enza viruses (Table 1) [31]. In addition, similar long infection model and in the measurement of virus titre retention of laninamivir was observed in the target reduction in mice lungs and brain [33]. tissues (trachea and lung) after intratracheal administration of CS-8958 to rats, and approximately 20% Two unique characteristics of CS-8958 and of the dose (0.2 mg/kg) was retained in the lungs at laninamivir as a long-acting drug 24 h [32]. These results demonstrated that CS-8958 administered intranasally or intratracheally to ani- The therapeutic and prophylactic efficacies achieved mals was converted/hydrolysed to laninamivir and after the single intranasal administration of CS-8958 retained as the hydrolysed form in the target tissues

Antiviral Chemistry & Chemotherapy 21.2 79 M Yamashita

for a long time. The long-lasting characteristics of 50% at 6 h, whereas no inhibitory effect was observed laninamivir in the target tissues for influenza virus at this time point for the other three NA inhibitors. NA infection result in superior efficacy by a single dose of activity of A/New Caledonia/20/99 (H1N1) treated CS-8958, whereas repeated doses of other NA inhibi- with laninamivir and peramivir was suppressed to tors are required for efficacy. approximately 30% at 6 h, whereas the NA activity treated with zanamivir and oseltamivir carboxylate Binding stability of laninamivir to viruses recovered to 80–90% at 6 h. These results suggest that Bantia et al. [45] demonstrated that peramivir, a selec- laninamivir bound to viral NA more stably than any tive NA inhibitor [46,47], binds to NA, with the N9 other NA inhibitors tested. This stable binding of lani- subtype more tightly than oseltamivir carboxylate or namivir might be another reason for the long-acting zanamivir. This characteristic is thought to be one of characteristics of CS-8958. the reasons why peramivir is efficacious by a single intravenous injection. To assess the binding stability of Clinical studies laninamivir to various virus NAs, similar experiments with whole influenza virions were performed, and the Phase I studies results are shown in Figure 9 [33]. In these experiments, The next important point is to confirm whether the virions were treated with excess NA inhibitors, then long-lasting characteristic of laninamivir after a sin- unbound forms of NA inhibitor were removed and the gle intranasal administration of CS-8958 observed in NA reaction was chased over time after adding the NA animals is applicable to humans or not. Healthy male substrate. Considerable differences in the dissociation volunteers participated in double-blinded randomized rates of the NA inhibitors among the test virus strains placebo-controlled trials and received a single dose were observed. NA activity of A/Panama/2007/99 of 5, 10, 20, 40, 80 or 120 mg CS-8958 or a twice- (H3N2) was severely inhibited by all test NA inhibitors daily dose of 20 or 40 mg CS-8958 for 3 days. The for at least 5 h. NA activity of B/Mie/1/93 treated with clinical and laboratory parameters and plasma and laninamivir was appreciably inhibited by approximately urinary concentrations of CS-8958 and laninamivir for the 144 h post-dosing were measured. CS-8958 disap-

peared from plasma with a t½ of approximately 2 h. By Figure 8. Pharmacokinetics in the mouse lungs after intranasal contrast, laninamivir was slowly eliminated from the administration of CS-8958 body, lasting for up to 144 h after administration with

a t½ of approximately 3 days. There were no adverse events related to the test drug in all groups, and it was 100 indicated that when CS-8958 was inhaled by healthy volunteers, it was well-tolerated [35]. CS-8958 Although the detection of the active metabolite in Laninamivir plasma for an extended period might be consistent with 10 a long half-life in the target tissues, such as the trachea and lung, it was not possible to demonstrate this directly through sampling of the target tissue because 1 of the limitations of the Phase I study; however, these data suggested that the long-lasting concentration of laninamivir after a single intranasal administration of Lung concentration, nmol/g 0.1 CS-8958 in animals might also be applicable to humans 0 24 48 72 96 120 who inhaled CS-8958. Time, h Clinical studies in influenza patients

Analyte t½, h Cmax, nmol/g tissue Tmax, h Phase II/III studies in paediatric patients were conducted CS-8958 0.833 NC NC as a double-blinded randomized controlled trial to Laninamivir 41.4 6.41 3.0 compare CS-8958 with oseltamivir in the 2008–2009 influenza season in Japan. During this season, almost all H1N1 viruses were oseltamivir-resistant viruses with the H274Y substitution. Eligible patients were chil- Reproduced with permission from the American Society for Microbiology [31]. Mice were intranasally administered 0.5 µmol/kg (0.236 mg/kg) of CS-8958, and dren, 9 years of age and under, who had febrile influ- the concentration of CS-8958 and laninamivir in lungs was determined. Based on enza symptoms of no more than 36 h duration. Patients the concentration–time profiles of CS-8958 and laninamivir, the pharmacokinetic were randomized to one of three treatment groups: parameters were calculated and are also shown. Cmax, maximum concentration in lungs; NC, not calculated; tmax, time to maximum concentration; t1/2, half-life. a CS-8958 40 mg group, a CS-8958 20 mg group or

Figure 9. Binding stability of NA inhibitors to viruses

A/New Caledonia/20/99 (H1N1) B/Mie/1/93 70,000 70,000

60,000 60,000 t t 50,000 50,000

40,000 40,000

escence uni escence 30,000 uni escence 30,000

Fluor 20,000 Fluor 20,000

10,000 10,000

0 0 060 120 180 240 300 360 060 120 180 240 300 360 Time, min Time, min

A/Panama/2007/99 (H3N2) 20,000

Laninamivir t 16,000 Peramivir Zanamivir 12,000 Oseltamivir carboxylate escence uni escence Without virus 8,000

Fluor Without NA inhibitor

4,000

0 060 120 180 240 300 360 Time, min

Reproduced from [33]. The various influenza viruses treated with excess neuraminidase (NA) inhibitors (10–20× the 90% inhibitory concentration values) and unbound NA inhibitors were removed from the drug–virus mixtures with a Bio-Spin P-6 column (Bio-Rad Laboratories, Hercules, CA, USA). The NA substrate was added to the virus–NA-inhibitor complex and the NA activities were chased for 6 h. A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2) and B/Mie/1/93 were used as virus sources for these experiments. Laninamivir, peramivir, zanamivir or oseltamivir carboxylate were used as NA inhibitors. NA activities without virus and without NA inhibitor are also shown. an oseltamivir group (n=61–62 patients per group). during the course of illness might be a beneficial regimen CS-8958 was administered as a single inhalation. Osel- in terms of therapeutic compliance. tamivir 2 mg/kg was orally administered twice daily for A Phase III study of adults, the Multinational Asian 5 days, which is the regular regimen for paediatric use. Clinical Research for Influenza Virus Extermination on CS-8958 markedly reduced the median time to allevia- Long-Acting Neuraminidase-Inhibitor Study (MAR- tion of influenza illness in comparison with oseltamivir VEL), was performed as a randomized double-blinded in patients infected with oseltamivir-resistant influenza active-controlled study in order to confirm the efficacy A (H1N1); the difference was -60.9 h in the 40 mg and safety of CS-8958 administered as a single inhaled group and -66.2 h in the 20 mg group (Figure 10) [36]. dose of 20 or 40 mg compared with oseltamivir 75 mg By contrast, there were no significant differences in orally administered twice daily for 5 days (a total of 10 the time to alleviation of illness between the CS-8958 times) in adult patients with influenza A or B virus infec- groups and the oseltamivir group against influenza A tion [38]. According to the results, non-inferiority to (H3N2) or B infection. CS-8958 was well tolerated oseltamivir was confirmed in both the 20 mg and 40 mg [36]. CS-8958 was an effective and well-tolerated treat- group of CS-8958 in terms of the primary end point, ment for children with oseltamivir-resistant influenza A which was the time to alleviation of influenza illness. (H1N1) virus infection. A single inhalation of CS-8958 Both CS-8958 doses were well-tolerated [37,38].

Antiviral Chemistry & Chemotherapy 21.2 81 M Yamashita

Figure 10. Therapeutic efficacy of CS-8958 in paediatric influenza patients

Subgroup according to virus type Full analysis set A/H1N1 (full analysis set) 100 100 90 CS-8958 20 mg (n=61) 90 CS-8958 20 mg (n=40) 80 CS-8958 40 mg (n=61) 80 CS-8958 40 mg (n=40) Oseltamivira (n=62) Oseltamivira (n=32) 70 70 60 60 oportion, % oportion, % 50 50 40 40 30 30 20 20 Cumulative pr Cumulative pr 10 10 0 0

0 0 24 48 72 96 24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 120 144 168 192 216 240 264 288 Time post-treatment, h Time post-treatment, h

Reproduced with permission from the American Society for Microbiology [36]. aDosage for oseltamivir was 2 mg/kg twice daily for 5 days. (A) The median time to alleviation of influenza illness was shorter in the CS-8958 groups than in the oseltamivir group, and the difference was -31.9 h in the 40 mg group (P=0.059) and -31.0 h in the 20 mg group (P=0.009) in the full analysis set. (B) The subgroup analyses showed that both dosages of CS-8958 markedly reduced the median time to illness alleviation in the H1N1-infected subpopulation in comparison with oseltamivir.

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Received 12 May 2010; accepted 31 August 2010; published online 9 November 2010