Efficacy of single intravenous injection of peramivir against influenza B virus infection in

AAC Accepts, published online ahead of print on 15 August 2011 Antimicrob. Agents Chemother. doi:10.1128/aac.00412-11 Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Kitano et al., Page 1 1 2 Efficacy of single intravenous injection of peramivir against influenza B virus infection in ferrets and cynomolgus macaques 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Mitsutaka Kitano, 1,3 Yasushi Itoh, 1 Makoto Kodama, 3 Hirohito Ishigaki, 1 Misako Nakayama, 1 Hideaki Ishida, 1 Kaoru Baba, 3 Takahiro Noda, 3 Kenji Sato 4, Yoichiro Nihashi 4, Takushi Kanazu 4, Ryu Yoshida, 3 Ryuzo Torii, 2 Akihiko Sato, 3 * Kazumasa Ogasawara 1 Department of Pathology, Shiga University of Medical Science, Otsu, Shiga 520-2192, 1 Research Center for Animal Life Science, Shiga University of Medical Science, Otsu, Shiga 520-2192, 2 Infectious Diseases, Medicinal Research Laboratories, Shionogi & Co., Ltd., Settsu, Osaka, 566-0022, 3 Drug Metabolism & Pharmacokinetics, Drug Developmental Research Laboratories, Shionogi & Co., Ltd., Toyonaka, Osaka, 561-0825 4, Japan Running title: In vivo effects of peramivir against influenza B virus infection *Corresponding author Infectious Diseases, Medicinal Research Laboratories Shionogi & Co., Ltd. 5-1 Mishima 2-chome, Settsu, Osaka 566-0022, Japan Tel: +81 (6) 6382 2612, Fax: +81 (6) 6382 2598, E-mail: akihiko.sato@shionogi.co.jp 22 1

Kitano et al., Page 2 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 ABSTRACT We evaluated the efficacy of single intravenous dose peramivir for treatment of influenza B virus infection in ferrets and cynomolgus macaques in the present study. A single dose of peramivir (30 and 60 mg/kg) given to ferrets on 1 day post-infection with influenza B virus significantly reduced virus titers in nasal washes and total virus shedding as measured by areas under the curve (AUC) by 2 log 10 compared with control group. Furthermore, nasal virus titers on day 2 post infection and AUCs of the body temperature raise of the ferrets singly injected with peramivir (30 and 60 mg/kg) were lower than those of ferrets orally administered with oseltamivir phosphate (30 and 60 mg/kg/day twice daily for 3 days). In macaques infected with influenza B virus, viral titers in nasal swab fluid on days 2 and 3 post infection and body temperature after single injection with peramivir (30 mg/kg) were lower than those after oral administration with oseltamivir phosphate (30 mg/kg/day for 5 days). Two animal models used in the present study demonstrated that inhibition of viral replication at the early time point after infection was critical in reduction of AUC of virus titers and IL-6 production, resulting in amelioration of symptoms. Our results shown in animal models suggest the early treatment with a single intravenous injection of peramivir is significantly effective for the treatment of influenza B infection. 2

Kitano et al., Page 3 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 INTRODUCTION Peramivir is a neuraminidase (NA) inhibitor for intravenous administration and it has been introduced into clinical practice for adult and child patients in Japan. Peramivir has potent activity against various influenza A and B viruses including highly pathogenic avian influenza (HPAI) viruses and pandemic (H1N1) 2009 influenza virus in vitro and in vivo mouse models (2, 6, 7, 10). Phase 2 clinical trials of intravenous peramivir in Japanese patients with seasonal influenza demonstrated the efficacy and safety of a single intravenous treatment (24). On the basis of these results, the United States Food and Drug Administration issued an emergency use authorization for intravenous peramivir exclusively for severe pandemic H1N1 hospitalized patients (4). Influenza B virus causes seasonal influenza infection and indistinguishable clinical symptoms from those caused by influenza A virus though pandemic influenza A virus infection attracts more attention than does influenza B virus infection (26, 29, 30). Influenza B virus was isolated more frequently and for a longer period after antiviral therapy than influenza A virus (21). Furthermore, influenza B virus epidemics did not diminish after the pandemic of 2009 H1N1 influenza A virus (34). Therefore, treatment for influenza B virus infection is important as well as for influenza A virus infection. NA inhibitors are major antiviral agents of influenza B virus infection because available M2 inhibitors, amantadine and rimantadine, are not active in influenza B viruses since the amino acid sequences of the B/M2 protein differ from those of the A/M2 protein (31). Nonetheless, some studies have claimed that an NA inhibitor oseltamivir was less effective in treating influenza B virus than influenza A virus with regard to duration of fever, irrespective of patient age or the timing of administration of the first dosing (21, 32). The 3

Kitano et al., Page 4 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 difference would be explained by higher 50% inhibitory concentration (IC 50 ) values of oseltamivir against NA activity of influenza B viruses than those of influenza A viruses (33). In addition, influenza B viruses with low susceptibility to NA inhibitors were isolated from immunocompromised children treated with oseltamivir or zanamivir (11, 12). Therefore, further development and evaluation of clinical efficacy of antiviral drugs against influenza B virus are required. Problems in the drug development are that the appearance of influenza B virus is variable season by season compared with influenza A virus that consistently appears almost every year (20) and that preexisting immunity against influenza B virus in humans may make evaluation of drug effects complicated. To solve the problems of shortage of human patients infected with influenza B virus and of preexisting immunity in clinical trials, studies using immunologically naïve animals against influenza B virus would offer the essential experimental approach for testing efficacy of antiviral drugs. Although mice are naturally resistant to most seasonal human influenza viruses unless the viruses are adapted, ferrets are susceptible to human influenza virus, including various influenza A and B viruses, without adaptation (14, 16, 17, 19, 28). Human influenza viruses infect upper respiratory tracts in ferrets and cause clinical symptoms, including fever, nasal congestion, anorexia and sneezing. Therefore, ferrets are often used for examining efficacy of antiviral agents in treatment and prevention of influenza infection (9, 25, 35). On the other hand, nonhuman primates are also susceptible to unadapted human influenza viruses (17). Since clinical symptoms by infection with influenza B viruses in cynomolgus macaque closely reflects the signs of diseases observed in humans (23), we investigated the therapeutic efficacy of a new NA inhibitor peramivir against influenza B virus infection using not only ferrets but also 4

Kitano et al., Page 5 83 84 85 86 87 88 89 90 cynomolgus macaques. In the present study, efficacy of single intravenous administration of peramivir was compared with those of multiple oral administration of oseltamivir phosphate in ferret and macaque models of influenza B virus infection. We demonstrated that a single intravenous injection of peramivir was more effective on reduction of viral titers and amelioration of symptoms in ferrets and cynomolgus macaques after influenza B virus infection than administration with oseltamivir phosphate. These results suggest that peramivir has potential as a treatment against influenza B virus infection. Downloaded from http://aac.asm.org/ on May 13, 2018 by guest 5

Kitano et al., Page 6 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 MATERIALS AND METHODS Compounds. Peramivir was synthesized by BioCryst Pharmaceuticals (Birmingham, AL). Oseltamivir phosphate was purchased from Sequoia Research Products (Oxford, UK), and oseltamivir carboxylic acid was purchased from Toronto Research Chemicals (Ontario, Canada). Viruses and cells. Influenza B viruses, B/SendaiH/1051/2007 and B/Kadoma/1/2005, were kindly provided by Sendai Medical Center and Osaka Prefectural Institute of Public Health, respectively. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and were grown in minimum essential medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen), 100 µg/ml kanamycin sulfate (Invitrogen) in a humidified atmosphere of 5% CO 2 at 37 o C. Ferrets. Approximately nine- to eleven-month-old female ferrets (Japan SLC Inc., Shizuoka, Japan) were used. All ferret studies were conducted under applicable laws and guidelines and after approval from the Shionogi Animal Care and Use Committee. Under anesthesia at least one week before virus inoculation, a data logger (DS1921H-F5, Maxim Integrated Products, Inc., Sunnyvale, CA) was implanted in the peritoneal cavity of each ferret to monitor body temperature every 15 min. The absence of influenza B-specific antibody in sera was confirmed before experiments using the hemagglutination inhibition (HI) test. Cynomolgus macaques. Approximately two- to five-year-old female cynomolgus macaques from the Philippines (Ina Research Inc., Ina, Japan) were used for challenge experiments. Macaque studies were conducted under applicable laws and guidelines and after approval from the Shiga University of Medical Sciences Animal Experiment Committee and Biosafety Committee. Under anesthesia at least one week before virus inoculation, a telemetry probe 6

Kitano et al., Page 7 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 (TA10CTA-D70, Data Sciences International, St. Paul, MN) was implanted in the peritoneal cavity of each macaque to monitor body temperature every 15 min. The absence of influenza B-specific antibody in sera was confirmed before experiments using the HI test. Individual macaques are distinguished by identification numbers. The macaques used in this study did not carry herpes B virus, hepatitis E virus, Mycobacterium tuberculosis, Shigella spp., Salmonella spp., or Entamoeba histolytica. NA inhibition assay. Whole viruses inactivated by NP-40 were used as a source of NA activity. 2 -(4-methylumbelliferyl)-α-D-N-acethylneuraminic acid (MUNANA, Sigma-Aldrich, St. Louis, MO) was used as substrate. The virus, a compound and MUNANA (final concentration 10 mm) were mixed in reaction buffer, and incubated at 37 o C for 30 min. The fluorometric intensity of 4-methylumbelliferon released from MUNANA was measured, the percent inhibition at each concentration of drug was determined, and the 50% inhibitory concentration (IC 50 ) was determined. The results are reported as the average of three experiments. Pharmacokinetic (PK) analysis. Female ferrets and cynomolgus macaques (three animals per group) were given peramivir (30 mg/kg) intravenously or oseltamivir phosphate (30 mg/kg) orally. Blood samples were collected from 0.083 h to 24 h after dosing and centrifuged to obtain plasma. The concentration of peramivir and oseltamivir carboxylate in plasma was determined by liquid chromatography-tandem mass spectrometry (LC/MS/MS). PK parameters were calculated and modeling performed using WinNonlin software ver. 4.0 (Pharsight Corp., Mountain View, CA). Antiviral study in a ferret model. Under anesthesia, ferrets were inoculated intranasally with B/Kadoma/1/2005 (300 tissue culture infective dose (TCID 50 )) in 200 µl of PBS. Four and six 7

Kitano et al., Page 8 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 ferrets were used into treated groups and an untreated control group, respectively. Six groups were compared in the study using ferrets as followings: (1) Peramivir (30 mg/kg) in saline was administered once intravenously one day after infection (P30x1, abbreviations were described in Fig. 2). (2) Peramivir (60 mg/kg) in saline was administered once intravenously one day after infection (P60x1). (3) Peramivir (30 mg/kg/day) in saline was administered once a day for 3 days from day 1 to day 3 after infection (P30x3). (4) Oseltamivir phosphate (30 mg/kg/day) in 0.5% methylcellulose solution (MC) was administered orally twice a day for 3 days from day 1 to day 3 (O30x3). (5) Oseltamivir phosphate (60 mg/kg/day) in 0.5% MC was administered orally twice a day for 3 days from day 1 to day 3 (O60x3). The first administration of peramivir and oseltamivir phosphate was performed on day 1 post infection (pi). (6) Control animals inoculated with virus but not treated with any reagents were orally administered with 0.5% MC twice a day from day 1 to day 3. To monitor virus replication in nasal cavities, nasal washes were collected from infected ferrets on days 1 to 4 pi. Collected samples were stored at a temperature below -80 o C until use. For virus titration, serial dilutions of nasal washes were inoculated onto confluent MDCK cells in 96-well plates. After 1-h incubation, the suspension was removed, and the cells were cultured in MEM including 0.5% BSA (Sigma-Aldrich) and 3 µg/ml trypsin. The plates were incubated at 37 o C in 5% CO 2 for 3 days. The presence of cytopathic effects (CPE) was determined under a microscope and viral titers were calculated as log 10 TCID 50 /ml. When no CPE was observed using undiluted viral solution, it was defined as the undetectable level that was considered to be lower than 1.4 log 10 TCID 50 /ml. Inflammatory cells in nasal washes were counted microscopically in a hemocytometer. The protein concentration in cell-free nasal washes was 8

Kitano et al., Page 9 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 measured by using a protein reagent (Wako Pure Chemicals, Osaka, Japan). Body temperature was expressed by calculating the average temperatures during nighttime (10 pm 8 am) for avoiding the effect of anesthesia and was compared with that before virus inoculation. Antiviral study in a cynomolgus model. Under anesthesia, cynomolgus macaques were inoculated intranasally with B/SendaiH/1051/2007 (2 x 10 5 TCID 50 ) in 1 ml of PBS. Three macaques were used in each group. Four groups were compared in the macaque study as followings: (1) Peramivir (30 mg/kg) in saline was administered intravenously once immediately after virus inoculation (P30-d0, abbreviations were described in Fig. 5). (2) Peramivir (30 mg/kg) in saline was administered intravenously on day 1 pi (P30-d1). (3) Oseltamivir phosphate (30 mg/kg/day) in 0.5% MC was administered orally once a day for 5 days from day 0 to day 4 (O30x5). The first administration of oseltamivir phosphate was performed immediately after virus inoculation. (4) Control animals inoculated with virus but not treated with any reagents were orally received with 0.5% MC from day 0 to day 4. On day 0 before virus inoculation and days 1 to 12 after virus inoculation, the macaques were anesthetized and then nasal swab samples were collected with two cotton sticks (TE8201, Eiken Chemical, Ltd., Tokyo, Japan) and the sticks were subsequently immersed in 1 ml of PBS containing 0.1% BSA and Penicillin-Streptomycin. Blood samples were collected for measuring anti-ha antibody responses in sera. Collected samples were stored at a temperature below 80 ºC until use. Virus titers in nasal swab fluid were determined using MDCK cells as described above. Levels of inflammatory cytokines and chemokines (interleukin (IL)-6, tumor-necrotizing factor (TNF)-α and monocyte chemotactic protein (MCP)-1) in the nasal swab fluid were assessed using the Cytometric Bead Assay (Becton, Dickinson and Company, Franklin Lakes, NJ) 9

Kitano et al., Page 10 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 according to the manufacturer's instructions. Results of assays were analyzed using FCAP Array software (Becton, Dickinson and Company). Body temperature was expressed by calculating the averages of the highest and lowest temperatures during 1 day and the body temperature after the virus inoculation was compared with that before the virus inoculation. Hemagglutination inhibition (HI) assay. Sera were treated with receptor destroying enzyme (RDEII, Denka Seiken, Tokyo, Japan). Serially diluted sera were mixed with 4 HA units of virus antigen for 1 h at room temperature. The mixture was then incubated with 0.5% chicken red blood cells for 30 min at room temperature. The HI titers were expressed as reciprocals of the highest dilution of serum samples that completely inhibited hemagglutination. Statistical analysis. Virus titers, cytokine levels and body temperature of each animal were calculated as the area under the curve (AUC) by the trapezoidal method. Differences of AUC in virus titers, cytokine levels and body temperature change were analyzed by the Dunnett s multiple comparison method. The Dunnett s test was also carried out for comparison in the virus titers, body temperature, body weight and the number of inflammatory cells on each day. The efficacy of peramivir was compared with that of oseltamivir by using Student's t-test. Statistical analysis was performed using the statistical analysis software, SAS version 9.2 for Windows (SAS Institute, Cary, NC). P values below 0.05 were considered as statistically significant. Sequence analysis of NA genes. Viral RNA was isolated directly from nasal swab fluid of infected macaques or nasal wash fluid of infected ferrets by using the RNeasy Mini Kit (Qiagen, Duesseldorf, Germany). The NA region of influenza virus was amplified by PCR using OneStep RT-PCR kit (Qiagen) and specific primers. The primers for amplification of the NA included a forward primer, 5 -ATGCTACCTTCAACTGTACAAAC -3 or 10

Kitano et al., Page 11 201 202 203 204 205 5 -CCTAAGAACACAAGAAAGTGCC -3 and a reverse primer 5 - GCCAGCAATAAAACCATAGAATG-3 or 5 - CACAGGTGTTGATATGGCTCTGTAA-3. The amplified DNA was sequenced in Applied Biosystems 3730xl DNA Analyzer, by TAKARA sequencing service. The sequences of the NA region derived from isolated viruses were compared with that of inoculation viruses, and amino acid substitutions were analyzed. Downloaded from http://aac.asm.org/ on May 13, 2018 by guest 11

Kitano et al., Page 12 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 RESULTS Sensitivity of influenza B virus NA to NA inhibitors in vitro. Initially, we examined the sensitivity of B/Kadoma/1/2005 and B/SendaiH/1051/2007 neuraminidases to peramivir and oseltamivir carboxylate in vitro (Table1). Since virus sensitivity to NA inhibitors in vivo is not always correlated with that analyzed in cell culture, we determined enzyme 50 % inhibitory concentrations (IC 50 ) (27). IC 50 of peramivir against NA activity of B/Kadoma/1/2005 and B/SendaiH/1051/2007 were 1.78 ± 0.08 nm and 4.26 ± 0.17 nm, respectively. On the other hand, IC 50 values of oseltamivir carboxylate against NA activity of B/Kadoma/1/2005 and B/SendaiH/1051/2007 were 5.93 ± 0.51 nm and 13.1 ± 0.50 nm, respectively. These values were comparable to previous results that IC 50 s of peramivir against NA activity of other strains of influenza B virus were between 0.60 and 10.8 nm and IC 50 s of oseltamivir carboxylate were 5.0 to 24.3 nm (3). Regarding these two influenza B viruses, the inhibitory activity of peramivir was more potent than that of oseltamivir carboxylate (p < 0.001). Pharmacokinetics of peramivir and oseltamivir carboxylate in ferrets and cynomolgus macaques. Pharmacokinetic analysis of peramivir in ferrets and cynomolgus macaques showed rapid uptake into the circulation following intravenous injection (Fig.1). After administration of 30 mg/kg, peramivir concentrations on average was 126 µg/ml and 147 µg/ml at 0.083 h, then decreased to 0.14 µg/ml and 0.76 µg/ml at 8h in ferrets and macaques, respectively. C max were calculated as 194 µg/ml in ferrets and 160 µg/ml in macaques (Table 2). Area under the curve (AUC) of plasma concentration of peramivir after injection with 30 mg/kg in the macaques (234 µg hr/ml) 12

Kitano et al., Page 13 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 was higher than that observed in ferrets injected with 30 mg/kg peramivir (89.1 µg hr/ml). On the other hand, mean times to maximum concentration (T max ) of oseltamivir carboxylate were 2.67 h and 3 h in ferrets and macaques, respectively. AUC of plasma concentration of oseltamivir carboxylate after injection with 30 mg/kg in ferrets (23.7 µg hr/ml) was almost equivalent as that in macaques (25.4 µg hr/ml). We have confirmed low toxicity of peramivir in cynomolgus macaques. When peramivir was administered daily by intravenous infusion to macaques at a dose of 720 mg/kg/day for 30 days, which was the maximum feasible dose, no toxic findings were evident in the clinical observations, body weights, food consumption, hematology, urinalysis, organ weights or histopathology (data not shown). Efficacy of peramivir against influenza B virus infection in ferrets. To evaluate the efficacy of peramivir and oseltamivir against influenza B virus in vivo, peramivir or oseltamivir phosphate was administered on day 1 pi into ferrets infected intranasally with B/Kadoma/1/2005 (300 TCID 50 /ferret) of which titer in nasal samples of ferrets was highest among various clinical isolates examined (data not shown) and that induced fever in ferrets, thereafter virus titers in nasal washes were examined. No viruses were detected in nasal washes of 6 control ferrets on day 1 pi, the maximum virus titer in the control group was observed on day 3 pi and then the virus titer was declined (Fig. 2A). According to these results in control ferrets, we focused the analysis on days 2, 3 and 4 pi to examine the effects of peramivir and oseltamivir on virus replication in the early phase after infection. On day 2 pi, virus titers in all groups treated with peramivir were significantly reduced compared with that in the control group (p < 0.01), but no 13

Kitano et al., Page 14 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 significant difference on days 3 and 4 pi was observed in the group treated with peramivir (30 mg/kg) once (P30x1, see abbreviations in Fig. 2). On day 3 pi, significant reduction in virus titers was also observed in the groups P60x1 and P30x3 compared with control. Significant reduction in virus titers on day 2 pi was observed in the group treated with oseltamivir (60 mg/kg/day) (O60x3, also see abbreviations in Fig. 2) compared with control (p < 0.05) but not in O30x3. In addition, on day 3 pi, virus titers in two groups treated with oseltamivir phosphate were significantly lower than that in the control group (p < 0.05). In comparison of efficacy between peramivir and oseltamivir, the virus titer on day 2 pi in the group P30x1 was significantly lower than that in the group O30x3 (p < 0.05). To examine the effects of peramivir against virus replication during infection, the AUCs of the viral titers from days 2 to 4 pi were calculated. Virus titer AUCs of the groups treated with peramivir and oseltamivir phosphate were significantly reduced in a dose-dependent manner (Fig. 2B). In addition, the virus titer AUC in the group P60x1 was comparable to those in the groups P30x3 and O60x3. These results indicated that a single intravenous injection of peramivir had potent antiviral activity in the early phase after virus inoculation and that the efficacy of a single intravenous injection of peramivir against influenza B virus was comparable to that of oseltamivir phosphate administered for 3 days in the ferret model. To monitor the emergence of resistant variants after peramivir treatment and during oseltamivir phosphate treatment, we determined NA gene sequences of virus obtained from nasal wash fluid on day 4 pi. An isolated virus in the group O30x3 showed a mutation resulting in an amino acid change at position 31 (L to I) of the NA gene. This mutation is located around transmembrane domain and not associated with reported resistance or decreased susceptibility to 14

Kitano et al., Page 15 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 NA inhibitors (1, 8). In the other ferrets, no NA amino acid changes were detected in viruses from the nasal wash fluid (data not shown). We examined the effects of peramivir and oseltamivir on body temperature in ferrets after inoculation with B/Kadoma/1/2005. Higher body temperature than that before the inoculation was observed for 4 days in the control group (Fig. 3A). On day 2 pi, the groups P60x1 and P30x3 showed significantly lower body temperature than did the control group (p < 0.05). The group P30x1 showed slightly but not significant reduction in body temperature on day 2 pi. In contrast, two groups administered with oseltamivir phosphate showed no apparent reduction in body temperature. AUCs of the body temperature change of the groups P30x1 and P60x1 were lower than those of O30x3 and O60x3, respectively (p < 0.05 and p < 0.01) (Fig. 3B). To assess the effects of peramivir and oseltamivir on inflammation in nasal cavities of infected ferrets, the protein concentration and the number of inflammatory cells in nasal wash fluid were measured. The protein concentration and the number of inflammatory cells on day 3 pi were reduced in all groups treated with peramivir and the group O60x3 but not in the group O30x3 (Fig. 4A and B). In addition, body weights were measured as one of the clinical signs. A significant difference in the average percent body weight (weight on day 4 / weight on day 0) between the group P30x3 (102.0%) and the untreated control group (97.4%) was observed (p < 0.05) (Fig. 4C). While nasal signs (nasal discharge, sneezing and mouth breathing) induced by influenza B infection in the control group were relatively mild, ferrets treated with peramivir or oseltamivir phosphate exhibited little or no nasal sign (data not shown). 292 293 Efficacy of a single intravenous injection of peramivir against influenza B virus in 15

Kitano et al., Page 16 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 cynomolgus macaques. We have reported that cynomolgus macaques were susceptible to influenza B virus that replicated in the upper respiratory tracts. Especially, B/SendaiH/1051/2007 caused fever with loss of appetite and body weight more clearly than did B/Kadoma /1/2005 (23). Therefore, cynomolgus macaques were intranasally inoculated with B/SendaiH/1051/2007 (2 x 10 5 TCID 50 ) and 30 mg/kg of peramivir was administered intravenously once immediately after virus inoculation (P30-d0, see abbreviations in Fig. 5) or 24 h after virus inoculation (P30-d1). Thirty mg/kg/day of oseltamivir phosphate was administered orally once a day for 5 days as a comparison (O30x5). When the virus titers in nasal swab fluid were measured from day 1 to 12 pi, the maximum virus titer in the control group was observed on day 3 pi and then the virus titer declined after day 4 pi (Fig. 5A). On days 2 and 3 pi, virus titers in all groups treated with peramivir were significantly reduced compared with those in the control group (p < 0.01). On days 5 and 7 pi, significant reduction in virus titers was also observed in the group P30-d1. In contrast, no apparent reduction of virus titers was observed in the early phase after virus inoculation in the group O30x5 and the virus titer on day 7 pi in the group O30x5 was significantly lower than that of the control group (p < 0.05). In comparison between peramivir and oseltamivir on day 2 pi, virus titers of P30-d0 and P30-d1 were significantly lower than that of O30x5 (p < 0.05 and p < 0.01, respectively). On day3 pi, the virus titer of P30-d1 was also significantly lower than that of O30x5 (p < 0.05). When the statistical analysis was performed on the basis of the AUCs of the viral titers from day 1 to 7 pi, the virus titer AUCs of the groups treated with peramivir were significantly lower than that of the control group (p < 0.05), while that treated with oseltamivir was not (Fig. 5B). Furthermore, the virus titer AUC of P30-d1 was significantly lower than that of O30x5 (p < 16

Kitano et al., Page 17 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 0.05). We determined NA gene sequences of virus obtained from nasal swab fluid on day 5 pi. Viruses isolated from macaques treated with peramivir and oseltamivir showed no mutation in NA amino acid sequences (data not shown). Body temperatures were compared among the treated and untreated groups. Higher body temperatures than those before the virus inoculation were observed in the untreated control group for 1 to 4 days (Fig. 6). Body temperature of macaques treated with peramivir was substantially lower than those of macaques in the control group, whereas no significant suppression in body temperature was observed in all treated macaques using statistical analysis on the basis of the AUC of body temperature change (data not shown). However, the averages of maximal temperature rise in the groups P30-d0 (mean ± SD = 0.33 ± 0.15 o C) and P30-d1 (0.43 ± 0.31 o C) after infection were significantly lower than that in the control (1.13 ± 0.21 o C) (p < 0.05). In contrast, no significant suppression of maximal temperature raise was observed in the group O30x5 (0.60 ± 0.26 o C) compared with the control group. To examine the effects of peramivir and oseltamivir on immune responses in infected macaques, production of inflammatory cytokines and chemokines in nasal swab fluid was measured (Fig. 7A). Levels of cytokine and chemokine secretion were increased after infection. However, the kinetics of cytokine and chemokine production varied in individual macaques. Therefore, levels of cytokines and chemokines were expressed as AUC from day 1 to 12 and statistical analysis was conducted on the basis of the AUC (Fig. 7A). Levels of IL-6 production in groups P30-d0, P30-d1 and O30x5 were significantly lower than those in the control group. On the other hand, no significant effect on TNF-α and MCP-1 production was observed in treated groups compared with the control group. 17

Kitano et al., Page 18 338 339 340 341 342 343 To serologically confirm influenza B virus infection and to compare the effects of the reagents on production of anti-ha antibodies, sera were collected on indicated days after virus inoculation. HI activities against B/SendaiH/1051/2007 were observed in sera of groups treated with peramivir or oseltamivir phosphate on day 8 pi and increased until day 14 pi (Fig. 7B). These results indicate that peramivir and oseltamivir did not inhibit the production of serum anti-ha antibodies against B/SendaiH/1051/2007 in cynomolgus macaques. Downloaded from http://aac.asm.org/ on May 13, 2018 by guest 18

Kitano et al., Page 19 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 DISCUSSION Influenza B virus causes seasonal influenza even after newly emerging H1N1 influenza A virus infection has become pandemic in 2009 and the number of Russian H1N1 virus cases decreased (34). Since influenza B virus infection causes significant morbidity as influenza A virus does (26, 29, 30), antiviral agents against influenza B virus are required. The efficacy of peramivir against influenza B virus in vitro was more potent than that of oseltamivir and zanamivir (5, 15). Therefore, in the present study, we examined the efficacy of peramivir against influenza B virus in vivo using ferrets and macaques that were susceptible to infection with a number of unadapted human influenza isolates and showed clinical symptoms. Since absence of HI activity in sera was confirmed before experiments and animals were maintained under the specific-pathogen-free condition in the present study, use of immunologically naïve animals against influenza B virus might support the interpretation that change of virus titers in the early stage of infection was mainly caused by antiviral agents but not by memory immune responses against influenza B virus, which might exist in many humans. In addition, animal models enable to examine effective time points of initiation of treatments after virus exposure since it might be difficult to know when patients were infected with virus in clinical studies. These points might be critical if differences of effects among antiviral agents would be modest. In ferrets infected with human influenza B virus, viral titers and virus titer AUC were decreased with single intravenous administration of peramivir and repeated oral administration of oseltamivir phosphate compared with those of control (Fig. 2). However, single administration of 60 mg/kg of peramivir attenuated fever by about 1 o C, while no change in fever was observed in the groups treated with oseltamivir phosphate (Fig. 3). The difference of virus titers in between 19

Kitano et al., Page 20 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 ferrets treated with peramivir and ferrets treated with oseltamivir phosphate was observed on day 2 pi. This suggested that viral propagation in an early phase after virus infection contributed to severity of symptoms including fever and that suppression of virus replication in the early phase during infection was crucial to ameliorate symptoms in the ferret model. To further evaluate the therapeutic efficacy of peramivir against influenza B virus, we next compared the inhibitory effects of single intravenous administration of peramivir with those of repeated oral administration of oseltamivir phosphate using cynomolgus macaques. Virus titers, virus titer AUC, body temperature and the IL-6 AUC in nasal swabs of macaques treated with peramivir were lower than those of the control group (Figs. 5 to 7). These findings were concordant with our previous study that the levels of IL-6 in the nasal swabs were correlated with both virus replication and symptoms (23). These results observed in macaques and ferrets suggest that virus propagation in the nasal cavity in the early phase of infection is related to body temperature during infection. On the other hand, the virus titer on day 7 pi and the IL-6 AUC in nasal swabs of macaques treated with oseltamivir phosphate were significantly lower than those of the control group but body temperature and virus titer AUC were not. It was thought that reduction of virus titers in the late phase might cause a low IL-6 level on days 7 to 8 pi resulting in a decrease in the IL-6 AUC (data not shown). However, it did not affect suppression of fever in macaques treated with oseltamivir phosphate since the virus titer in the early phase was not suppressed. These results also support importance of suppression on virus replication in the early phase of infection to reduce symptoms of influenza and significance of the early treatment with antiviral agents. Although oseltamivir phosphate has been administered twice a day in humans, we 20

Kitano et al., Page 21 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 administered oseltamivir phosphate once a day for 5 days in the present macaque study for the following reasons. Firstly, since anesthesia was necessary for drug administration and it decreased body temperature, anesthesia twice a day would make evaluation of macaque s body temperature change and feeding of food to macaques difficult. Secondly, there was no significant difference in duration of virus shedding and total symptom scores after treatment with oseltamivir phosphate between once and twice a day in the previous study although a group treated with oseltamivir once a daily showed a higher virus titer AUC than did a group treated twice daily (13). Thirdly, we confirmed that the C max of oseltamivir carboxylate in plasma of macaques was 6440 ± 3180 ng/ml after oral administration of 30 mg/kg dose of oseltamivir phosphate, which was 1,570 times higher than enzyme inhibition IC 50 value (4.1 ng/ml = 13.1 nm) against B/SendaiH/1051/2007 (Tables 1 and 2). In addition, the plasma concentration of oseltamivir carboxylate after 24 h (36 ± 8.0 ng/ml) was still 9 times higher than the IC 50 value (4.1 ng/ml) (Fig. 1 and Table 1). Therefore, one dose administration of oseltamivir in the macaque model was expected to show pharmacological effects from the aspect of pharmacokinetics. Furthermore, we did not detect mutations in NA genes in nasal samples from macaques after treatment with oseltamivir once daily as well as after treatment with peramivir though substitution of an amino acid was detected in a nasal wash sample of a ferret treated with oseltamivir twice a daily. Nonetheless, low effectiveness of oseltamivir against influenza B virus was demonstrated by not only virus titer in nasal swab fluid but also symptom in the macaque model. These results partially coincide with clinical studies that showed the longer duration of virus shedding and fever in patients treated with oseltamivir phosphate in influenza B virus infection than in influenza A virus infection (21, 22, 32). On the other hand, in the macaque model, intravenous 21

Kitano et al., Page 22 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 administration of 30 mg/kg dose of peramivir can achieve C max of up to 160 µg/ml, which is 100,000 times higher than the IC 50 values (1.4 ng/ml = 4.26 nm) against B/SendaiH/1051/2007 (Fig. 1, Tables 1 and 2). The concentration of peramivir in plasma dropped quickly after injection but the level of peramivir at 24 h postdosing (52.4 ng/ml) was still 37 times higher than the IC 50 value (1.4 ng/ml). Therefore, the high concentration of peramivir in plasma during 24 h after injection is thought to be effective to reduce virus replication in the early phase of infection. In our previous study, HI and neutralization activities in sera of macaque were detected 6 to 8 days after infection and coincided with decreasing virus titers in swab samples (23). In the present study, HI activities against B/SendaiH/1051/2007 were observed in sera of macaques treated with peramivir or oseltamivir phosphate after day 8 pi at the comparable level of sera from untreated macaques. These results indicate that peramivir and oseltamivir did not inhibit production of anti-ha antibodies against B/SendaiH/1051/2007 in cynomolgus macaques. It is thought that the titers of anti-ha antibodies against B/SendaiH/1051/2007 in treated cynomolgus macaques may be sufficient to protect from challenge with the same virus strain since macaques possessing similar or lower HI titers of sera after vaccination with an H7 vaccine were protected from infection with a different H7 strain in the previous experiment (18). In the present study, we demonstrated that peramivir injected once intravenously had beneficial effects on viral titers and symptoms of ferrets and cynomolgus macaques with influenza B virus infection. The effects observed with peramivir treatment seem to be superior to those observed with once daily oseltamivir phosphate treatment in cynomolgus macaque when oseltamivir was given once daily. Therefore, single intravenous administration of peramivir could be an alternative to oseltamivir to treat patients with acute influenza B virus infection. 22

Kitano et al., Page 23 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 ACKNOWLEDGEMENTS We would like to thank Dr. Hidekazu Nishimura for providing B/SendaiH/1051/2007, Dr. Tetsuo Kase for providing B/Kadoma/1/2005, Drs. Hideaki Tsuchiya, Norio Okahara and Takahiro Nakagawa for animal care, Drs. Shinya Omoto and Shinobu Kawauchi-Miki for valuable discussions and Hiroko Iwasaki and Mayumi Kakui for help on enzyme assay. All work reported here was financially supported by Shionogi Co., Ltd. REFERENCES 1. Air, G. M., W. G. Laver, M. Luo, S. J. Stray, G. Legrone, and R. G. Webster. 1990. Antigenic, sequence, and crystal variation in influenza B neuraminidase. Virology 177:578-87. 2. Babu, Y. S., P. Chand, S. Bantia, P. Kotian, A. Dehghani, Y. El-Kattan, T. H. Lin, T. L. Hutchison, A. J. Elliott, C. D. Parker, S. L. Ananth, L. L. Horn, G. W. Laver, and J. A. Montgomery. 2000. BCX-1812 (RWJ-270201): discovery of a novel, highly potent, orally active, and selective influenza neuraminidase inhibitor through structure-based drug design. J Med Chem 43:3482-6. 3. Bantia, S., C. D. Parker, S. L. Ananth, L. L. Horn, K. Andries, P. Chand, P. L. Kotian, A. Dehghani, Y. El-Kattan, T. Lin, T. L. Hutchison, J. A. Montgomery, D. L. Kellog, and Y. S. Babu. 2001. Comparison of the anti-influenza virus activity of RWJ-270201 with those of oseltamivir and zanamivir. Antimicrob Agents Chemother 45:1162-7. 23

Kitano et al., Page 24 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 4. Birnkrant, D., and E. Cox. 2009. The Emergency Use Authorization of peramivir for treatment of 2009 H1N1 influenza. N Engl J Med 361:2204-7. 5. Boivin, G., and N. Goyette. 2002. Susceptibility of recent Canadian influenza A and B virus isolates to different neuraminidase inhibitors. Antiviral Res 54:143-7. 6. Boltz, D. A., N. A. Ilyushina, C. S. Arnold, Y. S. Babu, R. G. Webster, and E. A. Govorkova. 2008. Intramuscularly administered neuraminidase inhibitor peramivir is effective against lethal H5N1 influenza virus in mice. Antiviral Res 80:150-7. 7. Chand, P., Y. S. Babu, S. Bantia, N. Chu, L. B. Cole, P. L. Kotian, W. G. Laver, J. A. Montgomery, V. P. Pathak, S. L. Petty, D. P. Shrout, D. A. Walsh, and G. M. Walsh. 1997. Design and synthesis of benzoic acid derivatives as influenza neuraminidase inhibitors using structure-based drug design. J Med Chem 40:4030-52. 8. Collins, P. J., L. F. Haire, Y. P. Lin, J. Liu, R. J. Russell, P. A. Walker, J. J. Skehel, S. R. Martin, A. J. Hay, and S. J. Gamblin. 2008. Crystal structures of oseltamivir-resistant influenza virus neuraminidase mutants. Nature 453:1258-61. 9. Govorkova, E. A., N. A. Ilyushina, D. A. Boltz, A. Douglas, N. Yilmaz, and R. G. Webster. 2007. Efficacy of oseltamivir therapy in ferrets inoculated with different clades of H5N1 influenza virus. Antimicrob Agents Chemother 51:1414-24. 10. Govorkova, E. A., I. A. Leneva, O. G. Goloubeva, K. Bush, and R. G. Webster. 2001. Comparison of efficacies of RWJ-270201, zanamivir, and oseltamivir against H5N1, H9N2, and other avian influenza viruses. Antimicrob Agents Chemother 45:2723-32. 11. Gubareva, L. V., M. N. Matrosovich, M. K. Brenner, R. C. Bethell, and R. G. Webster. 1998. Evidence for zanamivir resistance in an immunocompromised child 24

Kitano et al., Page 25 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 infected with influenza B virus. J Infect Dis 178:1257-62. 12. Hatakeyama, S., N. Sugaya, M. Ito, M. Yamazaki, M. Ichikawa, K. Kimura, M. Kiso, H. Shimizu, C. Kawakami, K. Koike, K. Mitamura, and Y. Kawaoka. 2007. Emergence of influenza B viruses with reduced sensitivity to neuraminidase inhibitors. JAMA 297:1435-42. 13. Hayden, F. G., J. J. Treanor, R. S. Fritz, M. Lobo, R. F. Betts, M. Miller, N. Kinnersley, R. G. Mills, P. Ward, and S. E. Straus. 1999. Use of the oral neuraminidase inhibitor oseltamivir in experimental human influenza: randomized controlled trials for prevention and treatment. JAMA 282:1240-6. 14. Herlocher, M. L., S. Elias, R. Truscon, S. Harrison, D. Mindell, C. Simon, and A. S. Monto. 2001. Ferrets as a transmission model for influenza: sequence changes in HA1 of type A (H3N2) virus. J Infect Dis 184:542-6. 15. Hurt, A. C., P. Iannello, K. Jachno, N. Komadina, A. W. Hampson, I. G. Barr, and J. L. McKimm-Breschkin. 2006. Neuraminidase inhibitor-resistant and -sensitive influenza B viruses isolated from an untreated human patient. Antimicrob Agents Chemother 50:1872-4. 16. Hurt, A. C., S. S. Nor'e, J. M. McCaw, H. R. Fryer, J. Mosse, A. R. McLean, and I. G. Barr. 2010. Assessing the viral fitness of oseltamivir-resistant influenza viruses in ferrets, using a competitive-mixtures model. J Virol 84:9427-38. 17. Itoh, Y., K. Shinya, M. Kiso, T. Watanabe, Y. Sakoda, M. Hatta, Y. Muramoto, D. Tamura, Y. Sakai-Tagawa, T. Noda, S. Sakabe, M. Imai, Y. Hatta, S. Watanabe, C. Li, S. Yamada, K. Fujii, S. Murakami, H. Imai, S. Kakugawa, M. Ito, R. Takano, K. 25

Kitano et al., Page 26 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 Iwatsuki-Horimoto, M. Shimojima, T. Horimoto, H. Goto, K. Takahashi, A. Makino, H. Ishigaki, M. Nakayama, M. Okamatsu, K. Takahashi, D. Warshauer, P. A. Shult, R. Saito, H. Suzuki, Y. Furuta, M. Yamashita, K. Mitamura, K. Nakano, M. Nakamura, R. Brockman-Schneider, H. Mitamura, M. Yamazaki, N. Sugaya, M. Suresh, M. Ozawa, G. Neumann, J. Gern, H. Kida, K. Ogasawara, and Y. Kawaoka. 2009. In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses. Nature 460:1021-5. 18. Itoh, Y., H. Ozaki, H. Ishigaki, Y. Sakoda, T. Nagata, K. Soda, N. Isoda, T. Miyake, H. Ishida, K. Okamoto, M. Nakayama, H. Tsuchiya, R. Torii, H. Kida, and K. Ogasawara. 2010. Subcutaneous inoculation of a whole virus particle vaccine prepared from a non-pathogenic virus library induces protective immunity against H7N7 highly pathogenic avian influenza virus in cynomolgus macaques. Vaccine 28:780-9. 19. Jackson, S., N. Van Hoeven, L. M. Chen, T. R. Maines, N. J. Cox, J. M. Katz, and R. O. Donis. 2009. Reassortment between avian H5N1 and human H3N2 influenza viruses in ferrets: a public health risk assessment. J Virol 83:8131-40. 20. Kawai, N., H. Ikematsu, N. Iwaki, N. Hirotsu, and S. Kashiwagi. 2006. Prevalence of influenza B during the 2004-2005 season in Japan. Clin Infect Dis 43:1226-8. 21. Kawai, N., H. Ikematsu, N. Iwaki, T. Kawashima, T. Maeda, S. Mitsuoka, K. Kondou, I. Satoh, K. Miyachi, S. Yamaga, T. Shigematsu, N. Hirotsu, and S. Kashiwagi. 2007. Longer virus shedding in influenza B than in influenza A among outpatients treated with oseltamivir. J Infect 55:267-72. 22. Kawai, N., H. Ikematsu, N. Iwaki, T. Maeda, I. Satoh, N. Hirotsu, and S. Kashiwagi. 26

Kitano et al., Page 27 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 2006. A comparison of the effectiveness of oseltamivir for the treatment of influenza A and influenza B: a Japanese multicenter study of the 2003-2004 and 2004-2005 influenza seasons. Clin Infect Dis 43:439-44. 23. Kitano, M., Y. Itoh, M. Kodama, H. Ishigaki, M. Nakayama, T. Nagata, H. Ishida, H. Tsuchiya, R. Torii, K. Baba, R. Yoshida, A. Sato, and K. Ogasawara. 2010. Establishment of a cynomolgus macaque model of influenza B virus infection. Virology 407:178-184. 24. Kohno, S., H. Kida, M. Mizuguchi, and J. Shimada. 2010. Efficacy and safety of intravenous peramivir for treatment of seasonal influenza virus infection. Antimicrob Agents Chemother 54:4568-74. 25. Kubo, S., T. Tomozawa, M. Kakuta, A. Tokumitsu, and M. Yamashita. 2010. Laninamivir prodrug CS-8958, a long-acting neuraminidase inhibitor, shows superior anti-influenza virus activity after a single administration. Antimicrob Agents Chemother 54:1256-64. 26. Lee, N., P. K. Chan, D. S. Hui, T. H. Rainer, E. Wong, K. W. Choi, G. C. Lui, B. C. Wong, R. Y. Wong, W. Y. Lam, I. M. Chu, R. W. Lai, C. S. Cockram, and J. J. Sung. 2009. Viral loads and duration of viral shedding in adult patients hospitalized with influenza. J Infect Dis 200:492-500. 27. Matrosovich, M., T. Matrossovich, J. Carr, N. A. Roberts, and H.-D. Klenk. 2003. Overexpression of the alpha-2,6-sialyltransferase in MDCK cells increases influenza virus sensitivity to neuraminidase inhibitors. J Virol 77:8418-25. 28. Munster, V. J., E. de Wit, J. M. van den Brand, S. Herfst, E. J. Schrauwen, T. M. 27

Kitano et al., Page 28 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 Bestebroer, D. van de Vijver, C. A. Boucher, M. Koopmans, G. F. Rimmelzwaan, T. Kuiken, A. D. Osterhaus, and R. A. Fouchier. 2009. Pathogenesis and transmission of swine-origin 2009 A(H1N1) influenza virus in ferrets. Science 325:481-3. 29. Newland, J. G., J. R. Romero, M. Varman, C. Drake, A. Holst, T. Safranek, and K. Subbarao. 2003. Encephalitis associated with influenza B virus infection in 2 children and a review of the literature. Clin Infect Dis 36:e87-95. 30. Peltola, V., T. Ziegler, and O. Ruuskanen. 2003. Influenza A and B virus infections in children. Clin Infect Dis 36:299-305. 31. Pinto, L. H., and R. A. Lamb. 2006. The M2 proton channels of influenza A and B viruses. J Biol Chem 281:8997-9000. 32. Sugaya, N., K. Mitamura, M. Yamazaki, D. Tamura, M. Ichikawa, K. Kimura, C. Kawakami, M. Kiso, M. Ito, S. Hatakeyama, and Y. Kawaoka. 2007. Lower clinical effectiveness of oseltamivir against influenza B contrasted with influenza A infection in children. Clin Infect Dis 44:197-202. 33. Tashiro, M., J. L. McKimm-Breschkin, T. Saito, A. Klimov, C. Macken, M. Zambon, and F. G. Hayden. 2009. Surveillance for neuraminidase-inhibitor-resistant influenza viruses in Japan, 1996-2007. Antivir Ther 14:751-61. 34. World Health Organization. 2010. Influenza activity in the world, Global alert and response. (http://www.who.int/csr/disease/influenza/update/en/index.html). 35. Yun, N. E., N. S. Linde, M. A. Zacks, I. G. Barr, A. C. Hurt, J. N. Smith, N. Dziuba, M. R. Holbrook, L. Zhang, J. M. Kilpatrick, C. S. Arnold, and S. Paessler. 2008. Injectable peramivir mitigates disease and promotes survival in ferrets and mice infected 28

Kitano et al., Page 29 564 565 with the highly virulent influenza virus, A/Vietnam/1203/04 (H5N1). Virology 374:198-209. 29