US 20150.045318A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0045318 A1 Ralf et al. (43) Pub. Date: Feb. 12, 2015 (54) COMPONENT AND METHOD FOR Publication Classification TREATINGVIRAL DISEASE (51) Int. Cl. A 6LX3 L/7076 (2006.01) (71) Applicant: Institut Pasteur of Shanghai, Chinese A63L/85 (2006.01) Academy of Sciences, Shanghai (CN) A63/675 (2006.01) (52) U.S. Cl. (72) Inventors: Altmeyer Ralf, Shanghai (CN); Peijun CPC ........... A6 IK3I/7076 (2013.01); A61 K3I/675 Ren. Shanghai (CN (2013.01); A61 K3I/I85 (2013.01) en, Shanghai (CN) USPC ................................ 514/47, 514/89; 514/577 (21) Appl. No.: 14/384,075 (57) ABSTRACT A method for treating viral infection includes administering (22) PCT Filed: Mar. 11, 2013 to a subject in need thereof a composition containing P2X receptor antagonists. The methods may achieve preventive or (86). PCT No.: PCT/CN2013/072402 therapeutic effect on hand foot and mouth disease by inhib iting viruses. The P2X receptor antagonists can inhibit infec S371 (c)(1), tion by a positive-sense single-stranded RNA picornavirus. (2) Date: Sep. 9, 2014 The virus may be an enterovirus or a Coxsackie virus, such as human enterovirus 71. The P2X receptor antagonist may be (30) Foreign Application Priority Data PPADS, iso-PPADS, PPNDS, Suramin, NFO23, TNP-ATP, NF279, NF157, Evans Blue, an analog thereof, a derivative Mar. 9, 2012 (CN) ......................... 20121 OO6262O.9 thereof, or a pharmaceutically acceptable salt thereof. Patent Application Publication Feb. 12, 2015 Sheet 1 of 25 US 2015/0045318A1 10-fold reduction: 3.44 puM 100-fold reduction: 5.19 M 1000-fold reduction: 6.45 M 10,000-fold reduction: 7.19 M ECso = 6.93 uM (2511-fold reduction) 6 Patent Application Publication Feb. 12, 2015 Sheet 2 of 25 US 2015/0045318A1 EC50=4.7uM 500 OOO 400 OOO 3DO OOO 200 OOO EC50=2.6M 100 OOO Patent Application Publication Feb. 12, 2015 Sheet 3 of 25 US 2015/0045318A1 6 E O as se O EC50=8.07M 1. Ole 1 2 4. 8 16 32 uM FIG. 4 12,000,000 EC50=2.0LM 10,000,000 8,000,000 E O 6,000,000 4,000,000 2,000,000 Patent Application Publication Feb. 12, 2015 Sheet 4 of 25 US 2015/0045318A1 g O 2.5 5 10 20 40 23.OO 10 Patent Application Publication Feb. 12, 2015 Sheet 5 of 25 US 2015/0045318A1 § (wNHHOdwºVN89LVAD) ##uleug £4Kauppi ?ëjaar-1 LigaH fiun-1 FIG. 8 infection group & Drug group Placebo group Lae© | FIG. 9 Patent Application Publication Feb. 12, 2015 Sheet 6 of 25 US 2015/0045318A1 1. 5 1 2. 9 6 the control 8, 20mg/kkg 50mg/kg O 1 2 3 4 5 6 7 8 9 10 11 Post-injection time FIG 11 Patent Application Publication Feb. 12, 2015 Sheet 7 of 25 US 2015/0045318A1 C S O Cells not pre-treated Cells pre-treated - 7. With drug with drug s E. > 5 valN. S.e 5 ita & 4 1. { Cu { & {& Prior to infection r re s a- a During infection -- r -- -- w Post-infection an ar re r FIG. 12 * Relative EV71 viral load Ce viability Patent Application Publication Feb. 12, 2015 Sheet 8 of 25 US 2015/0045318A1 FIG. 14 10-fold 100-fold reduction reduction 7.14 4.41 -.5 . 1. 1. FIG. 15 Patent Application Publication Feb. 12, 2015 Sheet 9 of 25 US 2015/0045318A1 8.0 x 10 6.0 x 10 4.0 x 10 2.0 x 10 log1 nmol uM SH-TS SH-RS ECso 2.24 3.47 FIG. 16 C 6. f e S 5. Ea 9 c. 5 gC - 4. NO i We 4. 10-fold reduction: 5.89 uM 100-fold reduction: 15.49 puM 2O 3. 9 logo nmol F.G. 17 Patent Application Publication Feb. 12, 2015 Sheet 10 of 25 US 2015/0045318A1 E 40 * Control group CD 30 Drug group E 3 200 o O D - 10 N D Day post-infection FIG. 18 40. 10 Control drug 39 in s N' ' ' w8 o' A sesakorn 3 Day Post Infection FIG. 19 Patent Application Publication Feb. 12, 2015 Sheet 11 of 25 US 2015/0045318A1 NF 279 ". o - as 5 E SR d R S 9 S. L e c) R ... O R S. RNA Va i: Cell viability O 1.O 12 14 1S 1.8 2.O Logou FIG. 20A Evans blue ". s O - 5 s d S 9 S. se R 2 C. R RNA u & Cell viability 0.5 1. 15 2.0 Log10 FIM FIG. 20B Patent Application Publication Feb. 12, 2015 Sheet 12 of 25 US 2015/0045318A1 NF 157 6) -- RNA a Cell viability -0.5 . O).5 O 15 2.0 2.5 L Ogo puM FIG. 20 C iso-PPADS 120 -- iOO 8 O g - RNA 20 a Cell viability 0.5 O.O. 0.5 1. 1.5 2. log10 ul FIG. 20D Patent Application Publication Feb. 12, 2015 Sheet 13 of 25 US 2015/0045318A1 NF O23 to RNA a Cell viability 0.5 O.O. O.5 1.0 1.5 2.) g Logio ul FIG. 20E PPADS 12O OO 8 O 8- RNA 2O Cell viability -0.5 O) O5 1. S g Log 10 uM FIG. 20F Patent Application Publication Feb. 12, 2015 Sheet 14 of 25 US 2015/0045318A1 is logo EW.71 relative titter is viability dr viability 0505 1 1 O uM FIG. 21A BBG e logo EV71 relative titter I viability 1 1 O uM FIG 21B Patent Application Publication Feb. 12, 2015 Sheet 15 of 25 US 2015/0045318A1 MRS 2159 2 1oo 8. O s O. 1 1 1 O 1OO uM FIG. 21C MRS 2179 2O 1 OO SO s 4. O O. 3. 2 40 & is logo EW/1 relative titter 2O A viability W O1 1 10 1 OO uM FIG 21 D Patent Application Publication Feb. 12, 2015 Sheet 16 of 25 US 2015/0045318A1 RO O437626 7. 2O S 4. 3O s S. 60 9. 3 CD 2 40 C. g 1. to logo EW.71 relative titter 2O is viability O O O. 1 1 1 O 1 OO uM FIG 21E TNP-ATP wa9 were S it 3 2. Va g 1. st logo EW 71 relative titter : a viability W O.1 1. 1 O 1 OO uM FIG. 21F Patent Application Publication Feb. 12, 2015 Sheet 17 of 25 US 2015/0045318A1 2',5'-ADP m s v N D g S A) t 9. CD R k O Qa d to logo EW Z1 relative titter O is viability- O O.1 1 10 1 OO uM FIG. 21G A 438O79 7. 12O 6 a 1 OO w S 8O S old 4 A) 2 SO. O. 3 E 2 4o 2 Sp to logo EV71 relative titter 3. a viability 2O 5. 1 1 O 16 uM FIG. 21H Patent Application Publication Feb. 12, 2015 Sheet 18 of 25 US 2015/0045318 A1 5-BOBD ims wo t r N. ce y 2 se rib Fog o EW Z1 relative titter O viability O. 1 1 1 O LM FIG. 21 A 74OOO3 8. 12O c) E O 1 OO o go O wa 5 4 O a 3 e So 2 3? to log 1 O EV71 eduvalent genome - S. 1 is viablity 32 S4 FIG. 21. Patent Application Publication Feb. 12, 2015 Sheet 19 of 25 US 2015/0045318A1 A 839977 S S. 9. t 9 VC 5 to logo EVA 1 relative fitter O is viability O1 1. 1 O 1OO LM FIG. 21 K A-3 17491 e log10 EW71 equvalent genome in viablity LM FIG. 21. Patent Application Publication Feb. 12, 2015 Sheet 20 of 25 US 2015/0045318A1 AF-353 S s S. C. 9) VC se o O to logo EV71 relative titter wianity (505 O O. 1 1 1 O 1 OO LM FIG. 21M AZ 1 O6O612O S S c) A) 2 O SD Va 5 e logo EV71 relative titter O : viability O-1 1 1 O 1 OO uM FIG. 21N Patent Application Publication Feb. 12, 2015 Sheet 21 of 25 US 2015/0045318A1 AZ 1 1645373 to log1 O EV71 eduvalent genome viablity uM FIG. 21 O 4CN-62 2O O S 8. S CD A) 2 6O O. E Sb 40 g se 8 d no logo. EVf 1 relative titter & 2O O viability- a m r - O.1 1 1 O 1 OO LM FIG. 21P Patent Application Publication Feb. 12, 2015 Sheet 22 of 25 US 2015/0045318A1 OATP 7 12O 1 OO sa S 8O 4 S. 60 9. it 3 9 Va 2 d to Fogo EW71 relative titter O is viability O O.1 1 1 O 1 OO LM FIG. 21CR RO-3 S S t 2 O wa. 9. VC c g se logo FV 71 relative titter is viability O O1 1 1 O 1 OO LM FIG 21R Patent Application Publication Feb. 12, 2015 Sheet 23 of 25 US 2015/0045318A1 Spinorphin N C 2 Eleu-Val-Wai-yr-Pro-ir-ir o s re logo. EV? 1 retative titter O is viabilityr r O. 1 1 1 O LM FIG 21S bz ATP : E O s te m 2 is log10 EW 71 equivalent genome viability FIG. 21T Patent Application Publication Feb. 12, 2015 Sheet 24 of 25 US 2015/0045318A1 UTP 2.