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

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1 Antivirl Chemistry & Chemotherpy 21; 21:71 84 (doi: /IMP1688) Review Lninmivir nd its prodrug, CS-8958: long-cting neurminidse inhiitors for the tretment of influenz Mkoto Ymshit 1 * 1 Biologicl Reserch Lortories, Diichi Snkyo Co., Ltd, Tokyo, Jpn *Corresponding uthor e-mil: ymshit.mkoto.yr@diichisnkyo.co.jp Oseltmivir nd znmivir re currently licensed worldwide for influenz tretment nd chemoprophylxis. Both drugs require twice-dily dministrtion for 5 dys for tretment. A new influenz drug, lninmivir (code nme R ), nd its prodrug form, CS-8958 (lninmivir octnote or lninmivir prodrug), which re long-cting neurminidse inhiitors, re introduced in this review. Lninmivir potently inhiited the neurminidse ctivities of vrious influenz A nd B viruses, including sutypes N1 N9, pndemic (29) H1N1 virus, highly pthogenic vin influenz (HPAI) H5N1 viruses nd oseltmivir-resistnt viruses. Becuse of the long retention of lninmivir in mouse lungs fter n intrnsl dministrtion of CS-8958, therpeutic dministrtion of single dose of CS-8958 showed superior efficcy to repeted dministrtions of znmivir or oseltmivir in niml infection models for influenz A nd B viruses. These include pndemic (29) H1N1 virus nd HPAI H5N1 virus. Prophylctic single dministrtion of CS-8958, s erly s 7 dys prior to infection, lso showed superior efficcy. Finlly, the potentil of single inhltion of CS-8958 for influenz ptients ws demonstrted y clinicl studies, nd CS-8958 hs een pproved nd is commercilly ville s Invir (Diichi Snkyo Co., Ltd, Tokyo) in Jpn. Introduction Influenz is serious respirtory illness, which cn e deilitting, nd could cuse complictions tht led to hospitliztion nd deth, especilly in elderly individuls. This respirtory disese is cused y influenz A nd B viruses, which re pthogens tht re highly contgious mong humns. Influenz A viruses re clssified into sutypes on the sis of the ntigenicities of hemgglutinin (HA) nd neurminidse (NA) molecules. To dte, 16 HA sutypes (H1 H16) nd 9 NA sutypes (N1 N9) hve een reported. Sesonl influenz or influenz epidemics re cused y influenz A viruses H1N1 nd H3N2 nd influenz B virus [1]. The glol urden of influenz epidemics is elieved to e 3.5 million cses of severe illness nd 3, 5, deths nnully [2]; these numers were clculted efore the new pndemic in 29. In the pst 1 yers, humns hve experienced three influenz pndemics: the first in 1918 (H1N1), the second in 1957 (H2N2) nd the third in 1968 (H3N2) [1]. Since its emergence in the erly spring of 29, pndemic (29) H1N1 influenz A viruses hve een circulting worldwide [3]. Although most infected individuls hve exhiited n uncomplicted mild respirtory disorder, the pthogenicity of this virus is clerly higher thn tht of sesonl influenz viruses in niml models [4 6] nd humns, including those who do not hve underlying illnesses [7]. The World Helth Orgniztion (WHO) [8] hs reported >18, deths worldwide s of 2 July 21 nd the US Centers for Disese nd Prevention [9] estimted.2% mortlity rte of midlevel rnge depending on ge. Another possile concern is pndemic cused y highly pthogenic vin influenz (HPAI) H5N1 viruses. In 1997, humn infections with HPAI H5N1 viruses were first documented in Hong Kong [1 12]. Since 23, these viruses hve spred throughout Asi, Europe nd Afric with high moridity nd mortlity mong vin species nd with occsionl trnsmission to humns, resulting in high mortlity. A totl of 57 cses infected with the HPAI H5N1 virus nd s mny s 32 deths were reported s of 18 Octoer 21 [13]. Although humn-to-humn trnsmission is rre, once the H5N1 viruses cquire this ility, devstting pndemic might e inevitle. Two countermesures, vccintion nd tretment with ntivirls, re ville to control humn influenz. 21 Interntionl Medicl Press (print) (online) 71

2 M Ymshit Although vccintion plys crucil role in influenz prophylxis, it is insufficient oth for prophylxis nd tretment ginst pndemic virus; therefore, ntivirls re n importnt tool tht might e used to mitigte influenz pndemics. Currently, two types of nti-influenz virus drugs re ville: M2 ion chnnel lockers (dmntnes) [14] nd NA inhiitors. In Figure 1, life cycle of influenz virus nd ction points of the inhiitors re explined; however, in terms of dmntnes, dmntne-resistnt viruses redily emerge nd re lredy prevlent worldwide mong the sesonl influenz viruses (oth the H1N1 nd the H3N2 sutypes) [15,16]. The dmntne drugs hve not een recommended for use for the tretment or chemoprophylxis of influenz in the US since the 25 influenz seson [15,17]. The recently emerged pndemic (29) H1N1 virus is lredy dmntne-resistnt [3]. Moreover, the emergence of dmntne-resistnt HPAI H5N1 viruses in Vietnm, Cmodi nd Thilnd [18] hs prompted the WHO to recommend oseltmivir for the tretment nd prophylxis of humn H5N1 influenz virus infections [19]. Accordingly, mny countries hve stockpiled oseltmivir in nticiption of n H5N1 pndemic. The second nd most recently developed clss of drugs with ctivities ginst influenz A nd B viruses re the NA inhiitors, which ind to the NA of newly formed virus prticles nd prevent their efficient relese from the host cell [2]. Two NA inhiitors, znmivir (inhled drug, 1 mg/dose; Relenz) nd oseltmivir (orl drug, 75 mg/ dose; Tmiflu), re currently licensed worldwide. Both drugs require twice-dily dministrtion for tretment. Another NA inhiitor, permivir (single intrvenous Figure 1. Life cycle of the influenz virus nd the inhiition step of neurminidse inhiitors I J H G E F A B H + D vrna mrna +RNA C A. The hemgglutinin (HA) of influenz virus recognizes neurminic cid linked to glycoproteins or glycolipids on the cell surfce, nd viruses thus ind to trget cells. B. The viruses ound to cells penetrte into the cell vi endocytosis. C. Protons in the endosome enter the virion vi the M2 ion chnnel protein, nd the HA structure ltered y cidifiction triggers memrne fusion, followed y the relese of virl RNAs (vrnas) into the cytoplsm. Admntne drugs lock the M2 ion chnnel. D. From vrnas, messenger RNAs (mrnas) for protein synthesis nd positive-strnd RNA (+RNA) for repliction of vrnas re synthesized in the nucleus. E. vrnas for progeny viruses re trnsported close to the cell memrne. F. Virl proteins re synthesized. G. vrnas nd virl proteins re ssemled nd generte progeny viruses on the cell surfce. H. Progeny viruses form ggregtes of ech other or with cells through the inding of HA to neurminic cid on viruses nd/or cells. I nd J. Virl neurminidse (NA) cleves the neurminic cid linkge, relesing progeny viruses from the virus ggregtes nd expnding infection. NA inhiitors inhiit this step to stop the relese of progeny viruses from virus ggregtes. The inset shows virus ggregtes (rrow) when cultured cells were infected with influenz viruses in the presence of lninmivir Interntionl Medicl Press

3 Lninmivir nd its prodrug CS-8958 for treting influenz drip infusion, 3 mg/dose; Rpict), ws licensed in 21 in Jpn. Oseltmivir is predominnt nd is used worldwide for the tretment of influenz, nd the genertion nd circultion of oseltmivir-resistnt sesonl influenz viruses hve ecome mjor concerns [21 25]. In prticulr, the worldwide prevlence of oseltmivirresistnt mutnts of sesonl H1N1 virus with the H274Y sustitution (histidine to tyrosine t position 274, numering sed on the N2 sutype) in NA hs een reported. In ddition, 95% of H1N1 isoltes tested from the fourth qurter of 28 to Jnury 29 [26] nd lmost ll the H1N1 isoltes of the seson in the US [27] were reported to e oseltmivir-resistnt. Additionlly, numer of oseltmivir-resistnt pndemic 29 H1N1 viruses [28] nd HPAI H5N1 viruses [25] hve lredy ppered, lthough their ppernce is still spordic. These epidemics of oseltmivir-resistnt influenz viruses therefore necessitte the development of lterntive ntivirl gents. Ymshit et l. [29] nd Kuo et l. [3] found new potent NA inhiitor, lninmivir (code nme R ), nd reported tht CS-8958 (lninmivir octnote or lninmivir prodrug) cts s long-cting NA inhiitor. The results of in vitro nd in vivo experiments of lninmivir nd CS-8958 [4,29 34] nd the clinicl studies of CS-8958 [35,36] re introduced in this review. The potentil of the single dministrtion of CS-8958 for influenz ptients ws confirmed y the clinicl studies conducted for the influenz seson [36 38]. CS-8958 hs een pproved nd is commercilly ville s Invir (Diichi Snkyo Co., Ltd, Tokyo) in Jpn. Compound Lninmivir (R ) is potent NA inhiitor of vrious influenz viruses. Its chemicl structure is (2R,3R,4S)-3-cetmido-2-[(1R,2R)-2,3-dihydroxy- 1-methoxypropyl]-4-gunidino-3,4-dihydro-2Hpyrn-6-croxylic cid, s shown in Figure 2 [29]. It demonstrted prolonged survivl effect in mouse infection model with influenz virus. Moreover, the esterified forms of lninmivir, with cyl chins of vrious lengths, significntly improved the prolonged survivl effect in the sme model. Among these forms, 3-(O)-octnoyl lninmivir (CS-8958; Figure 2) is the est compound in terms of its life-prolonging effect [29]. Both compounds were first discovered y Diichi Snkyo Co., Ltd. In vitro ctivities Neurminidse inhiitory ctivity A summry of NA inhiitory ctivities is shown in Tle 1. Lninmivir hd potent inhiitory ctivity to NAs of vrious influenz viruses, such s the sesonl Figure 2. Chemicl structures of lninmivir nd CS-8958 H 3 C H 3 C OH OH NH O OCH 3 H H1N1 [29], pndemic (29) H1N1 [4], H3N2 [29], HPAI H5N1 viruses [33] nd influenz B viruses [29]. Lninmivir showed 5% inhiitory concentrtion (IC 5 ) of.41 nm to pndemic (29) H1N1, A/ Cliforni/4/29, nd exhiited medin IC 5 of 1.55 nm to nine pndemic (29) H1N1 strins isolted in Jpn. The vlues were comprle to two other NA inhiitors, znmivir nd oseltmivir croxylte, n ctive metolite of oseltmivir. Also, lninmivir inhiited NA ctivity of HPAI H5N1 viruses with n IC 5 of nm. This IC 5 ws comprle to tht of znmivir nd oseltmivir croxylte, with the exception of A/Indonesi/UT36/5 (H5N1), which exhiited reduced susceptiility to oseltmivir croxylte (IC 5 =1 nm). Lninmivir showed less potent inhiitory ctivities ginst NA of H3N2 nd influenz B viruses compred with znmivir nd oseltmivir croxylte. The IC 5 of lninmivir to H3N2 ws pproximtely 1 lower thn tht of oseltmivir croxylte. To dte, 16 HA sutypes (H1 H16) nd 9 NA sutypes (N1 N9) of influenz A viruses hve een descried, ut only limited numer of sutypes O HN H 2 N CO 2 H NH Lninmivir (R ) O O H 3 C OH HN O OCH 3 H O HN H 2 N Lninmivir prodrug (CS-8958) CO 2 H NH Reproduced with permission from the Americn Society for Microiology [29]. Antivirl Chemistry & Chemotherpy

4 M Ymshit Tle 1. Inhiitory ctivities of lninmivir to neurminidses of vrious influenz viruses Numer of Medin IC 5, nm (rnge) Virus type Virus strin strins investigted Lninmivir Znmivir Oseltmivir croxylte Sesonl H1N ( ) 1.38 ( ) 1.26 ( ) H3N ( ) 7.33 ( ) 1.33 ( ) B ( ) 6.41 ( ) 9.55 ( ) Pndemic (H1N1) 29 A/Cliforni/4/ Jpnese isoltes ( ) 1.35 ( ).679 ( ) HPAI H5N (.28.32).15 (.7.72).35 (.31 1.) N1 N9 9 ( ) ( ) ( ) H5N1 A/R(duck/Mongoli/54/ duck/mongoli/47/1) H2N2 A/Singpore/1/ H5N3 A/duck/Hokkido/84/ H8N4 A/turkey/Ontrio/6118/ H12N5 A/duck/Alert/6/ H11N6 A/duck/Englnd/1/ H7N7 A/sel/Msschusetts/1/ H3N8 A/duck/Ukrine/1/ H11N9 A/duck/Memphis/546/ Dt for nine viruses with sutype N1 N9 re descried. Ressortnt virus generted from A/duck/Mongoli/54/1 (H5N2) nd A/duck/Mongoli/47/1 (H7N1). HPAI, highly pthogenic vin influenz; IC 5, 5% inhiitory concentrtion. (H1N1, H2N2 nd H3N2) hve circulted in humns [1]. In the future, new viruses with other NA sutypes will possily generte nd circulte in humns. As shown in Tle 1, lninmivir inhiited ll NA sutypes nd might e effective ginst emerging viruses with NA sutypes other thn N1 nd N2 [29]. As shown in Tle 2, lninmivir inhiited NA ctivity of the oseltmivir-resistnt viruses of sesonl H1N1, HPAI H5N1 nd H3N2, which were isolted from infected ptients [29]. Also, lninmivir mintined inhiitory ctivity to oseltmivir-resistnt pndemic (29) H1N1 virus, which possessed the H274Y sustitution [34]. According to these results, it should e noted tht the degrees of resistnce of oseltmivir croxylte nd permivir to H274Y mutnts were comprle to ech other, nd were 63-fold nd 564- fold, respectively (Tle 2). Virus susceptiility in cell culture The inhiitory effect of lninmivir to virl repliction in cultured cells ws investigted. Also, the 5% effective concentrtion (EC 5 ) determined y plque ssy for sesonl H1N1, H3N2 nd influenz B viruses, nd the 9% effective concentrtion on the sis of the medin tissue culture infective dose vlue of virus titre for the pndemic (29) H1N1 virus re shown in Tle 3. Lninmivir strongly inhiited the repliction of these viruses. The EC 5 vlues hve very lrge rnge. It ws reported tht the susceptiility of influenz viruses to NA inhiitors ws vrile, indicting tht NA inhiitory ctivity is currently the only in vitro ssy tht is predictive of in vivo susceptiility to NA inhiitors [39]. This phenomenon could e prtly explined y receptor specificity of the virus. HA is receptor recognition protein of humn influenz virus, which minly recognizes silic cid with n α-2-6 linkge s receptor on the cells [4,41]. Menwhile, the mjor silic cid linkge on the Mdine Dry cnine kidney (MDCK) cells, which ws used for the plque ssy, hs een reported to e n α-2-3 linkge [42,43]. This recognition/receptor mismtch induces wek inding of viruses to the cells, enling progeny viruses to e esily relesed from the infected cells without NA ction. In this cse, influenz viruses could replicte in the cells in the presence of NA inhiitors. In vivo efficcy Therpeutic efficcy fter single dministrtion of CS-8958 in niml infection models In vitro inhiitory ctivities of lninmivir were not lrgely different from those of two other NA inhiitors. However, octnoyl lninmivir, CS-8958, showed good efficcy y single intrnsl dministrtion in n niml infection model. The licensed drugs, znmivir (inhled drug, 1 mg/dose) nd oseltmivir (orl drug, 75 mg/dose), require twice-dily dministrtion for 5 dys for tretment of influenz. The virus titres in the lungs of mice infected with influenz virus A/PR/8/34 were mesured fter single intrnsl dministrtion of CS-8958, twice-dily orl dministrtion of oseltmivir nd twice-dily intrnsl dministrtion of znmivir. Groups treted with CS-8958 t doses of.27 nd.8 mg/kg oth Interntionl Medicl Press

5 Lninmivir nd its prodrug CS-8958 for treting influenz Tle 2. Inhiitory ctivity of lninmivir, znmivir nd oseltmivir croxylte ginst oseltmivir-resistnt influenz viruses Oseltmivir Lninmivir Znmivir croxylte Virus type Strin Muttion in NA IC 5, nm Rtio IC 5, nm Rtio IC 5, nm Rtio Sesonl H1N1 A/Yokohm/67/26 (clone 1) WT A/Yokohm/67/26 (clone 11) H274Y H3N2 A/Kwski/IMS22A-954/23 WT A/Kwski/IMS22B-955/23 R292K ,4 8,4 A/Yokohm/IMS9A-229/23 WT A/Yokohm/IMS9B-25/23 E119V A/Kwski/MS31A-13/22 WT A/Kwski/MS31B-126/22 N294S HPAI H5N1 A/Hnoi/348/5 WT H274Y ,2 N294S A/Vietnm/123/4 WT H274Y ,1 3,5 N294S Pndemic (H1N1) 29 cd Three strins WT Four strins H274Y , The left mino cid mutted to the right one t the indicted residue numer, which is sed on the N2 neurminidse (NA) numering. Amino cids re descried using one-letter symol. The rtio of the 5% inhiitory concentrtion (IC 5 ) of mutnt virus to tht of wild-type virus is indicted. c Men IC 5 vlues to three wild-type strins of A/New York/18/29, A/Wshington/29/29 nd A/Singpore/91/29 nd four H274Y mutnts of A/Osk/18/29, A/Wshington/29/29, A/Hong Kong/2369/29 nd A/Singpore/57/29 re shown. d Dt from [34]. The IC 5 vlues were determined y fluorescent ssy using 2-(4-methylumelliferyl)-α-N-cetylneurminic cid s fluorogenic sustnce. The IC 5 vlues of permivir to WT nd H274Y mutnts were.22 nm nd 124 nm, respectively. HPAI, highly pthogenic vin influenz. Tle 3. Inhiitory ctivities of lninmivir, znmivir nd oseltmivir croxylte ginst repliction of vrious influenz viruses in cell cullture Numer of EC 5, nm Virus type/sutype Virus strin strins investigted Lninmivir Znmivir Oseltmivir croxylte Sesonl H1N H3N B Pndemic (H1N1) 29 A/Cliforni/4/ N1 N H5N1 A/R(duck/Mongoli/54/ duck/mongoli/47/1) H2N2 A/Singpore/1/ H5N3 A/duck/Hokkido/84/ H8N4 A/turkey/Ontrio/6118/ H12N5 A/duck/Alert/6/ H11N6 A/duck/Englnd/1/ H7N7 A/sel/Msschusetts/1/ H3N8 A/duck/Ukrine/1/ H11N9 A/duck/Memphis/546/ Dt re rnges of the strins investigted or mens of three different ssys. The 5% effective concentrtion (EC 5 ) vlues were determined y plque ssy, nd re descried on the sis of the medin tissue culture infective dose. Dt for nine viruses with sutype N1 N9 re descried. showed sttisticlly significnt reductions in virus titres compred with the oseltmivir group t dose of 1 mg/kg (Figure 3A). The CS-8958 group t dose of.24 mg/kg (.5 µmol/kg) showed similr reduction in virus titres compred with the znmivir group t dose of.17 mg/kg (.5 µmol/kg; Figure 3B). The superior efficcy of the single dministrtion of CS-8958 ws confirmed in lethl mouse infection model [3]. The efficcy of CS-8958 ws investigted in ferrets, which re susceptile to influenz virus nd lso develop n upper respirtory trct infection s result of influenz virus infection. At 4 h post-infection, ferrets Antivirl Chemistry & Chemotherpy

6 M Ymshit Figure 3. Therpeutic efficcy of single dministrtion of CS-8958 in the mouse infection model Virus titre, log 1 PFU/lungs A infected with B/Mlysi/256/24 received dose of sline intrnslly,.24 mg/kg (.5 µmol/kg) dose of CS-8958,.17 mg/kg (.5 µmol/kg) dose of znmivir or 25 mg/kg of twice-dily orl dministrtion of oseltmivir from 4 hpi. The virus titres in the nsl wshes re shown in Figure 4 [3]. The single intrnsl dministrtion of CS-8959 significntly decresed virus titres in the ferret infection model. Therpeutic efficcy fter single dministrtion of CS-8958 in HPAI H5N1 virus nd in the pndemic (29) H1N1 infection mouse model The therpeutic efficcy of CS-8958 ginst HPAI H5N1 influenz viruses ws evluted using mouse lethl infection model. Mice were infected with three different strins of HPAI H5N1 viruses, nd were then given single dose of CS-8958 intrnslly or were treted with oseltmivir dministered orlly twice-dily for 5 dys. Survivl of the infected mice ws monitored B 9. Time post-infection, h Figure 4. Therpeutic efficcy of single dministrtion of CS-8958 in the ferret infection model Virus titre, log 1 PFU/lungs 8. NS Time post-infection, h CS mg/kg Oseltmivir 1. mg/kg CS mg/kg Oseltmivir 1 mg/kg CS µmol/kg Znmivir.5 µmol/kg Virl titre in nsl wsh, log 1 PFU/ml Time post-infection, dys Znmivir.5 µmol/kg Oseltmivir 25 mg/kg CS µmol/kg Reproduced with permission from the Americn Society for Microiology [3]. (A) Virus titres in the mouse lungs fter sline dministrtion, repeted orl dministrtions of oseltmivir (1. mg/kg or 1 mg/kg) nd single intrnsl dministrtion of CS-8958 (.27 mg/kg or.8 mg/kg). (B) Virus titres in the mouse lungs fter sline dministrtion, repeted intrnsl dministrtions of znmivir (.5 µmol/kg, corresponding to.17 mg/kg) nd single intrnsl dministrtion of CS-8958 (.5 µmol/kg, corresponding to.24 mg/kg). Mice were infected with A/PR/8/34 (H1N1; 3 plque-forming units [PFU]) t h. The dministrtions were performed t the indicted time points, once for CS-8958 (lck rrow) nd twice dily for other NA inhiitors (gry rrows). The virus titres were mesured t three time points. Sttisticlly significnt differences were oserved etween oth groups of CS-8958 nd the 1 mg/kg dministrtion group of oseltmivir (A). Sttisticl nlysis ws performed y two-wy ANOVA. P<.1. NS, non-significnt. Reproduced with permission from the Americn Society for Microiology [3]. Ferrets were intrnslly infected with B/Mlysi/256/24 (1, plqueforming units [PFU]). Virus titres re shown for the nsl wsh fter sline dministrtion, repeted orl dministrtions of oseltmivir (25 mg/kg), single intrnsl dministrtion of znmivir (.5 µmol/kg, corresponding to.17 mg/kg) nd single intrnsl dministrtion of CS-8958 (.5 µmol/kg, corresponding to.24 mg/kg). The dministrtions were performed once for znmivir nd CS-8958 nd twice dily for oseltmivir. Virus titres were mesured t three time points. The virus titres in the nsl wshes from the untreted ferrets decresed fter 2 dys post-infection. This ws minly result of the limited numer of cells in the noses of the ferrets tht were susceptile to virus infection, nd ws less likely to e cused y innte immunity, if ny. Therefore, sttisticl nlysis ws performed on the sis of the re under the curve of the virl titres from 1 2 dys postinfection y two-wy ANOVA. P<.1 nd P<.1 versus control Interntionl Medicl Press

7 Lninmivir nd its prodrug CS-8958 for treting influenz for 21 dys nd survivl dt of mice infected with A/Vietnm/123/4 re shown in Figure 5A. A single dose of CS-8958 ws efficcious for mice infected with the HPAI H5N1 viruses [33]. This therpeutic efficcy of single dose of CS-8958 ginst H5N1 viruses ws lso confirmed y the determintion of virus titres in Figure 5. Therpeutic efficcy of CS-8958 ginst HPAI H5N1 virus nd pndemic (29) H1N1 virus in the mouse infection model A 1 HPAI H5N1 virus Survivl rte, % the lungs nd rin of HPAI virus A/Hnoi/348/5 clone 7- or A/Indonesi/UT36/5-infected mice. CS-8958 t.75 or 1.5 mg/kg drmticlly inhiited the repliction of oth viruses, prticulrly in the rin, compred with oseltmivir [33]. These results indicte tht CS-8958 confers more potent nd long CS mg/kg CS mg/kg CS mg/kg Oseltmivir 5 mg/kg Oseltmivir 5 mg/kg Time post-infection, dys B 8 Dy 3 8 Dy 6 Oseltmivir 4 mg/kg Virl titre, log 1 PFU/g lungs Virl titre, log 1 PFU/g lungs Oseltmivir 4 mg/kg Znmivir.8 mg/kg Znmivir 8 mg/kg CS mg/kg 4 Oseltmivir Znmivir CS Oseltmivir Znmivir CS-8958 (A) Reproduced from [33]. Mice were intrnslly infected with A/Vietnm/123/4 (H5N1; 4 5% mouse lethl dose) nd were then given CS-8958 intrnslly once (.75 mg/kg,.75 mg/kg or 1.5 mg/kg), oseltmivir orlly twice dily for 5 dys (5 mg/kg or 5 mg/kg) or distilled wter/sline s control. Survivl ws monitored dily for 21 dys. Sttisticl nlysis ginst the control ws done y log-rnk test using joint rnking method ( P<.5). (B) Reprinted y permission from Mcmilln Pulishers, Ltd [4]. Mice were intrnslly infected with A/Cliforni/4/9 (pndemic [29] H1N1; 1, plque-forming units [PFU]) nd dministered oseltmivir (orlly, twice dily for 5 dys), znmivir (intrnslly, once dily for 5 dys), CS-8958 (intrnslly, once) or distilled wter/phosphte-uffered sline (control). Doses re indicted in the figure. The virus titres in lungs on dys 3 nd 6 were determined y plque ssys in Mdine Dry cnine kidney cells. The sttisticl significnce of differences ginst control mice ws ssessed y the Student s t-test ( P<.5 nd c P<.1). HPAI, highly pthogenic vin influenz. Antivirl Chemistry & Chemotherpy

8 M Ymshit Figure 6. Therpeutic efficcy of CS-8958 ginst oseltmivir-resistnt viruses with the H274Y sustitution in the mouse infection model A 5. Sesonl H1N1 with H274Y B 1 HPAI H5N1 virus with H274Y Virl titre, log 1 PFU/lungs Survivl rte, % Time post-infection, h Time post-infection, dys CS mg/kg CS mg/kg Oseltmivir 1 mg/kg CS mg/kg CS mg/kg CS mg/kg Oseltmivir 5 mg/kg Oseltmivir 5 mg/kg (A) Reproduced with permission from the Americn Society for Microiology [3]. Mice were infected with oseltmivir-resistnt A/Yokohm/67/26 (H1N1) with the H274Y sustitution (1, plque-forming units [PFU]) nd were then intrnslly dministered once with CS-8958 (.27 mg/kg or.8 mg/kg) or orlly dministered twice dily with oseltmivir (1 mg/kg) or control. Virl titres in the lungs were mesured t three time points. Sttisticlly significnt differences were oserved etween CS-8958 nd control ( P<.1). (B) Reproduced from [33]. Mice were intrnslly infected with A/Vietnm/123/4 (H5N1) with the H274Y sustitution (4 5% mouse lethl dose) nd then were intrnslly dministered once with CS-8958 (.75 mg/kg,.75 mg/kg or 1.5 mg/kg) or orlly dministered twice dily for 5 dys with either oseltmivir (5 mg/kg or 5 mg/kg) or control. Survivl ws monitored dily for 21 dys. Sttisticl nlysis ginst the control ws done y logrnk test using joint rnking method ( P<.5). lsting protection to mice ginst H5N1 influenz viruses thn oseltmivir. Next, efficcy ginst pndemic (29) H1N1 A/ Cliforni/4/9 virus ws investigted in the mouse infection model. Mice infected with the virus were given single dose of CS-8958 intrnslly, oseltmivir ws orlly dministered twice-dily for 5 dys or znmivir ws dministered intrnslly once-dily for 5 dys. The virus titres in the mice lungs were mesured on dys 3 nd 6 fter infection (Figure 5B) [4]. Agin, single dministrtion of CS-8958 showed significnt efficcy nd stronger decrese in virl titres compred with repeted dministrtion of oseltmivir or znmivir [4]. Therpeutic efficcy fter single dministrtion of CS-8958 in oseltmivir-resistnt H1N1 nd HPAI H5N1 virus infection mouse model The efficcy fter single dose of CS-8958 ws confirmed in mouse model infected with the oseltmivirresistnt sesonl H1N1 virus monitored y virus titres in lungs (Figure 6A) [3] nd with the HPAI H5N1 virus monitored y the survivl rte (Figure 6B) [33]. Both viruses hve the H274Y sustitution. In the sesonl A/ Yokohm/67/26 (H1N1) H274Y infection model, sttisticlly significnt virus titre reduction ws oserved in single dose of CS-8958 t.8 mg/kg compred with control. No reduction in virus titres ws oserved in twice-dily dministrtion of oseltmivir t 1 mg/ kg. In the HPAI A/Vietnm/123/4 (H5N1) H274Y virus infection model, only 12.5% nd 5% of mice survived t dose of 5 nd 5 mg/kg of oseltmivir (twice dily for 5 dys), respectively. By contrst, one-hlf of the mice survived with single dose of.75 mg/kg of CS It should e noted tht for one-hlf of the mice to survive, the dose of CS-8958 ws comprle to presumed clinicl dose of CS-8958, wheres s much s 5 mg/kg (twice dily for 5 dys) of oseltmivir (clinicl dose, 75 mg/dose) ws required. Thus, CS-8958 lso showed good efficcy to oseltmivir-resistnt virus. Prophylctic efficcy fter single dministrtion of CS-8958 in niml models Once-dily dministrtion of znmivir (inhled drug, 1 mg/dose) nd of oseltmivir (orl drug, 75 mg/dose) hve een licensed s prophylctic drugs. This suggests tht the hlf life (t 1/2 ) of ech drug in trget tissue is Interntionl Medicl Press

9 Lninmivir nd its prodrug CS-8958 for treting influenz Figure 7. Prophylctic efficcy of CS-8958 in the mouse infection model A 1 B Survivl, % 6 4 Survivl, % 6 4 c Time post-infection, dys Time post-infection, dys 12 h 4 dys 7 dys 1 dy Reproduced with permission from the Americn Society for Microiology [3]. Mice were infected with A/PR/8/34 (H1N1; 1 plque-forming units) on dy. (A) Oseltmivir t 11 mg/kg nd (B) CS-8958 t.17 mg/kg were dministered t 12 h, 1 dy, 4 dys or 7 dys efore infection. Sline ws dministered intrnslly s control t 7 dys efore infection. Survivl ws monitored dily for 2 dys. Sttisticl nlysis ginst control ws done y log-rnk test sed on joint rnking method ( P<.1, P<.5 nd c P<.1). not long; therefore, they require once-dily dministrtion. By comprison, the t 1/2 of CS-8958 fter intrnsl dministrtion to mice is quite long. Prophylctic efficcy of single dose of CS-8958 ws investigted in mouse infection model. A single dministrtion of CS-8958 prior to infection with A/PR/8/34 showed life-prolonging effect in the mouse infection model. One-hlf of the mice dministered CS-8958 t.17 mg/kg 7 dys efore infection survived. By contrst, only one-qurter of the mice treted with oseltmivir t s much s 11 mg/kg 1 dy efore infection survived. The dose of oseltmivir ws much higher thn the clinicl setting (Figure 7) [3], proly ecuse of short t 1/2 of 3 h nd time of mximum concentrtion of.5 h of oseltmivir croxylte in mouse plsm fter n intrvenous dministrtion of oseltmivir croxylte nd orl dministrtion of oseltmivir, respectively [44]. Prophylctic efficcies ginst H5N1 virus fter the single intrnsl dministrtion of CS-8958 were lso confirmed in oth the lethl mice infection model nd in the mesurement of virus titre reduction in mice lungs nd rin [33]. Two unique chrcteristics of CS-8958 nd lninmivir s long-cting drug The therpeutic nd prophylctic efficcies chieved fter the single intrnsl dministrtion of CS-8958 could e result of the unique chrcteristics of the compounds descried elow. Long-lsting drug in respirtory orgns of nimls The lung concentrtion time profiles of CS-8958 nd lninmivir fter single intrnsl dministrtion of CS-8958 t dose of.5 µmol/kg (.236 mg/kg) to mice re shown in Figure 8 [31]. After dministrtion, the lung CS-8958 concentrtions decresed with t 1/2 of.833 h nd, t 12 h post-dose or lter, the CS-8958 concentrtions were elow the lower limit of quntifiction. By contrst, the lung lninmivir concentrtions incresed soon fter dministrtion, nd, susequently, lninmivir retined t 1/2 of s long s 41.4 h. Even t 12 h post-dose, lninmivir remined in lungs t concentrtion of.915 nmol/g [31], which is considerly higher thn the IC 5 of lninmivir ginst the NA ctivities of vrious influenz viruses (Tle 1) [31]. In ddition, similr long retention of lninmivir ws oserved in the trget tissues (trche nd lung) fter intrtrchel dministrtion of CS-8958 to rts, nd pproximtely 2% of the dose (.2 mg/kg) ws retined in the lungs t 24 h [32]. These results demonstrted tht CS-8958 dministered intrnslly or intrtrchelly to nimls ws converted/hydrolysed to lninmivir nd retined s the hydrolysed form in the trget tissues Antivirl Chemistry & Chemotherpy

10 M Ymshit for long time. The long-lsting chrcteristics of lninmivir in the trget tissues for influenz virus infection result in superior efficcy y single dose of CS-8958, wheres repeted doses of other NA inhiitors re required for efficcy. Binding stility of lninmivir to viruses Bnti et l. [45] demonstrted tht permivir, selective NA inhiitor [46,47], inds to NA, with the N9 sutype more tightly thn oseltmivir croxylte or znmivir. This chrcteristic is thought to e one of the resons why permivir is efficcious y single intrvenous injection. To ssess the inding stility of lninmivir to vrious virus NAs, similr experiments with whole influenz virions were performed, nd the results re shown in Figure 9 [33]. In these experiments, virions were treted with excess NA inhiitors, then unound forms of NA inhiitor were removed nd the NA rection ws chsed over time fter dding the NA sustrte. Considerle differences in the dissocition rtes of the NA inhiitors mong the test virus strins were oserved. NA ctivity of A/Pnm/27/99 (H3N2) ws severely inhiited y ll test NA inhiitors for t lest 5 h. NA ctivity of B/Mie/1/93 treted with lninmivir ws pprecily inhiited y pproximtely Figure 8. Phrmcokinetics in the mouse lungs fter intrnsl dministrtion of CS-8958 Lung concentrtion, nmol/g Time, h CS-8958 Lninmivir Anlyte t ½, h C mx, nmol/g tissue T mx, h CS NC NC Lninmivir Reproduced with permission from the Americn Society for Microiology [31]. Mice were intrnslly dministered.5 µmol/kg (.236 mg/kg) of CS-8958, nd the concentrtion of CS-8958 nd lninmivir in lungs ws determined. Bsed on the concentrtion time profiles of CS-8958 nd lninmivir, the phrmcokinetic prmeters were clculted nd re lso shown. C mx, mximum concentrtion in lungs; NC, not clculted; t mx, time to mximum concentrtion; t 1/2, hlf-life. 5% t 6 h, wheres no inhiitory effect ws oserved t this time point for the other three NA inhiitors. NA ctivity of A/New Cledoni/2/99 (H1N1) treted with lninmivir nd permivir ws suppressed to pproximtely 3% t 6 h, wheres the NA ctivity treted with znmivir nd oseltmivir croxylte recovered to 8 9% t 6 h. These results suggest tht lninmivir ound to virl NA more stly thn ny other NA inhiitors tested. This stle inding of lninmivir might e nother reson for the long-cting chrcteristics of CS Clinicl studies Phse I studies The next importnt point is to confirm whether the long-lsting chrcteristic of lninmivir fter single intrnsl dministrtion of CS-8958 oserved in nimls is pplicle to humns or not. Helthy mle volunteers prticipted in doule-linded rndomized plceo- controlled trils nd received single dose of 5, 1, 2, 4, 8 or 12 mg CS-8958 or twicedily dose of 2 or 4 mg CS-8958 for 3 dys. The clinicl nd lortory prmeters nd plsm nd urinry concentrtions of CS-8958 nd lninmivir for the 144 h post-dosing were mesured. CS-8958 disppered from plsm with t ½ of pproximtely 2 h. By contrst, lninmivir ws slowly eliminted from the ody, lsting for up to 144 h fter dministrtion with t ½ of pproximtely 3 dys. There were no dverse events relted to the test drug in ll groups, nd it ws indicted tht when CS-8958 ws inhled y helthy volunteers, it ws well-tolerted [35]. Although the detection of the ctive metolite in plsm for n extended period might e consistent with long hlf-life in the trget tissues, such s the trche nd lung, it ws not possile to demonstrte this directly through smpling of the trget tissue ecuse of the limittions of the Phse I study; however, these dt suggested tht the long-lsting concentrtion of lninmivir fter single intrnsl dministrtion of CS-8958 in nimls might lso e pplicle to humns who inhled CS Clinicl studies in influenz ptients Phse II/III studies in peditric ptients were conducted s doule-linded rndomized controlled tril to compre CS-8958 with oseltmivir in the influenz seson in Jpn. During this seson, lmost ll H1N1 viruses were oseltmivir-resistnt viruses with the H274Y sustitution. Eligile ptients were children, 9 yers of ge nd under, who hd ferile influenz symptoms of no more thn 36 h durtion. Ptients were rndomized to one of three tretment groups: CS mg group, CS mg group or 8 21 Interntionl Medicl Press

11 Lninmivir nd its prodrug CS-8958 for treting influenz Figure 9. Binding stility of NA inhiitors to viruses 7, A/New Cledoni/2/99 (H1N1) 7, B/Mie/1/93 6, 6, Fluorescence unit 5, 4, 3, 2, Fluorescence unit 5, 4, 3, 2, 1, 1, Time, min Time, min , A/Pnm/27/99 (H3N2) Fluorescence unit 16, 12, 8, 4, Lninmivir Permivir Znmivir Oseltmivir croxylte Without virus Without NA inhiitor Time, min Reproduced from [33]. The vrious influenz viruses treted with excess neurminidse (NA) inhiitors (1 2 the 9% inhiitory concentrtion vlues) nd unound NA inhiitors were removed from the drug virus mixtures with Bio-Spin P-6 column (Bio-Rd Lortories, Hercules, CA, USA). The NA sustrte ws dded to the virus NA-inhiitor complex nd the NA ctivities were chsed for 6 h. A/New Cledoni/2/99 (H1N1), A/Pnm/27/99 (H3N2) nd B/Mie/1/93 were used s virus sources for these experiments. Lninmivir, permivir, znmivir or oseltmivir croxylte were used s NA inhiitors. NA ctivities without virus nd without NA inhiitor re lso shown. n oseltmivir group (n=61 62 ptients per group). CS-8958 ws dministered s single inhltion. Oseltmivir 2 mg/kg ws orlly dministered twice dily for 5 dys, which is the regulr regimen for peditric use. CS-8958 mrkedly reduced the medin time to llevition of influenz illness in comprison with oseltmivir in ptients infected with oseltmivir-resistnt influenz A (H1N1); the difference ws -6.9 h in the 4 mg group nd h in the 2 mg group (Figure 1) [36]. By contrst, there were no significnt differences in the time to llevition of illness etween the CS-8958 groups nd the oseltmivir group ginst influenz A (H3N2) or B infection. CS-8958 ws well tolerted [36]. CS-8958 ws n effective nd well-tolerted tretment for children with oseltmivir-resistnt influenz A (H1N1) virus infection. A single inhltion of CS-8958 during the course of illness might e eneficil regimen in terms of therpeutic complince. A Phse III study of dults, the Multintionl Asin Clinicl Reserch for Influenz Virus Extermintion on Long-Acting Neurminidse-Inhiitor Study (MAR- VEL), ws performed s rndomized doule-linded ctive-controlled study in order to confirm the efficcy nd sfety of CS-8958 dministered s single inhled dose of 2 or 4 mg compred with oseltmivir 75 mg orlly dministered twice dily for 5 dys ( totl of 1 times) in dult ptients with influenz A or B virus infection [38]. According to the results, non-inferiority to oseltmivir ws confirmed in oth the 2 mg nd 4 mg group of CS-8958 in terms of the primry end point, which ws the time to llevition of influenz illness. Both CS-8958 doses were well-tolerted [37,38]. Antivirl Chemistry & Chemotherpy

12 M Ymshit Figure 1. Therpeutic efficcy of CS-8958 in peditric influenz ptients Cumultive proportion, % Full nlysis set 12 Time post-tretment, h CS mg (n=61) CS mg (n=61) Oseltmivir (n=62) Cumultive proportion, % Sugroup ccording to virus type A/H1N1 (full nlysis set) Time post-tretment, h CS mg (n=4) CS mg (n=4) Oseltmivir (n=32) Reproduced with permission from the Americn Society for Microiology [36]. Dosge for oseltmivir ws 2 mg/kg twice dily for 5 dys. (A) The medin time to llevition of influenz illness ws shorter in the CS-8958 groups thn in the oseltmivir group, nd the difference ws h in the 4 mg group (P=.59) nd -31. h in the 2 mg group (P=.9) in the full nlysis set. (B) The sugroup nlyses showed tht oth dosges of CS-8958 mrkedly reduced the medin time to illness llevition in the H1N1-infected supopultion in comprison with oseltmivir. Conclusions In contrst to the currently ville drugs, which require multiple dosing, for exmple, twice dily for 5 dys, it ws confirmed tht the single inhltion of CS-8958 is sufficient to tret influenz in humns. This CS-8958 dosing regimen will llow etter complince for ptients with influenz. Furthermore, individuls with influenz might spred influenz virus from up to 2 dys efore to pproximtely 5 dys fter the onset of symptoms, nd children cn spred the virus for 1 dys or longer. Therefore, continuous suppression of influenz virus repliction in influenz ptients is importnt. The dosing regimen of CS-8958, which is single dministrtion, hs potentil enefit compred with tht of currently ville drugs, which require twice-dily dministrtion for 5 dys, prticulrly ecuse interruption of dministrtion of current drugs fter relief or forgetfulness might increse the risk of trnsmission to others. We lerned from the pndemic in 29 tht nti-influenz drugs re importnt for tretment nd prophylxis of pndemic influenz. Also, in cse of epidemic of influenz, line-up of drugs with different chrcteristics will e eneficil for oth ptients nd clinicins. The ese of dministrtion of CS-8958 is especilly noteworthy ecuse only single inhltion is required during the course of illness. In ddition to currently ville drugs, including znmivir nd oseltmivir, it is expected tht CS-8958 will e nother option for tretment of influenz. Disclosure sttement The uthor declres no competing interests. References 1. Wright PF, Wester RG. Orthomyxoviruses. In Knipe DM, Howley PM, Griffin DE, et l. (Editors). Fields virology. 4th Ed. Phildelphi: Lippincott Willims & Wilkins 21; pp Fiore AE, Shy DK, Her P, et l. Prevention nd control of influenz. Recommendtions of the Advisory Committee on Immuniztion Prctices (ACIP), 27. MMWR Recomm Rep 27; 56: Novel Swine-Origin Influenz A (H1N1) Virus Investigtion Tem. Emergence of novel swine-origin influenz A (H1N1) virus in humns. N Engl J Med 29; 36: Itoh Y, Shiny K, Kiso M, et l. In vitro nd in vivo chrcteriztion of new swine-origin H1N1 influenz viruses. Nture 29; 46: Munster VJ, de Wit E, vn den Brnd JM, et l. Pthogenesis nd trnsmission of swine-origin 29 A (H1N1) influenz virus in ferrets. Science 29; 325: Mines TR, Jyrmn A, Belser JA, et l. Trnsmission nd pthogenesis of swine-origin 29 A (H1N1) influenz viruses in ferrets nd mice. Science 29; 325: Jin S, Kmimoto L, Brmley AM, et l. Hospitlized ptients with 29 H1N1 influenz in the United Sttes, April June 29. N Engl J Med 29; 361: Interntionl Medicl Press

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