The effect of intravenous peramivir, compared with oral oseltamivir, on the outcome of post-influenza pneumococcal pneumonia in mice

Transcription

1 Antivirl Therpy 215; 2:11 19 (doi: /IMP2744) Originl rtile The effet of intrvenous permivir, ompred with orl oseltmivir, on the outome of post-influenz pneumool pneumoni in mie Akitk Tnk 1, Shigeki Nkmur 1 *, Msfumi Seki 2, Noki Iwng 1, Toshiki Kjihr 1, Mitsutk Kitno 3, Tomoyuki Homm 3, Shintro Kurihr 4, Yoshifumi Immur 1, Tig Miyzki 1, Koihi Izumikw 1, Hiroshi Kkey 1, Ktsunori Yngihr 5, Shigeru Kohno 1 1 Deprtment of Moleulr Miroiology nd Immunology, Ngski University Grdute Shool of Biomedil Sienes, Ngski, Jpn 2 Division of Infetious Diseses nd Prevention, Osk University Hospitl, Suit, Jpn 3 Infetious Diseses, Mediinl Reserh Lortories, Shionogi & Co., Ltd, Osk, Jpn 4 Ngski University Infetion Control nd Edution Center, Ngski, Jpn 5 Deprtment of Lortory Mediine, Ngski University Grdute Shool of Biomedil Sienes, Ngski, Jpn *Corresponding uthor e-mil: moju516@ngski-u..jp Bkground: Pneumool pneumoni often ours seondry to influenz infetion nd ounts for lrge proportion of the moridity nd mortlity ssoited with sesonl nd pndemi influenz outreks. Permivir is novel, intrvenous neurminidse inhiitor tht exhiits potent ntivirl tivity ginst influenz A nd B viruses. We investigted the effiy of permivir for modulting the severity of seondry pneumool pneumoni. Methods: CBA/JNCrlj mie, infeted with influenz virus nd superinfeted with Streptoous pneumonie, were treted with either intrvenous permivir (single or multiple doses of 6 mg/kg/dy) or orl oseltmivir t doses of 1 or 4 mg/kg/dy in divided doses. The survivl rte, vile teril ount nd virus titre in the lungs, s well s ytokine/hemokine onentrtion nd histopthologil findings were ompred etween oth groups. Results: The medin durtion of survivl of oinfeted mie ws signifintly prolonged y tretment with multiple doses of permivir, reltive to mie treted with oseltmivir t either dose. Vile teril ounts nd virus titres in the lungs were signifintly redued y intrvenous permivir tretment ompred with no tretment or orl oseltmivir tretment. The prodution of inflmmtory ytokines/ hemokines ws lso signifintly suppressed y multiple dosing of permivir ompred with oseltmivir. Inresed survivl ppered to e medited y deresed inflmmtion, mnifested s lower levels of inflmmtory ells nd proinflmmtory ytokines in the lungs nd less severe histopthologil findings. The lungs of mie treted with multiple doses of permivir showed mild inflmmtory hnges ompred to oseltmivir. Conlusions: This study demonstrted tht multipledose regimen of intrvenous permivir ws more effiious thn single permivir dose or multiple doses of oseltmivir for improving outomes in pneumool pneumoni following influenz virus infetion in mie. Introdution Influenz nd pneumoni re listed together s the eighth most ommon use of deth in the United Sttes, with most of the mortlity ourring mong young hildren, the elderly nd immunoompromised ptients [1]. Severl oservtionl studies suggest tht erly ntivirl tretment of infeted ptients with oseltmivir n redue the severity nd durtion of symptoms, hospitliztions nd omplitions [2 8]. Antivirl tretment is urrently reommended y the Centers for Disese Control nd Prevention (CDC) nd the Infetious Diseses Soiety of Ameri (IDSA) for suspeted or proven influenz in those with serious or progressive illness nd in outptients with high-risk medil onditions [9]. Two lsses of ntivirl drugs re urrently pproved for use: M2 proton hnnel inhiitors nd neurminidse inhiitors (NAIs). Most existing NAIs re for orl or inhlnt use, nd their delivery my e unrelile or diffiult in severely ill ptients, espeilly those requiring mehnil ventiltion. This hs resulted in 215 Interntionl Medil Press (print) (online) 11

2 A Tnk et l. the need for n injetle nti-influenz gent for use in high-risk ptients. Permivir is the first intrvenous NAI to e pproved nd it hs shown superior ntivirl tivity ginst the 29 pndemi influenz virus (H1N1pdm9) in vitro, s well s good tivity ginst vin H5N1 influenz in niml experiments [1,11]. The CDC found tht permivir exhiited higher inhiitory tivity ginst the H1N1pdm9 virus thn other NAIs in vitro [12] nd the US Food nd Drug Administrtion (FDA) issued n emergeny-use uthoriztion for permivir exlusively for severe ph1n1 infetions [13]. In 42 ptients hospitlized with severe H1N1pdm9 influenz nd ssoited pneumoni, intrvenous permivir ppered to e well tolerted nd ssoited with reovery in most ptients [14]. Rndomized linil studies to eluidte the linil effetiveness of intrvenous permivir showed tht there were signifint redutions in the durtion of illness in oth high-risk ptients treted with repeted doses of permivir nd otherwise helthy individuls [15 17]. However, other oservtionl studies hve questioned permivir s effetiveness nd sfety in severely ill ptients [7,18]. Although influenz infetion itself n e lethl, influenz infetion is often omplited y teril invsion, prtiulrly y S pneumonie, Stphyloous ureus, Steptoous pyogenes nd Klesiell pneumonie [19 21]. Seondry teril pneumoni is mjor use of the exessive mortlity tht ours during influenz epidemis [22]. Multiple ftors ontriute to the inresed disese severity during oinfetion of influenz nd teri. Influenz infetion n derese muoiliry lerne of pneumooi [23]. In ddition, pneumooi show inresed dherene to pulmonry epithelium following influenz, whih ould e medited y virl neurminidse tivity [24]. In ddition, influenz hs the ility to indue neutrophil poptosis nd dysfuntion [25]. Previous studies demonstrted tht severl proinflmmtory ytokines, inluding type I interferons (IFNs), IFN-g, tumour nerosis ftor (TNF)- nd interleukin (IL)-6 re signifintly elevted during oinfetion nd inhiit the reruitment of immune ells suh s mrophges nd neutrophils [26 29]. MCullers et l. [3,31] reported tht prophylxis or erly tretment with oseltmivir deresed influenz virl lod nd weight loss nd signifintly redued seondry pneumool teril pneumoni severity in n experimentl murine model. Tretment with protein synthesis inhiitors, zithromyin nd lindmyin, improved the outomes of seondry pneumool pneumoni [32]. Previous studies reported tht mrolide ntiiotis ted s immunomodultors to suppress exessive ytokine prodution during n influenz infetion [33 35], in prt due to n ntiioti-indued redution in the expression of sili ids with n lph-2,6 linkge (SAlph2,6Gl) on the irwy epithelil ells, the reeptor for humn influenz virus [33]. Tken together, these findings provide preedent for the study of these phrmeutil gents to improve outomes relted to teril pneumoni, seondry to influenz infetion. The ility of permivir to inhiit the progression of ftl pneumool pneumoni following influenz virus infetion remins unproven. Therefore, the purpose of this study ws to evlute the inhiitory effet of intrvenous permivir, in omprison with orl oseltmivir, on the progression of pneumool pneumoni following influenz infetion in mouse model. Methods Mie CBA/JNCrlj mie (6-week-old mles) were purhsed from Chrles River Lortories Jpn (Yokohm, Jpn). All niml experiments were performed in ordne with the guidelines of the Lortory Animl Center for Biomedil Reserh, Ngski University Shool of Mediine, Ngski, Jpn. Viruses nd teri The H1N1 influenz virus, strin A/Puerto Rio 8/34 (PR8), ATCC VR-95, ws purhsed from Amerin Type Culture Colletion (ATCC, Mnsss, VA, USA). The virus ws ultured in Mdin Dry nine kidney (MDCK) ells for 3 dys nd stored with the ulture superntnt t -8 C. Stored virus solution ws thwed nd diluted with phosphte-uffered sline (PBS) to the desired density just efore inoultion. Fifty perent inhiitory onentrtion (IC 5 vlues) of permivir nd oseltmivir determined in enzyme inhiition ssys [36] were.5 ng/ml nd 1.4 ng/ml, respetively. S pneumonie (ATCC 49619), linil isolte with psulr serotype 19F, ws used in these studies. Mintenne nd storge of teri were performed s reported previously [37]. Bteri were ultured in Mueller-Hinton II Broth (Eiken Chemil, Tokyo, Jpn) with Strepto Hemo supplement (Eiken Chemil) t 37 C for 6 h until rehing log phse. The teril density in the roth ws determined y mesuring the sorne t 66 nm nd plotting the optil density on stndrd urve generted with known olony-forming unit (CFU) vlues. The teril ulture ws diluted to the desired density for oinfetion studies. Tissue ulture infetive dose The infetious virus titres were determined in MDCK ells y previously desried methods [36]. Mouse oinfetion studies One hundred 5% tissue ulture infetive doses (TCID 5 ) of A/PR/8(H1N1) (PR8) suspended in 5 ml Interntionl Medil Press

3 Permivir effiy ginst seondry pneumool pneumoni of PBS, ws intrnslly inoulted into mie nesthetized with pentoritl. This dose represented pproximtely one 5% lethl dose (LD 5 ). To indue pneumool superinfetion, the re-nesthetized mie were intrnslly inoulted with CFUs of S pneumonie, in 5 ml of PBS, 2 dys fter PR8 inoultion. Smpling of lung tissue nd exudte ws performed 2 dys fter pneumool inoultion. The study sheme is shown in Figure 1. Anti-influenz tretment Permivir nd oseltmivir phosphte were donted y Shionogi & Co. (Osk, Jpn). Mie were divided into four tretment groups: ontrol (untreted), orl oseltmivir (5 mg/kg, twie dy for 5 dys) [38], high dose of orl oseltmivir (oseltmivir-h; 2mg/kg, twie dy for 5 dys), single dose of intrvenous permivir (permivir-s; 6 mg/kg) nd multiple doses of intrvenous permivir (permivir-m; 6 mg/kg, one dy for 5 dys). All ompounds were prepred in sterile sline nd dministered (oseltmivir y orl gvge, permivir y intrvenous injetion in til vein) 2 h fter influenz virus inoultion without nesthesi. Lung CFU determintions nd histopthology Whole lungs were removed under septi onditions nd homogenized in 1. ml of PBS using Shke Mster NEO (Bio Medil Siene, Tokyo, Jpn). S pneumonie ws quntified y pling seril dilutions of the lung homogentes onto lood gr pltes nd inuting them t 37 C in 5% CO 2 tmosphere nd ounting the resultnt teril olonies. The remining homogentes were entrifuged t 1, g for 3 min nd the superntnts were used for enzyme-linked immunosorent ssy (ELISA) determintions of ytokines nd hemokines. Lung tissue setions were prffin-emedded, setioned nd stined with hemtoxylin nd eosin (HE) using stndrd proedures [34]. ELISA Conentrtions of TNF-, IL-6, kertinoyte-derived ytokine (KC), mrophge inflmmtory protein-2 (MIP-2) nd IFN-g in lung homogentes were ssyed using mouse Quntikine ELISA kits (R&D Systems, Minnepolis, MN, USA). These ytokine nlyses were performed ording to the mnufturer s protool. Sttistis The omprison of teril titres etween two groups ws performed y Mnn Whitney U test. We evluted differenes of teril titres, virl titres nd inflmmtory ytokines/hemokine mong groups s whole y the onewy nlysis of vrine (ANOVA). If signifint differene ws found y the ANOVA, pirwise omprison ws performed with the use of the Mnn Whitney U test. All nlyses were onduted using GrphPd Prism4 (Grph- Pd Softwre, Sn Diego, CA, USA). Survivl urves were estimted y the Kpln Meier method nd their homogeneities were evluted y the log-rnk test. Vlues with P<.5 were onsidered sttistilly signifint. Results Influenz enhned suseptiility of mie to seondry pneumool infetion To hrterize our murine model of post-influenz pneumool pneumoni, CBA/JNCrlj mie were infeted intrnslly with the PR8 strin of influenz Figure 1. Experimentl set-up for oinfetion model of CBA/JNCrlj mie nd the dministrtion shedule of the ntivirl gents Single dose of intrvenous permivir Multiple doses of intrvenous permivir Orl oseltmivir -2 2 Infetious dys, dy Influenz virus infetion, in (1 TCID 5 /5 µl) S pneumonie infetion, in (1 1 5 CFU/5 µl) Anlysis Mie reeived intrnsl (in) influenz (PR8 strin, 1 5% tissue ulture infetive doses [TCID 5 ]) or sline, followed y in S pneumonie 2 dys lter. Administrtion of orl oseltmivir (5 mg/kg, twie dy for 5 dys) or intrvenous permivir (6 mg/kg, single dose or dily for 5 dys) ws initited 2 h fter influenz inoultion. Lungs were smpled 2 dys fter pneumool inoultion. CFU, olony-forming unit. Antivirl Therpy

4 A Tnk et l. virus, followed two dys lter y S pneumonie. As shown in Figure 2A, signifint mortlity ws oserved in PR8/S pneumonie-infeted nimls, ut not in those inoulted with sline followed y S pneumonie. Furthermore, in nimls with preeding influenz infetion, teril hllenge with S pneumonie resulted in lmost 3-log inrese in the pulmonry teril urden within 2 dys (Figure 2B). Tken together, enhned mortlity ws oserved in nimls with prior influenz infetions when susequently hllenged with S pneumonie. Repeted doses of permivir signifintly prolonged the medin dte of survivl in oinfeted mie The effetiveness of intrvenous permivir for reduing the severity of seondry pneumool pneumoni ws ompred with tht of orl oseltmivir. As shown in Figure 3A, ll untreted mie died within 5 dys of seondry pneumool infetion. Tretment with oseltmivir or permivir (oth single nd multiple dosing) signifintly prolonged the medin durtion of survivl in oinfeted mie, reltive to untreted nimls. Additionlly, multiple dosing of permivir signifintly prolonged the medin durtion of survivl when ompred with single dose of permivir or with oseltmivir in oinfeted mie, ut no differene ws oserved etween mie treted with single dose of permivir nd those treted with oseltmivir (P=.136; Figure 3A). In dditionl studies shown in Figure 3B, multiple doses of permivir signifintly prolonged survivl when ompred with fourfold higher dose of oseltmivir in oinfeted mie, lthough tretment with oseltmivir signifintly prolonged the medin dte of survivl in oinfeted mie, reltive to untreted nimls. These results indite tht multiple doses of permivir ppered to improve the survivl of mie with seondry pneumool pneumoni, ompred with those treted with 5-dy regimen of oseltmivir or single dose of permivir. Intrvenous permivir redued teril urden nd virus titre in oinfeted mie The impt of the different drug-tretment regimens on vile teril ounts nd virl titres, fter oinfetion, were ompred. Tretment with oseltmivir or permivir signifintly redued vile teril ounts in the lungs 2 dys fter pneumool hllenge, ompred with untreted nimls. Furthermore, teril ounts in the intrvenous permivir-treted groups with multiple dosing deresed signifintly s ompred with the orl oseltmivir-treted groups (Figure 4A). Signifint differenes were oserved in the lung virl titres etween the oinfeted, untreted mie Figure 2. Prior influenz infetion enhnes sensitivity to seondry pneumool infetion A 1 B 1 8 Perentge survivl Flu+Sp Flu Sp Log CFU/ml Time fter infetion, dys Sp PR8+Sp (A) The survivl rte ws signifintly lower in mie infeted with oth influenz virus (Flu) nd S pneumonie (Sp) thn in singly infeted mie (P<.1). Pulmonry teril urden ws exmined in oinfeted (n=7) nd Sp-only infeted (n=4) mie 2 dys fter Sp inoultion. (B) The numers of vile teri in oinfeted lungs were signifintly higher thn in mie infeted only with Sp (P<.1). Survivl urves were estimted y the Kpln Meier method. Indites signifint differene (P<.1) in vile teril ounts etween singly Sp infeted mie nd Sp Flu-oinfeted mie. Eh experiment ws performed in duplite. CFU, olony-forming unit Interntionl Medil Press

5 Permivir effiy ginst seondry pneumool pneumoni Figure 3. Perentge survivl of mie oinfeted with influenz virus nd S pneumonie A 1 B Perentge survivl Permivir-M Permivir-S Perentge survivl Oseltmivir-H Oseltmivir Permivir-M Untreted Oseltmivir Time fter S pneumonie infetion, dys Untreted Time fter S pneumonie infetion, dys Multiple dosing of permivir signifintly prolonged the medin durtion of survivl when ompred with single dose of permivir or with oseltmivir in oinfeted mie. Mie were untreted (n=13) or treted with orl oseltmivir (5 mg/kg, twie per dy for 5 dys; n=8), single dose of intrvenous permivir (permivir-s: 6 mg/kg; n=9) or multiple doses of intrvenous permivir (permivir-m: 6 mg/kg, one dy for 5 dys; n=9). (A) Sttistil omprisons: untreted mie versus oseltmivir-treted mie, P=.3; untreted mie versus mie treted with permivir-s, P<.1; untreted mie versus mie treted with permivir-m, P<.1; mie treted with permivir-m versus oseltmivir-treted mie, P=.136. Permivir-M ppered to improve the survivl of mie with seondry pneumool pneumoni ompred with those treted with 5-dy regimen of oseltmivir or permivir-s. Mie were untreted (n=5) or treted with orl oseltmivir (5 mg/kg, twie per dy for 5 dys; n=8), high dose of orl oseltmivir (oseltmivir-h: 2 mg/kg, twie per dy for 5 dys; n=7) or permivir-m (6 mg/kg, one dy for 5 dys; n=8). (B) Sttistil omprisons: untreted mie versus oseltmivir-treted mie, P=.2; untreted mie versus mie treted with oseltmivir-h, P<.23; oseltmivir-treted mie versus permivir-m-treted mie, P=.23; oseltmivir-h-treted mie versus permivir-m-treted mie, P=.18. Survivl urves were estimted y the Kpln Meier method nd their homogeneity ws evluted y the log-rnk test. Indites signifint differene (P<.5) in survivl ompred with the oseltmivir group. Eh experiment ws performed in duplite. nd mie treted with oseltmivir (oth t 5 mg/kg nd 2 mg/kg) or permivir (oth single nd multiple dosing). Additionlly, the redution in lung virl titres with multiple-dose permivir ws signifintly greter thn with oseltmivir t oth dose levels (Figure 4B). These results suggest tht intrvenous permivir, espeilly when dministered in multiple doses, redues oth the teril nd virl urdens during oinfetion. The redued miroil urden results in the improvement of outomes ssoited with pneumool pneumoni seondry to influenz infetion. Intrvenous permivir suppressed prodution of proinflmmtory ytokines nd hemokines in oinfeted mie Our previous study showed tht the synergisti prodution of proinflmmtory ytokines during oinfetion plyed ruil role in the severity of seondry pneumool pneumoni [3]. Building on tht oservtion, in the urrent study, we exmined the onentrtions of TNF-, IL-6, KC, MIP-2 nd IFN-g in lung homogentes 2 dys fter infetion with influenz nd S pneumonie. The levels of proinflmmtory ytokines two dys fter infetion y influenz or S pneumonie lone were extremely low ompred with the oinfeted mie (dt not shown). As shown in Figure 5, the onentrtions of TNF-, KC nd MIP-2 were signifintly redued y oth single mpred with those ssoited with orl oseltmivir tretment. However, no signifint redution in the levels of IL-6 or IFN-g ws found; lthough, there ws trend towrds redution. These dt suggest tht intrvenous permivir might hve greter inhiitory effet on the prodution of proinflmmtory ytokines during oinfetion when ompred with oseltmivir tretment. Pulmonry inflmmtion ws redued in the lungs of mie treted with multiple doses of intrvenous permivir Two dys fter teril infetion, the lungs of slinetreted, influenz-infeted mie were found to e infiltrted y inflmmtory ells, whih reted mssive onsolidtion throughout the lungs. In mie treted with ntivirl gents, some inflmmtory ells were present, ut the exudtive lesions were redued. Surprisingly, the histology of lungs otined from oinfeted Antivirl Therpy

6 A Tnk et l. Figure 4. Pulmonry teril urden nd virl titres A B Log CFU/ml 6 4 Log TCID 5 /ml Untreted Oseltmivir Oseltmivir-H Permivir-S Permivir-M Untreted Oseltmivir Oseltmivir-H Permivir-S Permivir-M Pulmonry teril urden nd virl titres were mesured in oinfeted nimls treted with sline, orl oseltmivir, oth usul doses (oseltmivir) nd higher doses (oseltmivir-h), single dose of intrvenous permivir (permivir-s) or multiple doses of intrvenous permivir (permivir-m). (A) Vile teril ounts nd (B) virl titres in the superntnt of lung homogentes otined 2 dys fter seondry pneumool infetion. Eh experiment ws performed in duplite. P<.1; P<.5. CFU, olony-forming unit; TCID 5, 5% tissue ulture infetive dose. mie treted with multiple doses of intrvenous permivir showed few hnges (dt not shown). These findings suggest tht repeted doses of intrvenous permivir tretment leds to redution in the extent nd qulity of pulmonry inflmmtion, ontriuting to the improved outomes. Disussion Seondry pneumool pneumoni fter influenz infetion is leding use of deth worldwide. Although the effiy of ntivirl therpy with NAIs hs een reported for improving the mortlity nd omplitions of influenz, there is little evidene to eluidte the superiority for preventing the seondry pneumool pneumoni y NAIs [6,39]. In the present study, oth intrvenous permivir nd orl oseltmivir were tested to ompre the effet on the outome of seondry pneumool pneumoni in murine model. We hypothesized tht the effetiveness of intrvenous permivir on pneumool pneumoni following influenz infetion might e superior to orl oseltmivir due to permivir s exellent ntivirl tivity, inluding tight inding to virl NA nd phrmokinetis [15,4]. Our dt indite multiple doses of permivir signifintly prolonged survivl nd redued lung teril urden, virus titres nd pneumoni histopthologil severity ompred with oseltmivir. In ddition, s shown in Figure 5, the levels of inflmmtory ytokines treted with multiple doses of intrvenous permivir were signifintly lower thn tht of orl oseltmivir in the oinfeted mie. Tken together, they suggest redutions in oth virl nd teril growth might e required to inhiit exessive prodution of inflmmtory ytokines during virl teril oinfetion. The exellent effets of permivir demonstrted in this study my e due to the strong suppression of the initil influenz virus growth y the high lood onentrtion of the gent, immeditely fter dministrtion. Although permivir is usully used s single dose in unomplited influenz infetion, multiple dministrtions my e used for severely ill or highrisk ptients [17]. Furthermore, the effiy of multiple dosing of permivir in the lethl influenz infetion were reported in the murine model [41,42]. The present dt showed the effetiveness of multiple doses of permivir for preventing seondry pneumool pneumoni, lthough one limittion of our study ws tht drug dministrtion strted immeditely fter the mie were exposed to the influenz virus. Likewise, we did not exmine the virl nd teril titres t lter time points or the effet of delyed NAI tretment. Our niml model represented highly lethl one in whih the ontrol mie strted to die immeditely following S pneumonie inoultion nd ll mie were ded y 5 dys fter infetion. Additionl studies y using n lterntive oinfetion model mimiking Interntionl Medil Press

7 Permivir effiy ginst seondry pneumool pneumoni Figure 5. Conentrtions of proinflmmtory ytokines/hemokines in the lungs of oinfeted mie 5, 3, 4, TNF-α, pg/ml 3, 2, IL-6, pg/ml 2, 1, 1, Untreted Oseltmivir Permivir-S Permivir-M Untreted Oseltmivir Permivir-S Permivir-M 3, 15, KC, pg/ml 2, 1, MIP-2, pg/ml 1, 5, Untreted Oseltmivir Permivir-S Permivir-M Untreted Oseltmivir Permivir-S Permivir-M 1, 8, IFN-γ, pg/ml 6, 4, 2, Untreted Oseltmivir Permivir-S Permivir-M Conentrtions of proinflmmtory ytokines/hemokines in the lungs of oinfeted mie treted with sline, orl oseltmivir, single dose of intrvenous permivir (permivir-s) or multiple doses of intrvenous permivir (premmivir-m). Levels of tumour nerosis ftor (TNF)-, interleukin (IL)-6, kertinoyte-derived ytokine (KC), mrophge inflmmtory protein-2 (MIP-2) nd interferon (IFN)-g re shown. Eh experiment ws performed in duplite. P<.1; P<.1; P<.5. Antivirl Therpy

8 A Tnk et l. linil sitution further, for instne model showing intermedite lethlity, re wrrnted. Tretment of ute sesonl influenz in hospitlized dults with either permivir or oseltmivir, oth given in repeted doses, resulted in similr linil outomes [43]. The effets of oth drugs in ptients with severe seondry teril pneumoni hve not een previously ompred. In the urrent study we did not oserve the differenes etween the two oseltmivir doses tested (1 nd 4 mg/kg/dy in divided doses) in survivl or other outomes. Of note, pulished rndomized linil trils hve not found importnt differenes in outomes in out-lini ptients [44,45] or hospitlized ptients with influenz [46,47] ompring stndrd nd douled-dose oseltmivir regimens to dte. Comined therpy, involving permivir with ntiiotis would e expeted to improve the outome of seondry teril pneumoni. In the murine study y MCullers et l. [3], oseltmivir nd mpiillin demonstrted 1% survivl rte in oinfeted mie, wheres oseltmivir, lone, demonstrted only 75% survivl rte. If the results from the mie models trnslte to humns, intrvenous permivir might e n effetive tretment for the prevention of teril pneumoni seondry to n influenz infetion, prtiulrly in high-risk ptients. Antivirl therpy omined with ntiiotis during oinfetion might e le to improve the prognosis, espeilly in high-risk ptients, lthough nother set of niml experiments nd linil trils re required to exmine this hypothesis in the future. In onlusion, our study found tht multiple, ut not single, doses of intrvenous permivir showed greter effetiveness thn orl oseltmivir in reduing the severity of mixed influenz virl pneumool pneumoni in lethl murine model. Although further linil studies of intrvenous permivir re required, these oservtions my support, in prt, the prompt dministrtion of repeted dose regimen in seriously ill ptients, suh s hospitlized nd immunoompromised hosts, with influenz infetions. Aknowledgements This study ws prtilly supported y grnt from the Glol Centers of Exellene Progrm, Ngski University, Ngski, Jpn. Dislosure sttement S Kohno reeived n honorrium, leture fees nd reserh funds from Shionogi & Co., Ltd, nd Chugi phrmeutil Co., Ltd. The remining uthors delre no ompeting interests. Referenes 1. Hoyert DL, Xu J. Deths: preliminry dt for 211. Ntl Vitl Stt Rep 212; 61: Hsu J, Sntesso N, Mustf R, et l. Antivirls for tretment of influenz: systemti review nd met-nlysis of oservtionl studies. Ann Intern Med 212; 156: Chemly RF, Torres HA, Aguiler EA, et l. Neurminidse inhiitors improve outome of ptients with leukemi nd influenz: n oservtionl study. Clin Infet Dis 27; 44: Hernán MA, Lipsith M. Oseltmivir nd risk of lower respirtory trt omplitions in ptients with flu symptoms: met-nlysis of eleven rndomized linil trils. Clin Infet Dis 211; 53: Muthuri SG, Myles PR, Venktesn S, Leonrdi-Bee J, Nguyen-Vn-Tm JS. Impt of neurminidse inhiitor tretment on outomes of puli helth importne during the influenz A(H1N1) pndemi; systemti review nd met-nlysis in hospitlized ptients. J Infet Dis 213; 27: Yng SG, Co B, Ling LR, et l. Antivirl therpy nd outomes of ptients with pneumoni used y influenz A pndemi (H1N1) virus. PLoS ONE 212; 7:e Louie JK, Yng S, Aost M, et l. Tretment with neurminidse inhiitors for ritilly ill ptients with influenz A(H1N1)pdm9. Clin Infet Dis 212; 55: Yu H, Lio Q, Yun Y, et l. Effetiveness of oseltmivir on disese progression nd virl RNA shedding in ptients with mild pndemi 29 influenz A H1N1: opportunisti retrospetive study of medil hrts in Chin. BMJ 21; 341: Hrper SA, Brdley JS, Englund JA, et l. Sesonl influenz in dults nd hildren dignosis, tretment, hemoprophylxis, nd institutionl outrek mngement: linil prtie guidelines of the Infetious Diseses Soiety of Ameri. Clin Infet Dis 29; 48: Bnti S, Kellogg D, Prker C, Upshw R, Ilyushin NA, Bu YS. A single intrmusulr injetion of neurminidse inhiitor permivir demonstrtes ntivirl tivity ginst novel pndemi A/Cliforni/4/29 (H1N1) influenz virus infetion in mie. Antivirl Res 211; 9: Yun NE, Linde NS, Zks MA, et l. Injetle permivir mitigtes disese nd promotes survivl in ferrets nd mie infeted with the highly virulent influenz virus, A/ Vietnm/123/4 (H5N1). Virology 28; 374: Centers for Disese Control nd Prevention. Updte: drug suseptiility of swine-origin influenz A (H1N1) viruses, April 29. MMWR Mor Mortl Wkly Rep 29; 58: Birnkrnt D, Cox E. The Emergeny Use Authoriztion of permivir for tretment of 29 H1N1 influenz. N Engl J Med 29; 361: Hernndez JE, Adig R, Armstrong R, et l. Clinil experiene in dults nd hildren treted with intrvenous permivir for 29 influenz A (H1N1) under n Emergeny IND progrm in the United Sttes. Clin Infet Dis 211; 52: Kohno S, Kid H, Mizuguhi M, et l. Intrvenous permivir for tretment of influenz A nd B virus infetion in high-risk ptients. Antimiro Agents Chemother 211; 55: Kohno S, Kid H, Mizuguhi M, Shimd J. Effiy nd sfety of intrvenous permivir for tretment of sesonl influenz virus infetion. Antimiro Agents Chemother 21; 54: Kohno S, Yen MY, Cheong HJ, et l. Phse III rndomized, doule-lind study ompring single-dose intrvenous permivir with orl oseltmivir in ptients with sesonl influenz virus infetion. Antimiro Agents Chemother 211; 55: Interntionl Medil Press

9 Permivir effiy ginst seondry pneumool pneumoni 18. Sorello A, Jones SC, Crter W, et l. Emergeny use uthoriztion for intrvenous permivir: evlution of sfety in the tretment of hospitlized ptients infeted with 29 H1N1 influenz A virus. Clin Infet Dis 212; 55: LeVine AM, Koeningskneht V, Strk JM. Deresed pulmonry lerne of S. pneumonie following influenz A infetion in mie. J Virol Methods 21; 94: MCullers JA. Insights into the intertion etween influenz virus nd pneumoous. Clin Miroiol Rev 26; 19: Broug-Holu E, Toews GB, vn Iwrden JF, et l. Alveolr mrophges re required for protetive pulmonry defenses in murine Klesiell pneumoni: elimintion of lveolr mrophges inreses neutrophil reruitment ut dereses teril lerne nd survivl. Infet Immun 1997; 65: Morens DM, Tuenerger JK, Fui AS. Predominnt role of teril pneumoni s use of deth in pndemi influenz: implitions for pndemi influenz prepredness. J Infet Dis 28; 198: Pittet LA, Hll-Stoodley L, Rutkowski MR, Hrmsen AG. Influenz virus infetion dereses trhel muoiliry veloity nd lerne of Streptoous pneumonie. Am J Respir Cell Mol Biol 21; 42: Peltol VT, Murti KG, MCullers JA. Influenz virus neurminidse ontriutes to seondry teril pneumoni. J Infet Dis 25; 192: Colmussi ML, White MR, Crouh E, Hrtshorn KL. Influenz A virus elertes neutrophil poptosis nd mrkedly potentites poptoti effets of teri. Blood 1999; 93: Shhngin A, Chow EK, Tin X, et l. Type I IFNs medite development of postinfluenz teril pneumoni in mie. J Clin Invest 29; 119: Nkmur S, Dvis KM, Weiser JN. Synergisti stimultion of type I interferons during influenz virus oinfetion promotes Streptoous pneumonie oloniztion in mie. J Clin Invest 211; 121: Smith MW, Shmidt JE, Rehg JE, Orihuel CJ, MCullers JA. Indution of pro- nd nti-inmmtory moleules in mouse model of pneumool pneumoni fter influenz. Comp Med 27; 57: Sun K, Metzger DW. Inhiition of pulmonry ntiteril defense y interferon-during reovery from influenz infetion. Nt Med 28; 14: MCullers JA. Effet of ntivirl tretment on the outome of seondry teril pneumoni fter influenz. J Infet Dis 24; 19: MCullers JA, Brtmess KC. Role of neurminidse in lethl synergism etween influenz virus nd Streptoous pneumonie. J Infet Dis 23; 187: Krlström A, Boyd KL, English BK, MCullers JA. Tretment with protein synthesis inhiitors improves outomes of seondry teril pneumoni fter influenz. J Infet Dis 29; 199: Ymy M, Shiny K, Hthi Y, et l. Clrithromyin inhiits type sesonl influenz virus infetion in humn irwy epithelil ells. J Phrmol Exp Ther 21; 333:81 9. Aepted 17 Jnury 214; pulished online 12 Ferury Sto K, Sug M, Akike T, et l. Therpeuti effet of erythromyin on influenz virus-indued lung injury in mie. Am J Respir Crit Cre Med 1998; 157: Min JY, Jng YJ. Mrolide therpy in respirtory virl infetions. Meditors Inflmm 212; 212: Kitno M, Itoh Y, Kodm M, et l. Effiy of single intrvenous injetion of permivir ginst influenz B virus infetion in ferrets nd ynomolgus mques. Antimiro Agents Chemother 211; 55: Seki M, Yngihr K, Higshiym Y, et l. Immunokinetis in severe pneumoni due to influenz virus nd teri oinfetion in mie. Eur Respir J 24; 24: Wrd P, Smll I, Smith J, Suter P, Dutkowski R. Oseltmivir (Tmiflu) nd its potentil for use in the event of n influenz pndemi. J Antimiro Chemother 25; 55:i5 i Flgs ME, Koletsui PK, Vouloumnou EK, et l. Effetiveness nd sfety of neurminidse inhiitors in reduing influenz omplitions: met-nlysis of rndomized ontrolled trils. J Antimiro Chemother 21; 65: Bnti S, Arnold CS, Prker CD, Upshw R, Chnd P. Antiinfluenz virus tivity of permivir in mie with single intrmusulr injetion. Antivirl Res 26; 69: Boltz DA, Ilyushin NA, Arnold CS, Bu YS, Wester RG, Govorkov EA. Intrmusulrly dministered neurminidse inhiitor permivir is effetive ginst lethl H5N1 influenz virus in mie. Antivirl Res 28; 8: Kitno M, Kodm M, Itoh Y, et l. Effiy of repeted intrvenous injetion of permivir ginst influenz A (H1N1) 29 virus infetion in immunosuppressed mie. Antimiro Agents Chemother 213; 57: Ison MG, Hui DS, Clezy K, et l. A linil tril of intrvenous permivir ompred with orl oseltmivir for the tretment of sesonl influenz in hospitlized dults. Antivir Ther 213; 18: Niholson KG, Aoki FY, Osterhus AD, et l. Effiy nd sfety of oseltmivir in tretment of ute influenz: rndomized ontrolled tril. Neurminidse Inhiitor Flu Tretment Investigtor Group. Lnet 2; 355: Trenor JJ, Hyden FG, Vroomn PS, et l. Effiy nd sfety of the orl neurminidse inhiitor oseltmivir in treting ute influenz: rndomized ontrolled tril. US Orl Neurminidse Study Group. JAMA 2; 283: South Est Asi Infetious Disese Clinil Reserh Network. Effet of doule dose oseltmivir on linil nd virologil outomes in hildren nd dults dmitted to hospitl with severe influenz: doule lind rndomized ontrolled tril. BMJ 213; 346:f Lee N, Hui DS, Zuo Z, et l. A prospetive intervention study on higher-dose oseltmivir tretment in dults hospitlized with influenz A nd B infetions. Clin Infet Dis 213; 57: Antivirl Therpy