( I ) United States Patent ( 10 ) Patent No .: US 10,864,210 B2 Zou Et Al

US010864210B2

( I ) United States Patent ( 10 ) Patent No .: US 10,864,210 B2 Zou et al . (45 ) Date of Patent : Dec. 15 , 2020

( 54 ) COMPOSITION AND COMBINED ( 52 ) U.S. CI . MEDICATION METHOD FOR TREATING ??? A61K 31/4965 ( 2013.01 ) ; A61K 31/185 ENTEROVIRUS INFECTION ( 2013.01 ) ; A61K 31/422 ( 2013.01 ) ; A61K ( 7 ) Applicant: Institut Pasteur of Shanghai , Chinese 31/496 ( 2013.01 ) ; A61K 45/06 ( 2013.01 ) ; Academy of Sciences, Shanghai ( CN ) A61P 31/14 ( 2018.01 ) ; C12N 7700 ( 2013.01 ) ; GO1N 33/56983 ( 2013.01 ) ; ( 72 ) Inventors : Gang Zou , Shanghai ( CN ) ; Ralf (Continued ) Altmeyer, Shanghai ( CN ) ; Yizhuo ( 58 ) Field of Classification Search Wang , Shanghai ( CN ) None ( 73 ) Assignee : INSTITUT PASTEUR OF See application file for complete search history . SHANGHAI , CHINESE ACADEMY OF SCIENCES , Shanghai ( CN ) ( 56 ) References Cited ( * ) Notice : Subject to any disclaimer , the term of this FOREIGN PATENT DOCUMENTS patent is extended or adjusted under 35 CN 103145608 A 6/2013 U.S.C. 154 ( b ) by 0 days. CN 105664166 A 6/2016 ( 21 ) Appl . No .: 16 / 073,487 105688216 A 6/2016 ( 22 ) PCT Filed : Jan. 11 , 2017 OTHER PUBLICATIONS ( 8 ) PCTNO PCT /CN2017 / 070858 Sun et al . ( Antimicrobial Agents and Chemotherapy, Dec. 2015 , p . $ 371 ( c ) ( 1 ), 7782-7784 ) . * ( 2 ) Date : Jul . 27 , 2018 ( Continued ) ( PCT Pub . No.WO2017 / 128950 Primary Examiner Agnieszka Boesen ( 74 ) Attorney, Agent, or Firm - Panitch Schwarze PCT Pub . Date : Aug. 3 , 2017 Belisario & Nadel LLP ( 65 ) Prior Publication Data ( 57 ) ABSTRACT US 2019/0030027 A1 Jan. 31 , 2019 The present invention provides a composition and a com bined medication method for treating an enterovirus infec ( 30 ) Foreign Application Priority Data tion . In particular , the present invention provides a compo sition for inhibiting enteroviruses , wherein the composition Jan. 29 , 2016 ( CN ) 2016 1 0067164 at least contains an inhibitor of a 3D virus protein and an Jan. 29 , 2016 ( CN ) 2016 1 0067175 inhibitor of a capsid protein , or a combination of an inhibitor ( 51 ) Int . Ci . of a 3C protein and an inhibitor of a 3A protein . A61K 31/4965 ( 2006.01 ) A61K 31/185 ( 2006.01 ) 8 Claims, 5 Drawing Sheets ( Continued ) Specification includes a Sequence Listing .

itraconazo tik truconazole rupjmicivi: * Concile in 159 #jon ! -4 ) , e riviste y curvoligty . oslite inhibitioaraie% 16 ut ***##Vani) bibitio! UNSO 0.281 € .781 7.553 3.125 luDMSO 0.078 VT 0.373 0.625 "WMS Concept Gaqqqqqqqq 1.0 Cceration logo Coocabadon (100 M ) favipiravis favitiravir sonio suranui tcc 150 ***vvival racin 1272 121 Inne 192 service123e-14 19 Oi!Suvishrate) viraltiter onditiilo rateCale:00+5

1.5 2 : 0 2.5 2. 1 1.3 Congonisation ( agem Concentration togel UNSO 15.625 31:29 OMEN 0.377 1.053 3.596 7313 luiQQqqqqqqq ( L ) Coxiccarat GYON ishiditis 1964 -14* * ! * ! vat are W5074 00Drate ;?.3C1HWD%

10: POSTCOwym?

. **fiter CORCCIDERON ( logu EM !

ONSO 0.185 € .739 3.125 12.6 IIIClearom. ) US 10,864,210 B2 Page 2

( 51 ) Int . Ci. Gao et al . , “ Discovery of Itraconazole with Broad - Spectrum In Vitro C12N 7700 ( 2006.01 ) Antienterovirus Activity that Targets Nonstructural Protein 3A ," A61K 45/06 Antimicrobial Agents and Chemotherapy, vol . 59 , No. 5 , p . 2654 ( 2006.01 ) (May 2015 ) ( Abstract Only ) . A61P 31/14 ( 2006.01 ) Office Action dated Jan. 17 , 2018 in CN Application No. 201610067175 . A61K 31/422 ( 2006.01 ) 3 . A61K 31/496 ( 2006.01 ) Office Action dated Feb. 12 , 2018 in CN Application No. 201610067164 . GOIN 33/569 ( 2006.01 ) 5 . ( 52 ) U.S. CI . Office Action dated Jul. 31 , 2018 in CN Application No. 201610067164 . CPC C12N 2770/00021 ( 2013.01 ); GOIN 5 . Office Action dated Sep. 29 , 2018 in CN Application No. 201610067175 . 2333/085 ( 2013.01 ) ; GOIN 2500/04 ( 2013.01 ) 3 . References Cited Rocha - Pereira et al . , “ The Enterovirus Protease Inhibitor Rupintrivir ( 56 ) Exerts Cross -Genotypic Anti -Norovirus Activity and Clears Cells from the Norovirus Replicon ,” Antimicrobial Agents and Chemo OTHER PUBLICATIONS therapy, vol . 58 , No. 8 , pp . 4675-4681 ( Aug. 2014 ) ( Abstract Only ) . Zhang et al . , “ Rupintrivir is a promising candidate for treating Rhoden et al. ( Antiviral Research 2013 , p . 186-191 ) . * severe cases of enterovirus -71 infection: Evaluation of antiviral Albulascu et al . ( Antiviral Research , 2015 , p . 39-46 ) . * efficacy in a murine infection model, ” Antiviral Research , vol . 97 , Furuta et al , “ Favipiravir ( T -705 ), a novel viral RNA polymerase pp . 264-269 ( 2013 ) ( Abstract Only ) . inhibitor , ” Antiviral Res ., vol . 100 , pp . 446-454 ( 2013 ) . Zhang et al . , " Advances in research on anti - enteric virus 71 treat Prichard et al , “ Three - Dimensional Analysis of the Synergistic ment strategy ,” Chin . I Exp . Infect . Dis ( Electronic Edition ) , vol . 8 , Cytotoxicity of Ganciclovir and Zidovudine , ” Antimicrobial Agents No. 5 , pp . 715-718 ( Oct. 2014 ) . and Chemotherapy , vol . 35 , No. 6 , pp . 1060-1065 ( Jun . 1991 ) . Zhao et al . , “ A new antiviral drng - Favipiravir, ” Clinical Medication Int'l Search Report dated Apr. 20 , 2017 in Int'l Application No. Journal, vol . 13 , No. 4 , pp . 16-20 ( Jul. 2015 ) ( First Page English PCT / CN2017 / 070858 . Abstract ). Wang et al, “ In Vitro Assessment of Combinations of Enterovirus Shang et al ., “ An adenosine nucleoside analogue NITD008 inhibits Inhibitors against Enterovirus 71 , ” Antimicrobial Agents and Che EV71 proliferation ,” Antiviral Research , vol . 112 , pp . 47-58 ( 2014 ) . motherapy, vol . 60 , No. 9 , pp . 5357-5367 ( Sep. 2016 ) . English Translation of Written Opinion dated Dec. 19 , 2019 in SG Sadeghipour et al, “ Ribavirin - Resistant Mutants of Human Enterovirus Application No. 11201806433V . 71 Express a High Replication Fidelity Phenotype during Growth in Ren et al . , “ The approved pediatric drug suramin identified as a Cell Culture, ” Journal of Virology, vol . 87 , No. 3 , pp . 1759-1769 clinical candidate for the treatment of EV71 infection - suramin ( Feb. 2013 ) . inhibits EV71 infection in vitro and in vivo .” Emerging Microbes Qiao et al , “ Advances Research in 3D Protein of Enterovirus 71, " and Infections, vol . 3 , pp . 1-9 ( 2014 ) . Chinese Journal of Zoonoses , vol . 31 , No. 8 , pp . 763-765 ( Aug. 2015 ) . * cited by examiner U.S. Patent Dec. 15 , 2020 Sheet 1 of 5 US 10,864,210 B2

itraconazole rupintrivir inhibition rate % 150 - 150 inhibition rate -cell survival rate 6 1251 -125 cell survival rate

SO 70 73 -75 inhibitionrate% inhibitionrate% 50 Q Ex = 002 M raie%)sarvival'(cell ECS = 0.18 MM 50 20 25 survivalrate(%)cell 8 2 1.5 0.5 Concentration ( log : 0 M ) Concentration ( logiouM ) favipiravis suramin 128 151 inhibition rate % 188 +25 125 Urixton me -cell survival rate % H suvivale 89 181 100 ECS - 2934 1 ECE 687 M inhibitionrate% inhibitionrate% 50 50 rate)survival(%cell rate)survival(%cell 25 25

3.8 2.0 2.5 Concentration (logwxM ) Concentration ( logio UM )

GW8075 128 -jpbibition rate % 188 -cell survival rate %

inhibitionrate% 6C8 = 162 M survivalraie(%)cell

Concentration ( logio " M ) Figure 1A U.S. Patent Dec. 15 , 2020 Sheet 2 of 5 US 10,864,210 B2

itraconazole 8 rupintrivir

viraltiter

DMSO 0.391 0.781 1.563 3,325 InDMSO 0.078 0.156 0.313 0.625 Concentration ( um ) Concentration ( UM )

favipiravir suramin 8 DO viraltitar

DMSO 15.625 31.25 62.5 125 DMEM 0.977 1.953 3,906 7,813 Concentration ( M ) 10Concentration (UM ) GW5074 8

viraltiter )(log10TCIDso'm 6-5

DMSO 0.195 0.781 3,325 12.5 IIIConcentration. ( ?M )

Figure 1B U.S. Patent Dec. 15 , 2020 Sheet 3 of 5 US 10,864,210 B2

Viraltiter(logyPFUimli

itraconazole RUKAtivir

inhibitionrate(%) 23 inhibitionrate(%) HHHH EC 0.05 HM

5 Concentration loginpM Concentration logiouM

inhibitionrate(%) inhibitionrate(%) EC : . 19 EC . ( ) . 77 u M

Concentration log: M Concentration logiouM Figure 2 U.S. Patent Dec. 15 , 2020 Sheet 4 of 5 US 10,864,210 B2

A itraconazole -GW5074 B itraconazole - suramin

C suramin- rupintrivir D favipiravir- rupintrivir

@ 20-30 20 mus

E favipiravir - suramin F itraconazole - favipiravir

0 . 0 inn G itraconazole- rupintrivir

Figure 3 U.S. Patent Dec. 15 , 2020 Sheet 5 of 5 US 10,864,210 B2

Virat#ter(logoPFU)

Figure 4 US 10,864,210 B2 1 2 COMPOSITION AND COMBINED The first aspect of the present invention provides a MEDICATION METHOD FOR TREATING composition for inhibiting enterovirus, including a first ENTEROVIRUS INFECTION active ingredient and a second active ingredient, wherein the first active ingredient is an inhibitor of 3D CROSS - REFERENCE TO RELATED 5 protein of enterovirus ( eg , EV71 ) ; APPLICATION the second active ingredient is an inhibitor of capsid This application is a Section 371 of International Appli protein of enterovirus ( eg , EV71 ) ; cation No. PCT /CN2017 / 070858 , filed Jan. 11 , 2017 , which or was published in the Chinese language on Aug. 3 , 2017 , wherein the first active ingredient is an inhibitor of 3C under International Publication No. WO 2017/128950 A1 , 10 protein of enterovirus ( eg , EV71 ) ; which claims priority under 35 U.S.C. $ 119 ( b ) to Chinese the second active ingredient is selected from the group Application No. 2016-10067164.5 , filed Jan. 29, 2016 and consisting of: Chinese Application No. 2016-10067175.3 , filed Jan. 29 , an inhibitor of 3A protein of enterovirus ( eg , EV71 ) ; and 2016 , the disclosures of which are incorporated herein by 15 an inhibitor of 3D protein of enterovirus ( eg , EV71 ) . reference in their entirety . In another preferred embodiment, the composition REFERENCE TO SEQUENCE LISTING includes a first active ingredient and a second active ingre SUBMITTED ELECTRONICALLY dient, wherein the first active ingredient is an inhibitor of 3D This application contains a sequence listing , which is 20 protein of enterovirus ( eg , EV71 ) ; submitted electronically via EFS - Web as an ASCII format- the second active ingredient is an inhibitor of capsid ted sequence listing with a file name “ Sequence Listing ” , protein of enterovirus ( eg , EV71 ) . creation date of Jul . 25 , 2018 , and having a size of about 4.4 In another preferred embodiment, the first active ingre KB The sequence listing submitted via EFS - Web is part of the specification and is herein incorporated by reference in 25 dienta serine specifically residue) at binds position to the 121 amino of 3D acid protein residue of enterovirus( preferably, its entirety . wherein the amino acid residue is numbered based on SEQ ID NO : 1 . TECHNICAL FIELD In another preferred embodiment, the first active ingre dient includes favipiravir, an analog thereof, or a pharma The present invention belongs to the field of biomedicine , 30 ceutically acceptable salt thereof; and in particular, the present invention relates to a compo the second active ingredient comprises suramin , an analog sition and a combined medication method for treating thereof, or a pharmaceutically acceptable salt thereof. enterovirus infection . In another preferred embodiment, the analog of favipira BACKGROUND ART vir includes a substance which acts on the same target of 35 enterovirus as favipiravir. Enterovirus is a virus of the picornavirus family with a In another preferred embodiment, the analog of suramin single positive strand RNA , and more than 100 serotypes includes a substance which acts on the same target of have been found . Most enterovirus infections do not cause enterovirus as suramin . serious symptoms or only cause milder diseases , but often In another preferred embodiment, the enterovirus is have serious consequences in children and immunodefi- 40 selected from the group consisting of enterovirus 71 ( EV71 ) , ciency populations . Enterovirus 71 ( EV74 ) and Coxsacki- coxsackievirus A16 ( CVA16 ), CVB3 , PV1 or EV68 , and evirus A16 ( CVN16 ) in the genus Enterovirus are main rhinovirus. pathogens causing hand - foot -mouth disease in infants and In another preferred embodiment, the molar ratio of the young children in Asia -Pacific region. Symptoms of hand- first active ingredient to the second active ingredient is about foot- mouth disease are usually mild, such as fever, pharyn- 45 10-100 : 1-20 , preferably 10-100 : 1-10 , and more preferably galgia , diarrhea , local rash and the like, but some patients 10-100 : 1-5 . develop central nervous system ( CNS ) diseases such as In another preferred embodiment, the first active ingre aseptic meningitis, lethal encephalitis and even death . EV71 dient has an inhibitory activity towards 3D protein of is the main pathogen causing severe hand - foot -mouth dis- enterovirus ( e.g. , EV71 ) . ease . So far, there is no effective drug treatment for entero- 50 In another preferred embodiment, the second active ingre virus infection . The existing treatment is limited to support- dient has an inhibitory activity towards capsid protein of ive care , intravenous injection of immunoglobulin or enterovirus ( e.g. , EV71 ) . ribavirin . Therefore, it is extremely urgent to find antiviral In another preferred embodiment, the composition further medicaments . Enteroviruses use the virus's own RNA- includes a pharmaceutically acceptable carrier or excipient. dependent RNA polymerase to synthesize the genome, so 55 In another preferred embodiment, the dosage form of the progeny RNA is susceptible to mutation during RNA repli- pharmaceutical composition comprises tablet, granule , cap cation . sule , pill , injection, or oral solution . Therefore , in order to effectively prevent or treat entero- In another preferred embodiment, the composition is a virus infection , there is an urgent need in the art to develop unit dosage form , and the content of the first active ingre novel techniques for preventing and /or treating enterovirus 60 dient and the second active ingredient in each unit dosage infection . form is about 0.1-1 ( or 0.25-1 , or 0.5-1 ) of a daily dose , wherein the daily dose is 20-100 mg . SUMMARY OF THE PRESENT INVENTION In another preferred embodiment, the daily dose is 25-70 mg , such as 25 mg , 40 mg , 50 mg . The object of the present invention is to provide a 65 In another preferred embodiment, the composition component and a combined medication method for treating includes a first active ingredient and a second active ingre enterovirus infection . dient, US 10,864,210 B2 3 4 where, the first active ingredient is an inhibitor of 3C The fourth aspect of the present invention provides a protein of enterovirus ( eg , EV71) ; method for non - therapeutically inhibiting enterovirus the second active ingredient is selected from the group growth or killing enterovirus in vitro comprising the step of: consisting of: applying the composition for inhibiting enterovirus accord an inhibitor of 3A protein of enterovirus ( eg , EV71 ); and 5 ing to the first aspect of the present invention in a place that an inhibitor of 3D protein of enterovirus ( eg , EV71 ) . needs to be treated . In another preferred embodiment, the first active ingre The fifth aspect of the present invention provides use of dient includes rupintrivir, an analog thereof, or a pharma favipiravir , an analogue thereof, or a pharmaceutically ceutically acceptable salt thereof; acceptable salt thereof for the manufacture of a reagent used the second active ingredient is selected from the group 10 for: consisting of itraconazole , an analog thereof, or a pharma ( I ) inhibiting the synthesis of enterovirus 3D protein , ceutically acceptable salt thereof; and favipiravir, an analog and / or thereof, or a pharmaceutically acceptable salt thereof. ( II ) specifically binding to amino acid residue at position In another preferred embodiment, the analog of rupintrivir 15 residue121 of isenterovirus numbered 3Dbased protein on SEQ , wherein ID NO :the 1 . amino acid includes a substance which acts on the same target of In another preferred embodiment, the amino acid residue enterovirus as rupintrivir ( eg , AG7404 ) . at position 21 of enterovirus 3D protein is serine . In another preferred embodiment, the analog of itracona In another preferred embodiment, the agent is further used zole includes a substance which acts on the same target of for: ( III ) inhibiting the replication of enterovirus. enterovirus as itraconazole. 20 In another preferred embodiment, the enterovirus is In another preferred embodiment, the enterovirus is enterovirus 71 . selected from the group consisting of enterovirus 71 ( EV71 ) , The sixth aspect of the present invention provides a coxsackievirus A16 ( CVA16 ), CVB3 , PV1 or EV68 , and complex as shown in formula I , rhinovirus . In another preferred embodiment, the molar ratio of the 25 A - B first active ingredient to the second active ingredient is about wherein A is favipiravir or an analog thereof; and B is a 1-20 : 10-100 , preferably 1-10 : 10-100 , and more preferably 3D protein of enterovirus. 1-5 : 10-100 . In another preferred embodiment, in the complex , the In another preferred embodiment, the first active ingre- binding site of A and B includes the amino acid residue at dient has an inhibitory activity towards 3C protein of 30 position 121 of enterovirus 3D protein , wherein the amino enterovirus ( e.g. , EV71 ) . acid residue is numbered based on SEQ ID NO : 1 . In another preferred embodiment, the second active ingre- The seventh aspect of the present invention provides a dient has an inhibitory activity towards 3A protein and / or 3D drug - resistant enterovirus strain , wherein 3D protein of the of enterovirus ( e.g. , EV71 ) . In the present invention , itra- strain is mutated , and the mutation causes the enterovirus to conazole has a 3A protein inhibitory activity , and favipiravir 35 develop drug resistance. has a 3D protein inhibitory activity . In another preferred embodiment, the mutation occurs at In another preferred embodiment, the composition further the amino acid residue at position 121 of the 3D protein . includes a pharmaceutically acceptable carrier or excipient. In another preferred embodiment, the amino acid residue In another preferred embodiment, the dosage form of the at position 121 of the 3D protein is mutated from a serine pharmaceutical composition comprises tablet , granule, cap- 40 residue to an aspartic acid residue . sule , pill , injection , or oral solution . In another preferred embodiment, the strain is an entero In another preferred embodiment, the composition is a virus 71 strain . unit dosage form , and the content of the first active ingre- The eighth aspect of the present invention provides use of dient and the second active ingredient in each unit dosage drug - resistant enterovirus strain of the seventh aspect of the form is about 0.1-1 ( or 0.25-1 , or 0.5-1 ) of a daily dose , 45 present invention for screening an agent or a reagent for wherein the daily dose is 20-100 mg . inhibiting or killing enterovirus. In another preferred embodiment, the daily dose is 25-70 The ninth aspect of the present invention provides an mg , such as 25 mg , 40 mg , 50 mg . inhibitor of the drug -resistant enterovirus strain of the pres The second aspect of the present invention provides use of ent invention , and the inhibitor can inhibit or kill the the composition for inhibiting enterovirus according to the 50 drug - resistant enterovirus strain of the seventh aspect of the first aspect of the present invention for the manufacture of a present invention . medicament for preventing and /or treating an enterovirus The tenth aspect of the present invention provides a infection . method for screening a madicament comprising: contacting The third aspect of the present invention provides a a madicament to be screened with enterovirus or 3D protein method of preventing and / or treating an enterovirus infec- 55 of enterovirus, and detecting whether a complex of the tion , comprising the step of: formula II is formed , administering the composition for inhibiting enterovirus according to the first aspect of the present invention to a A - B ( II ) subject in need thereof, thereby inhibiting enterovirus in the wherein A ' is the madicament to be screened ; and B is the body of the subject. 60 3D protein of enterovirus . In another preferred embodiment, the subject comprises a In another preferred embodiment, in the complex , the human and a non - human mammal ( e.g. , a rodent ). binding site of A ' and B includes amino acid residue at In another preferred embodiment, the administration is position 121 of enterovirus 3D protein , wherein the amino carried out in an amount of 10 to 100 mg /kg body weight, acid residue at position 121 is based on SEQ ID NO : 1 . preferably 15 to 70 mg/ kg body weight, more preferably 10 65 In another preferred embodiment, the madicament to be to 50 mg/ kg body weight, based on the weight of the first screened includes , for example, favipiravir, an analog active ingredient. thereof, or a pharmaceutically acceptable salt thereof. US 10,864,210 B2 5 6 The eleventh aspect of the present invention provides an Before describing the present invention , it is to be under enterovirus inhibitor which targets 3D protein of enterovirus stood that this invention is not limited to the particular and inhibits the growth or reproduction of the enterovirus. methods and experimental conditions described as such In another preferred embodiment, the inhibitor targets methods and conditions may vary . It is also understood that amino acid residue at position 121 of enterovirus 3D protein , 5 the terms used herein is for the purpose of describing the wherein the amino acid residue at position 121 is based on particular embodiments, and is not intended to be used for SEQ ID NO : 1 . limit , and the scope of the invention is limited only by the In another preferred embodiment, the inhibitor is selected appended claims . from the group consisting of favipiravir, an analog thereof, 10 Unless otherwise defined , all technical and scientific or a pharmaceutically acceptable salt thereof. terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this It should be understood that within the scope of the invention belongs . As used herein , the term “ about ” , when present invention , the above each technical feature of the being used in reference to a particular recited value , means present invention and each technical feature specifically 15 that the value can vary by no more than 1 % from the recited described in the following ( such as the examples) can be value . For example , as used herein , the expression “ about combined with each other to constitute a new or preferred 1.00 ” includes all values between 99 and 101 ( for example, technical solution . Due to space limitations , it will not be 99.1 , 99.2 , 99.3 , 99.4 , etc. ) . described one by one here . Although any methods and materials similar or equivalent 20 to those described in the present invention can be used in the DESCRIPTION OF THE DRAWINGS examples or tests of the present invention , the preferred methods and materials are provided herein . FIG . 1 shows that itraconazole , rupintrivir, favipiravir, Favipiravir and Analog Thereof suramin , and GW5074 inhibit EV71 infection, wherein FIG . 1A shows the inhibitory effect of each compound on EV71 25 Favipiravir is a RdRP inhibitor with broad spectrum and toxic effects thereof on cells ; and FIG . 1B shows the antiviral activity . After intensive research , the inventors effect of different concentrations of each compound on viral unexpectedly found that favipiravir can inhibit EV71 repli titer. cation in vitro , and the mechanism studies have found that 30 its site of action is located in the 3D protein of EV71 . FIG . 2 shows that favipiravir inhibits virus replication by In a preferred embodiment of the present invention , the acting on 3D protein of enterovirus 71 . analog of favipiravir according to the present invention FIG . 3 shows the interaction effect of different medica nprises a substance which acts on the same target of ment combinations on the treatment of EV71 infection . enterovirus as favipiravir. FIG . 4. shows that the combination of itraconazole and 35 In a preferred embodiment of the present invention , the rupintrivir can synergistically inhibit EV71 titer. present invention provides an inhibitor of enterovirus, which targets the enterovirus 3D protein and inhibits the growth or DETAILED DESCRIPTION OF THE reproduction of the enterovirus . INVENTION 40 In a preferred embodiment, the inhibitor targets amino acid residue at position 121 of 3D protein of enterovirus , Through extensive and intensive research , the inventors wherein the amino acid residue is numbered based on SEQ firstly and unexpectedly discovered a composition for inhib ID NO : 1 iting enterovirus, the composition comprises favipiravir as In a preferred embodiment, the inhibitor is selected from the first active ingredient and suramin as the second active 45 the group consisting of favipiravir, an analog thereof, or a ingredient, or the composition comprises rupintrivir as the pharmaceutically acceptable salt thereof. first active ingredient and itraconazole and / or favipiravir as 3D protein is an enterovirus ( EV71 virus) RNA -depen the second active ingredient. The experiment results show dent RNA polymerase and is an important enzyme encoded that the medicament combination has a significant synergis- 50 by the viral genome , which catalyzes the replication and transcription of the viral genome . In a preferred embodiment tic effect, and no enhanced cytotoxicity is observed under of the present invention, the amino acid sequence of the 3D the tested concentration combination . protein is as follows: The present invention discloses a combined medication method for effectively inhibiting enterovirus infection . For 55 the inhibitory activity of enteroviruses, the inventors have ( SEQ ID NO : 1 ) unexpectedly discovered that favipiravir and suramin , rupin- GEIQWVKPNKETGRLNINGPTRTKLEPSVFHDIFEGNKEPAVLHSKDPRL trivir and itraconazole , rupintrivir and favipiravir show significant synergy through extensive tests . Moreover, no EVDFEQALFSKYVGNTLYEPDEYIKEAALHYANQLKOLEINTSQMSMEEA enhanced cytotoxicity is observed under the tested concen- 60 tration combination for above medicament combination . CYGTENLEAIDLHTSAGYPYSALGIKKRDILDPTTRDVSKMKFYMDKYGL The combination of favipiravir and suramin or the combi DLPYSTYVKDELRSIDKIKKGKSRLIEASSLNDSVYLRMTFGHLYEAFHA nation of rupintrivir and itraconazole can prevent the emer gence of resistant viruses. In addition , the inventors have NPGTITGSAVGCNPDTFWSKLPILLPGSLFAFDYSGYDASLSPVWFRALE also found that favipiravir inhibits viral replication by acting 65 on the 3D protein of EV71 , which can be used as a potential LVLREIGYSERAVSLIEGINHTHHVYRNKTYCVLGGMPSGCSGTSIFNSM target for the development of antiviral medicaments. US 10,864,210 B2 7 8 - continued INNIIIRALLIKTFKGIDLDELNMVAYGDDVLASYPFPIDCLELAKTGKE YGLTMTPADKSPCFNEVNWGNATFLIKRGFLPDEQFPFLIHPTMPMRIHE 5 SIRWTKDARNTQDHVRSLCLLAWHNGKQEYEKFVSTIRSVPVGRALAIPN YENLRRNWLELF Suramin and Analog Thereof Suramin is clinically used to treat trypanosomiasis and 10 acts with the capsid protein of EV71 to inhibit viral adsorp tion into cells . In a preferred embodiment of the present invention , the analog of suramin according to the present invention has the structure shown in the following formula IV :

$ O3Na SO3Na NaOzs . SO3Na .

NaO3S HN NH SO3Na

H R R

35 In a preferred embodiment of the present invention , the analog of suramin according to the present invention com prises a substance which acts on the same target of entero virus as suramin ( for example , NF449 , Nf110 and NM16 ) .

NF449 NaOzs

NaO3S NaO3S HN ? HN -SO3Na

HN -SO3Na NaO3S HN NaO3S

NaOzS US 10,864.210 B2 9 10 -continued NF110 NaOzS

HN HN -SO3Na

HN -SO3Na HN

NaOzs NM16 SO3 SO3

HN NH

N H 03S. SO3

45 Rupintrivir and Analog Thereof Itraconazole and Analog Thereof Rupintrivir was originally used to treat rhinovirus infec tion , and studies have shown that it inhibits viral replication Itraconazole is an oral triazole broad - spectrum antifungal primarily by inhibiting the 3C protein of EV71 . agent that inhibits Aspergillus and Candida albicans and is In a preferred embodiment of the present invention , the 50 effective in treating fungal infections in children . It is also a analog of rupintrivir according to the present invention , recently reported broad - spectrum enterovirus inhibitor that AG7404 , has the following structure : inhibits the viral life cycle by acting on the 3A protein of the virus and the oxidative sterol -binding protein of the host . In a preferred embodiment of the present invention , the NH 55 analog of itraconazole according to the present invention comprises a substance which acts on the same target of enterovirus as itraconazole . GW5074 and Analog Thereof 60 GW5074 is a Raf signaling pathway kinase inhibitor that has an inhibitory effect on enterovirus replication but does not act on this cellular pathway . It exerts antiviral activity by acting on the 3A protein of poliovirus . In a preferred embodiment of the present invention , the analog of rupintrivir according to the present invention 65 The structural formulas of the compounds involved in the comprises a substance which acts on the same target of present application are as follows : 1 is itraconazole , 2 is enterovirus as rupintrivir ( eg AG7404 ) . rupintrivir, 3 is favipiravir, 4 is GW5074 , and 5 is suramin . US 10,864.210 B2 11 12

Cy. 1

N - N

N Pornool 2 HN N NH

Br H NH2 OH

OH Br

H 5

OH OH H H N O S = 0 = 0 N

OH HO

OE -OH HOS =

In the combined medication method , suramin and favipi- 55 virus, and a pharmaceutically acceptable carrier . The active ravir as well as itraconazole and rupintrivir show significant ingredient for inhibiting enterovirus comprises a first active synergistic effect, and no significant enhanced cytotoxicity is ingredient and a second active ingredient, wherein the first detected at the measured concentration ; rupintrivir and active ingredient comprises favipiravir, an analog thereof, or suraminfavipiravir show show an a weakadditive synergistic effect; andeffect itraconazole; rupintrivir and 60 a pharmaceutically acceptable salt thereof; and the second suramin , itraconazole and favipiravir, GW5074 and itra active ingredient is selected from the group consisting of conazole show strong antagonistic effect. suramin , an analog thereof, or a pharmaceutically acceptable Composition salt thereof; and rupintrivir, an analog thereof, or a pharma The term “ composition , ” as used herein , includes a phar- ceutically acceptable salt thereof. maceutical composition . 65 The pharmaceutical composition of the present invention The composition of the first aspect of the present inven- may further comprise various pharmaceutical excipients tion comprises an active ingredient which inhibits entero- compatible with the compound or composition contained US 10,864,210 B2 13 14 therein , and is prepared into a dosage form which is advan- buffalo , bull , sheep , goat , geese , chicken and the like . The tageous for administration by a conventional method , such " patient” or “ organism ” to be treated preferably is a mam as , but not limited to , aqueous solution injection , powder mal , especially a human . injection , pill , powder, tablet, patch , suppository, emulsion , As used herein , the term “ preventing” means that various cream , gel , granule, capsule , aerosol, spray , powder spray , 5 means or measures are used for preventing the occurrence or sustained release preparation, controlled release preparation development of a disease, including medical, physical or and the like . The pharmaceutical excipients may be conven tionally used in various preparations, such as , but not limited chemical means , to prevent and reduce occurrence or devel to , isotonic agent, buffer, flavoring agent, excipients, filler, opment of symptoms of various diseases before the disease binder, disintegrator, lubricant, and the like ; or may be 10 is not recognized by clinical standards. selected for use in accordance with the substance , such as : As used herein , the term “ treating” refers to the inhibition, but not limited to emulsifier, solubilizer, bacteriostatic agent, suppression , reduction , amelioration , mitigation , cessation, analgesic, antioxidants and the like . Such excipients can delay or reversal of the progression or aggravation of a effectively improve the stability and solubility of the com disease for the prevention and suppression of the develop pound contained in the composition, or change the release 15 ment or progression of the disease, and the described various rate and absorption rate of the compound to improve the indicators of disease , disorder, or pathology at the time of metabolism of various compounds in the organism , thereby retention and / or administration include alleviating or reduc enhancing the administration effect of the composition. In ing symptoms or complications, or curing or eliminating addition , excipients such as , but not limited to , gelatin , disease , disorder, or condition . albumin , chitosan , polyethers and polyesters ( such as , but 20 The term “ medicament ” as used herein refers to a single not limited to , polyethylene glycol, polyurethane , polycar- compound or a composition formed by a plurality of com bonate and copolymers thereof) may be used for achieving pounds which can be used for preventing or treating a specific administration purposes or modes such as sustained disease ; to a composition or a formulation having a single release administration , controlled release administration , compound as a main active ingredient; and also to a com pulse administration and the like . 25 position or formulation that is composed of a plurality of The main manifestations to facilitate administration are : compounds as active ingredients . The “ medicament ” should but not limited to , improving the therapeutic effect, improv- be understood to mean not only the products examined and ing bioavailability , reducing toxic side effect, improving approved for production by the administrative organization patient compliance and the like . In the case of aqueous injection , the excipient generally 30 establishedforms containing by the a lawssingle of compound a country , asbut the also active various ingredient matter includes isotonic agent and buffer, as well as the necessary formed in the process for obtaining approval and approval of emulsifier ( eg , Tween - 80 , Pluronic , and Poloxamer, etc. ) production. The “ formation ” is understood to refer to obtain solubilizer, bacteriostatic agent and the like . In addi on , it ment by chemical synthesis, biotransformation or purchase . also includes other pharmaceutically acceptable excipient The administration route of pharmaceutical composition such as antioxidant, adjuster, analgesic and the like . 35 provided by the present invention includes , but is not limited The excipient used in the preparation of oral liquid to , administration through oral, nasal , buccal , transdermal, preparation generally includes solvent, as well as the nec- pulmonal, vaginal , subcutaneous or intravenous route to the essary flavoring, bacteriostatic , emulsifying, and coloring organism . agent . The main advantages of the present invention are as The excipient used in the preparation of tablet generally 40 follows . includes filler ( eg , starch , powdered sugar , dextrin , lactose , ( 1 ) The present invention firstly reveals the synergistic compressible starch , microcrystalline cellulose , calcium sul- inhibitory effect of favipiravir and suramin , rupintrivir and fate , calcium hydrogen phosphate , mannitol, etc. ) , binder itraconazole or favipiravir on enterovirus. ( eg ethanol, starch slurry, sodium carboxymethyl cellulose , ( 2 ) The composition of the present invention takes effect hydroxypropyl cellulose , methylcellulose, ethyl cellulose , 45 with a low amount. hydroxypropylmethyl cellulose , gelatin solution , sucrose ( 3 ) The composition of the present invention can greatly solution and aqueous solution or alcohol solution of poly- reduce the clinical dosage , which can reduce the production vinylpyrrolidone, etc. ), disintegrant ( such as dry starch , cost and reduce the burden on the patient. sodium carboxymethyl starch , low - substituted hydroxypro- ( 4 ) The composition of the present invention can prevent pyl cellulose , cross - linked polyvinylpyrrolidone and cros- 50 the development of drug - resistant strains. carmellose sodium ) and lubricant ( such as magnesium stear- ( 5 ) The present invention firstly discloses a novel target ate , micronized silica gel , talc , hydrogenated vegetable oil , for enterovirus . polyethylene glycol 4,000 , polyethylene glycol 6,000 , mag- The present invention will be further described in detail nesium lauryl sulfate , etc. ) . below with reference to specific examples. It should be The excipient used in the preparation of emulsion is 55 understood that these examples are only for illustrating the generally water , oil ( e.g. , fatty acid ) , emulsifier, and neces- present invention and are not intended to limit the scope of sary preservative and flavoring agent. the present invention . The experimental methods in the The excipient used in the preparation of granule is similar following examples, which do not specify the detailed to that for tablet, but the granulation process is different. The conditions , are usually in accordance with conventional prepared granules are mixed with a glidant as needed , and 60 conditions such as the conditions described in Guide to then filled into capsules to obtain capsules . Molecular Cloning ( Sambrook, J. et al . , translated by Huang As used herein , the term “ subject, ” “ organism , ” “ animal, " Peitang et al . , Beijing: Science Press , 2002 ) , or in accor or " patient” includes human , wild animal , and livestock . dance with the conditions recommended by the manufac Wild animal is an animal that is in natural state and has not turer . Unless otherwise indicated, percentages and parts are been artificially domesticated . Livestock is an animal that is 65 by weight. The experimental materials and reagents used in artificially raised to provide a source of food , such as , but not the following examples are available from commercially limited to , dog , cat , mouse , rat, hamster, pig , rabbit, cow , available sources unless otherwise specified . US 10,864,210 B2 15 16 Materials and Method containing 2 % FBS and 1 % P / S is added . The experimental Cells , Viruses and Compounds results are analyzed using MacSynergy II software and a 3D RD ( human rhabdomyomas) and Vero (African green schematic is obtained . monkey kidney ) cells in DMEM medium containing 1 % Determination of Viral Titer penicillin / streptomycin ( P / S ) and 10 % fetal bovine serum 5 The titers of EV71 G082 strain and the recombinant virus ( FBS ) are cultured in a 37 ° C. , 5 % CO2 incubator. EV71 are measured, and 1 ml of DMEM containing 3x10 Vero FY573 strain (GenBank Accession No. HM064456 ) is used cells is added to each well of a 12 - well plate (Corning in the evaluation experiment for the antiviral activity of the Costar ), and cultured for 24 hours. The virus is diluted in compound , the viral titer reduction experiment, and com- 10 - fold , that is , 27 ul of the virus solution is mixed with 243 bined medication experiment. EV71 G082 is used in the 10 ul DMEM containing 2 % FBS and 1 % P / S . The medium in viral titer reduction experiment, mutant virus screening and the 12 - well plate is aspirated , and 200 ul of virus solution is evaluation experiment. Compounds itraconazole , rupintrivir, added to each well , then placed in a 37 ° C. , 5 % CO2 favipiravir and GW5074 are purchased from Sigma- Aldrich , incubator for 1 h of infection, and gently shaken every 15 Santa Cruz and Chembest, respectively, and dissolved in minutes . Then , the virus solution is aspirated , and 1 ml DMSO for experiments. Suramin is purchased from Bayer 15 DMEM containing 0.8 % methylcellulose ( Aquacide II , Cal and dissolved in the medium . biochem ) and 2 % FBS is added, cultured in a 37 ° C. , 5 % Evaluation Experiment for Compounds CO2 incubator for 6 days, and placed in 3.7 % formalin . After Itraconazole purchased from Sigma - Aldrich is dissolved fixed for 1 h , it is stained with 1 % crystal violet . in DMSO to a final concentration of 10 mM ; suramin The titer of EV71 FY573 strain is determined by half of purchased from Bayer is dissolved in a medium containing 20 the tissue culture infectious dose ( TCID50 ) . 20,000 RD cells 2 % FBS to a final concentration of 50 mM ; favipiravir are added to each well of a 96 - well transparent plate . After purchased from Chembest is dissolved in DMSO to a final 24 hours of culture, 100 ul of 10 - fold diluted virus ( from concentration of 400 mM ; rupintrivir purchased from Santa 10-1 to 10-8 ) is added to 10 wells for each dilution . After 1 Cruz is dissolved in DMSO to a final concentration of 2 mM ; h of infection , the virus is aspirated and DMEM containing and GW5074 purchased from Sigma is dissolved in DMSO 25 2 % FBS is added . After 7 days of culture in a 37 ° C. , 5 % to a final concentration of 10 mM . To evaluate the inhibition CO2 incubator, the mixture is placed in 3.7 % formalin for 1 of EV71 - induced CPE activity by five compounds, the h of fixation and then stained with 1 % crystal violet . The inventors perform dose dependent experiments. 50 ul of viral titer is measured by Reed - Muench method and DMEM containing 10000 RD cells added to each well of expressed as TCIDs /ml . a 96 - well white plate ( Corning Costar ), and after 24 hours of 30 Viral Titer Reduction Experiment culture in a 37 ° C. , 5 % CO2 incubator, a gradient dilution of RD cells are seeded in a 12 - well plate with 3x109 cells / the test compound is added to each well ( for the compound well , cultured overnight at 37 ° C. for 24 hours . Then EV71 dissolved in MSO the final concentration of DMSO is virus solution with MOI = 0.1 and 2 - fold dilution of itracona 0.25 % ) , and 5 ul of 0.25 % DMSO or medium is added to the zole , rupintrivir, favipiravir, suramin and GW5074 are added control group . Then , 45 ul of dilution containing 150 PFU 35 and cultured at 37 ° C. for 48 h . The supernatant is collected , virus is added , and the final volume of each well is 100 After frozen in a -80 ° C. refrigerator, and then determined for 96 hours of culture , the plate is taken out and equilibrated at viral titer . To determine the resistance of the passaged virus room temperature for 30 minutes. Then , 50 ul of CellTiter- to favipiravir, the inventors inoculate Vero cells in a 12 - well Glo ( Promega ) reagent is added to each well , and allowed to plate , 3x10 cells /well and culture overnight at 37 ° C. After stand at room temperature for 10 to 30 minutes, and detected 40 24 hours, EV71 virus solution with MOI = 0.1 is added and using a Veritas Microplate Luminometer ( Turner BioSys- 300 uM and 600 uM favipiravir are separately added . After tem ) . In order to determine the effect of the compound on the cultured at 37 ° C. for 48 hours, the supernatant is collected cells , the inventors perform a cytotoxicity experiment in the and frozen in a -80 ° C. refrigerator, and then the viral titer same manner as the dose - dependent experiment except that is measured . no virus solution is added and an equal volume of DMEM 45 Virus Screening Experiment containing 2 % FBS and 1 % P / S is added . The present inventors conduct a passage experiment to Combined Medication Experiment screen a favipiravir resistant strain . 3x105 Vero cells are In order to evaluate the inhibitory effect of the combina- seeded in a 12 - well plate , cultured overnight at 37 ° C. After tion medication method on EV71 infection , the inventors 24 hours, EV71 G082 strain virus solution with MOI = 0.1 carry out the experiment by checkerboard method (1-2 ) . 50 ul 50 and favipiravir are added . When a significant cytopathic of DMEM containing 10000 RD cells is added to each well effect is observed , the supernatant is collected . The pre of a 96 - well white plate ( Corning Costar) and incubated in cultured Vero cells are infected with the collected virus and a 37 ° C. , 5 % CO2 incubator for 24 h , and then 5 ul of two favipiravir is added . During the passage , the concentration 2 - fold diluted test compounds is added to the central 60 of favipiravir is gradually increased , and one to three rounds wells of a 96 - well plate and 5 ul of 0.25 % DMSO or medium 55 of screening are carried out for each concentration with one is added to the control group . Then , 40 ul dilution containing control group for each round . After 16 continuous passages , 150 PFU virus is added , and the final volume of each well the titer of the virus in the supernatant, the resistance to the is 100 ul . After 96 hours of culture , the plate is taken out and compound, and sequencing are determined . equilibrated at room temperature for 30 minutes . Then , 50 ul Mutant Virus Resistance Experiment of CellTiter -Glo ( Promega ) reagent is added to each well , 60 To determine the resistance of the selected mutant virus and allowed to stand at room temperature for 10 to 30 and recombinant virus to the compound , the inventors minutes , and detected using a Veritas Microplate Luminom- conduct experiments based on cytopathic effects and viral eter ( Turner BioSystem ). In order to determine the effect of titer reduction experiments. In the experiment based on the the simultaneous addition of two compounds on the cells , cytopathic effect, 5000 Vero cells are added to each well of the inventors perform cytotoxicity experiment in the same 65 a 96 - well white plate , and after incubated in a 37 ° C. , 5 % manner as combined medication experiment except that no CO2 incubator for 24 hours , a gradient dilution of the test virus solution is added and an equal volume of DMEM compound is added to each well ( for the compound dis US 10,864,210 B2 17 18 solved in DMSO , the final concentration of DMSO is the cell control group , and o , represents the standard devia 0.25 % ) , and 5 ul of 0.25 % DMSO or medium is added to the tion of the signal of the virus control group ; Z = 1 - ( ( 30c + control group . Then , 45 ul dilution containing 250 HU 30 , ) / uc - 4,1 ) , when Z factor is between 0.5 and 1 , it indi mutant virus or recombinant virus is added and the final cates that the experimental method can effectively volume of each well is 100 ul After 96 hours of culture , the 5 distinguish the differences between control groups . The plate is taken out and equilibrated at room temperature for antiviral activity of the compound CPE inhibition rate = 30 minutes . Then, 50 ul of CellTiter -Glo reagent is added to (tcpd - H )/ (uc - u , ) * 100 % , wherein Hcpd represents the aver each well , allowed to stand at room temperature for 10 to 30 age signal intensity of the test compound; and the effect of minutes, and detected using a Veritas Microplate Luminom the compound on the cell, cell survival rate = tepd /Hex100 % . eter. EV71 G082 strain is served as a control. 10 The half maximum effect concentration ( EC50 ) is a concen In the viral titer reduction experiment, Vero cells are tration that causes a 50 % maximum effect. The half -cyto inoculated in a 12 - well plate at 3x103 cells /well , and cul toxic concentration ( CC50 ) refers to a concentration of the tured overnight at 37 ° C. After 24 hours, EV71 virus solution medicament that causes 50 % cytotoxicity , which is with MOI = 0.1 and 300 uM of favipiravir are added . The expressed in this experiment as a 50 % reduction in the supernatant is collected after 48 hours of culture , and the 15 fluorescence intensity of the experimental group compared viral titer is examined . with the control group . In the analysis of the interaction Construction of Mutant Virus effects of the two compounds using Macsnergy II , at 95 % A plasmid containing the mutated DNA fragment is confidence , a volume greater than zero represents that the constructed using Fast Site - directed Mutagenesis kit (Trans- interaction of the two compounds is a synergistic effect, and Gen Biotech ) according to the manufacturer's instructions 20 a negative value represents an antagonistic effect. Value and verified by sequencing. The correct cDNAs are linear- between -25 and +25 indicates that the effect between the ized and transcribed into RNA according to in vitro tran- two compounds is not significant, value between 25 and 50 scription kit (MEGAscript T7 Kit , Ambion ), which then represents significant but weak synergy , value between 50 transferred to Vero cells by electroporation . After the obvi- and 100 means moderate synergy, and value greater than 100 ous CPE is observed , the supernatant is collected and the 25 indicates strong synergy . plaque assay is used to determine the viral titer. RT -PCR Example 1 Single Component Activity Assay The viral RNA is extracted with reference to the instruc tion manual of QIAamp viral RNA minikit ( Qiagen ) and Itraconazole , rupintrivir, favipiravir, suramin , and stored at -80 degrees, and the PCR reaction is carried out 30 GW5074 are effective in inhibiting EV71 infection of cells using a SuperScript III One - Step RT - PCR System with in a dose -dependent manner . Platinum Tap DNA Polymerase ( Invotroge ). FIG . 1 shows that itraconazole, rupintrivir , favipiravir, Immunostaining Experiment suramin , and GW5074 inhibit EV71 infection . ( A ) 2 - fold The present inventors perform immunostaining for quali- dilution of itraconazole , rupintrivir, favipiravir, suramin , and tative detection of viruses in screening experiment of resis- 35 GW5074 were separately added to RD cells , and virus or tant strains. 3x10 % Vero cells are pre - inoculated in 24 - well medium was added and cultured for 96 hours . The cell plates , and cultured in a 37 ° C. , 5 % CO2 incubator for 24 h , viability was measured by CellTiter - Glo kit. The inhibitory then virus stock and 10 - fold diluted virus are added to each effects of four compounds on EV71 and the toxic effects on well and placed in a 37 ° C. , 5 % CO2 incubator for 1 h of cells were examined . ( B ) Virus and 2 - fold dilution of infection , and gently shaked every 15 minutes . The virus 40 itraconazole, rupintrivir , favipiravir, suramin , and GW5074 solution is then aspirated , and 1 ml of DMEM containing were separately added into RD cells . After 48 hours of 0.8 % methylcellulose ( Aquacide II , Calbiochem ) and 2 % culture , the supernatant was collected and the viral titer was FBS is added , and cultured in an incubator at 37 ° C. , 5 % determined by TCID50 method . The results were processed CO2 for 6 days. The cells are fixed with 4 % formaldehyde using Graphpad Prism5 . The data in the figure were obtained solution , and the fixed cells are washed twice with PBS 45 from two independent parallel experiments, and the error containing 0.05 % Tween - 20 ( PBS - T ) , incubated with pri- bars represent the standard deviation of the two parallel mary antibody against enterovirus 71 ( MAB979 , Merck experiments . Milipore ) for 1 h at room temperature , then washed for three times with PBS - T and incubated with horseradish peroxi Example 2 Favipiravir Acts on 3D Protein of dase -conjugated secondary antibody ( goat anti -mouse , 50 Enterovirus 71 Bethyl, Montgomery, Tex .) for 1 h at room temperature . After the plate is washed for 3 times with PBS , TrueBlue The present inventors obtained two strains of resistant peroxidase substrate ( KPL® , 50-78-02 ) is added for devel- viruses by screening viruses resistant to favipiravir. The viral opment. When clear blue spots are formed in the control titer reduction experiment showed that both strains were group , the reaction is stopped with distilled water . The plate 55 resistant to favipiravir, and the inventors found that the same is dried , and the results are recorded . Cells that produce blue mutation occurred on the 3D protein by sequence analysis . are positive , and cells that do not produce blue do not The drug -resistant virus phenotype experiment demon contain viruses . strated that EV71 having amino acid residue mutation at Data Analysis position 121 in the 3D protein was resistant to favipiravir. The raw data are input into an Excel sheet to calculate the 60 Using the reverse genetics system , the inventors constructed signal - to - background ratio ( S / B ) , the signal -to -noise ratio a virus carrying such mutation and had its resistance to ( S / N ) , Z factor, and the inhibition rate of the test compound favipiravir verified . The results showed that the mutation of against the virus . The calculation formulas are as follows: serine to aspartic acid at position 121 of 3D protein of S / B = uduy , wherein ue represents the average value of the enterovirus 71 conferred drug - resistance to EV71, and the signal of the cell control group , and u , represents the average 65 present inventors inferred that favipiravir acted on 3D value of the signal of the virus control group ; S /N = (ue - ) protein of EV71 . Furthermore , the present inventors tested ( 0. - 0 , ), o represents the standard deviation of the signal of the inhibitory effect of itraconazole, rupintrivir, suramin , and US 10,864,210 B2 19 20 GW5074 on the mutant virus, and compared with the wild a multiplicity of infection ( MOI ) of 0.1 and different con type virus ( G082 ) , no cross -resistance was found . The centrations of itraconazole , rupintrivir or DMSO were added results were shown in FIG . 2 . to Vero cells , and the supernatant was collected after 48 FIG . 2 shows that favipiravir inhibits viral replication by hours of culture , and the viral titer was detected by plaque acting on 3D protein of enterovirus 71. ( A ) Enterovirus 71 5 formation assay . The results were processed using Graphpad was incubated with favipiravir and the concentration of Prism5. The data in the figure were obtained from two favipiravir was continually increased . The supernatant from independent parallel experiments, and the error bars repre the 16th passage culture was collected and sequenced . EV71 sent the standard deviation of the two parallel experiments . containing 3D protein mutation was constructed by reverse genetics system . The viral titer was determined by plaque 10 Example 5 Analysis of the Interaction of formation assay , the virus resistance to favipiravir was Rupintrivir and Itraconazole in Combined detected by viral titer reduction experiment. The experimen Medication by Chou - Talalay Method tal method was the same as the compound verification Using the Compusyn software , the interaction between experimentsuramin and. (GW5074 B ) The effectson a virusof itraconazole containing , 3Drupintrivir protein, 15 itraconazole and rupintrivir was analyzed according to the mutation were determined using an experiment based on Chou - Talalay method , and it was found that the two com cytopathic effect . The results were processed using Graph pounds produced synergistic effect. And the synergistic pad Prism5 . The data in the figure were obtained from two effect was most obvious when the molar concentration ratio independent parallel experiments , and the error bars repre was itraconazole / rupintrivir = 10 / 1. The results are shown in sent the standard deviation of the two parallel experiments. 20 Table 2 . Example 3 Combined Activity Assay TABLE 2 molar % The combination of itraconazole , rupintrivir, favipiravir, concentration Inhi suramin , and GW5074 produces synergistic , additive or 25 ratio of bition DRI antagonistic effects in the treatment of EV71 infection. The results are shown in FIG . 3 . itraconazole / rate itracon FIG . 3 shows that the interaction effect of different rupintrivir ( EDn) CI azole rupintrivir medicament combinations in the treatment of EV71 infec 1 : 0.1 50 0.01 ( very strong synergy ) 285.689 138.384 75 0.02 ( very strong synergy ) 212.177 89.313 tion . 3D plot was made using MacSynergy II software and 30 90 0.02 ( very strong synergy ) 157.582 57.642 the data in the plot were obtained from at least three 95 0.03 (very strong synergy ) 128.717 42.795 independent parallel experiments. ( A ) combination of itra 1 : 0.05 50 0.21 (strong synergy ) 9.514 9.217 conazole and GW5074 , ( B ) combination of itraconazole and 75 0.21 (strong synergy ) 10.341 8.706 suramin , ( C ) combination of suramin and rupintrivir, ( D ) 90 0.21 (strong synergy ) 11.241 8.224 combination of favipiravir and rupintrivir , ( E ) combination 35 95 0.21 (strong synergy ) 11.897 7.911 of favipiravir and suramin , ( F ) combination of favipiravir and itraconazole , and ( 6 ) combination of itraconazole and Table 2. EV71 was added to Vero cells and co - cultured rupintrivir. The horizontal plane represents the additive with different concentrations of itraconazole and rupintrivir effect of the two medicaments, the points above the hori- or DMSO . After 48 hours, the supernatant was collected and zontal plane represent the synergistic effect of the two 40 the viral titer was determined by plaque formation assay . medicaments , and the points below the horizontal plane The inhibitory effect of different concentration combinations represent the antagonistic effect. on EV71 infection was obtained by comparison with the control group . The interaction between the two medicaments TABLE 1 at the n % inhibition effect was analyzed using Compusyn 45 software , and CI ( combination index ) and DRI ( dose reduc MacSynergy II analysis tion index ) were used as evaluation parameters. CI < 0.1 Synergy /antagonism indicates a very strong synergistic effect, and 0.1 < CI < 0.3 Medicament combination (UM2 % ) Effect indicates a strong synergistic effect. DRI indicates the itraconazole rupintrivir itraconazole favipiravir 434.4434.517-88.11 / -4.4 strong antagonismsynergy degree ( fold ) of the decrease in the concentration required itraconazole suramin 0.14 / –246.23 strong antagonism 50 for the two compounds in combined medication to achieve rupintrivir favipiravir 64.9 / 0 weak synergy the same inhibitory effect as compared with one compound rupintrivir suramin 4.96 / 0 additive effect used in antiviral therapy. The data in the figure were favipiravir suramin 337.59 / 0 strong synergy obtained from two independent parallel experiments . itraconazole GW5074 53.91 / -167.68 strong antagonism 55 Example 6 Combination of Itraconazole and Table 1 shows the synergistic , additive or antagonistic Rupintrivir can Prevent the Emergence of results calculated according to MacSynergy II software . Drug -Resistant Viruses and Eliminate all Viruses Example 4 Itraconazole and Rupintrivir EV71 , itraconazole , and 0.5 uM or 1 uM of rupintrivir Synergistically Inhibit Viral Titer 60 were simultaneously added to Vero cells , the virus was subcultured , and the supernatant was collected . After 20 Itraconazole and rupintrivir are not only able to inhibit the passages , the inventors examined the viruses in the super cytopathic effects produced during viral infection , but also natants of the 10th, 16th and 20th passage by immunostaining synergistically inhibit viral production . The results are experiments, and no virus was detected . The viral titer shown in FIG . 4 . 65 reduction experiment proved that itraconazole could not FIG . 4 shows that the combination of itraconazole and remove all the viruses in the cell culture . The experimental rupintrivir can synergistically inhibit EV71 titer . EV71 with results showed that the combination of itraconazole and US 10,864,210 B2 21 22 rupintrivir prevented the emergence of drug -resistant viruses REFERENCES and completely inhibit the replication of EV71 in vitro . All references mentioned in this application are incorpo rated by reference in this application , as if each were Furuta Y , Gowen B B , Takahashi K , Shiraki K , Smee D F , incorporated by reference individually . In addition , it should 5 Barnard D L. Favipiravir ( T -705 ), a novel viral RNA be understood that after reading the above teachings of the polymerase inhibitor . Antiviral Res 2013 ; 100 : 446-454 . present invention , those skilled in the art can make various Prichard M N , Prichard L E , Baguley W A , Nassiri M R , changes or modifications to the present invention , and these Shipman C , Jr. Three - dimensional analysis of the syner equivalent forms also fall within the scope defined by the gistic cytotoxicity of ganciclovir and zidovudine . Antimi appended claims of the present application . crob Agents Chemother 1991 ; 35 : 1060-1065 .

SEQUENCE LISTING

< 160 > NUMBER OF SEQ ID NOS : 1 < 210 > SEQ ID NO 1 < 211 > LENGTH : 462 < 212 > TYPE : PRT < 213 > ORGANISM : Enterovirus < 400 > SEQUENCE : 1 Gly Glu Ile Gin Trp Val Lys Pro Asn Lys Glu Thr Gly Arg Leu Asn 1 5 10 15 Ile Asn Gly Pro Thr Arg Thr Lys Leu Glu Pro Ser Val Phe His Asp 20 25 30 Ile Phe Glu Gly Asn Lys Glu Pro Ala Val Leu His Ser Lys Asp Pro 35 40 45 Arg Leu Glu Val Asp Phe Glu Gin Ala Leu Phe Ser Lys Tyr Val Gly 50 55 60 Asn Thr Leu Tyr Glu Pro Asp Glu Tyr Ile Lys Glu Ala Ala Leu His 65 70 75 80 Tyr Ala Asn Gin Leu Lys Gin Leu Glu Ile Asn Thr Ser Gin Met Ser 85 90 95 Met Glu Glu Ala Cys Tyr Gly Thr Glu Asn Leu Glu Ala Ile Asp Leu 100 105 110 His Thr Ser Ala Gly Tyr Pro Tyr Ser Ala Leu Gly Ile Lys Lys Arg 115 120 125 Asp Ile Leu Asp Pro Thr Thr Arg Asp Val Ser Lys Met Lys Phe Tyr 130 135 140 Met Asp Lys Tyr Gly Leu Asp Leu Pro Tyr Ser Thr Tyr Val Lys Asp 145 150 155 160 Glu Leu Arg Ser Ile Asp Lys Ile Lys Lys Gly Lys Ser Arg Leu Ile 165 170 175 Glu Ala Ser Ser Leu Asn Asp Ser Val Tyr Leu Arg Met Thr Phe Gly 180 185 190 His Leu Tyr Glu Ala Phe His Ala Asn Pro Gly Thr Ile Thr Gly Ser 195 200 205 Ala Val Glycys Asn Pro Asp Thr Phe Trp Ser Lys Leu Pro Ile Leu 210 215 220 Leu Pro Gly Ser Leu Phe Ala Phe Asp Tyr Ser Gly Tyr Asp Ala Ser 225 230 235 240 Leu Ser Pro Val Trp Phe Arg Ala Leu Glu Leu Val Leu Arg Glu Ile 245 250 255 Gly Tyr Ser Glu Arg Ala Val Ser Leu Ile Glu Gly Ile Asn His Thr 260 265 270 His His Val Tyr Arg Asn Lys Thr Tyr Cys Val Leu Gly Gly Met Pro 275 280 285 Ser Gly Cys Ser Gly Thr Ser Ile Phe Asn Ser Met Ile Asn Asn Ile 290 295 300 US 10,864,210 B2 23 24 - continued

Ile Ile Arg Ala Leu Leu Ile Lys Thr Phe Lys Gly Ile Asp Leu Asp 305 310 315 320 Glu Leu Asn Met Val Ala Tyr Gly Asp Asp Val Leu Ala Ser Tyr Pro 325 330 335 Phe Pro Ile Asp Cys Leu Glu Leu Ala Lys Thr Gly Lys Glu Tyr Gly 340 345 350 Leu Thr Met Thr Pro Ala Asp Lys Ser Pro Cys Phe Asn Glu Val Asn 355 360 365 Trp Gly Asn Ala Thr Phe Leu Lys Arg Gly Phe Leu Pro Asp Glu Gln 370 375 380 Phe Pro Phe Leu Ile His Pro Thr Met Pro Met Arg Glu Ile His Glu 385 390 395 400 Ser Ile Arg Trp Thr Lys Asp Ala Arg Asn Thr Gln Asp His Val Arg 405 410 415 Ser Leu Cys Leu Leu Ala Trp His Asn Gly Lys Gin Glu Tyr Glu Lys 420 425 430 Phe Val Ser Thr Ile Arg Ser Val Pro Val Gly Arg Ala Leu Ala Ile 435 440 445 Pro Asn Tyr Glu Asn Leu Arg Arg Asn Trp Leu Glu Leu Phe 450 455 460

The invention claimed is : 3. The composition of claim 1 , wherein the analog of 1. A composition for inhibiting enterovirus, wherein the 30 rupintrivir includes a substance which acts on the same composition includes a first active ingredient and a second target of enterovirus as rupintrivir ( eg , AG7404 ) ; and / or active ingredient, the analog of itraconazole includes a substance which acts on the same target of enterovirus as itraconazole . wherein the first active ingredient is an inhibitor of 3C 4. The composition of claim 1 , wherein the molar ratio of protein of enterovirus ( eg , EV71 ) ; 35 the first active ingredient to the second active ingredient is the second active ingredient is an inhibitor of 3A protein about 1-10 : 10-100 . of enterovirus ( eg , EV71 ) ; 5. The composition of claim 1 , wherein the molar ratio of wherein the inhibitor of 3C protein of enterovirus ( eg , the first active ingredient to the second active ingredient is EV71 ) is rupintrivir, an analog thereof, or a pharma 1-5 : 10-100 . 40 6. The composition of claim 1 , wherein the dosage form ceutically acceptable salt thereof; and of the pharmaceutical composition is a tablet, granule, the inhibitor of 3A protein of enterovirus ( eg , EV71 ) is capsule , pill , injection, or oral solution . itraconazole , an analog thereof, or a pharmaceutically 7. The composition of claim 1 , wherein the composition acceptable salt thereof, and is a unit dosage form , and the content of the first active wherein a molar ratio of the first active ingredient to the ingredient and the second active ingredient in each unit second active ingredient is about 1-20 : 10-100 . 45 dosage form is 0.1-1 of a daily dose , wherein the daily dose 2. The composition of claim 1 , wherein the first active is 20-100 mg . ingredient specifically binds to an amino acid residue at 8. The composition of claim 2 , wherein the amino acid position 121 of 3D protein of enterovirus, wherein the amino residue is a serine residue. acid residue is numbered based on SEQ ID NO : 1 .