USOO8445533B2 (12) United States Patent (10) Patent No.: US 8.445,533 B2 Liu et al. (45) Date of Patent: May 21, 2013 (54) ANDROGRAPHOLIDE DERIVATIVESTO FOREIGN PATENT DOCUMENTS TREATVIRAL INFECTIONS CN 1293955 A 5, 2001 CN 1433756 A * 8, 2003 (75) Inventors: Rui Hai Liu, Ithaca, NY (US); James R. CN 1433,757 A 8, 2003 CN 1433758 A 8, 2003 Jacob, Cortland, NY (US); Bud CN 1437939. A 8, 2003 Tennant, Ithaca, NY (US) CN 145.0059 A 10, 2003 CN 1478774 A 3, 2004 (73) Assignee: Cornell Research Foundation, Inc., CN 1554337 A 12, 2004 Ithaca, NY (US) WO WO96, 17605 A1 * 6, 1996 WO O1,57026 A1 8, 2001 (*) Notice: Subject to any disclaimer, the term of this WO 01f85710 A1 11, 2001 patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 922 days. Puri et al. “Immunostimulantagents from Andrographis paniculata.” (21) Appl. No.: 11/269,942 J. Nat. 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(Continued) Primary Examiner — Shengjun Wang (51) Int. C. A6 IK3I/34 (2006.01) (74) Attorney, Agent, or Firm — LeClairRyan, a (52) U.S. C. Professional Corporation USPC .......................................................... S14/473 (57) ABSTRACT (58) Field of Classification Search USPC .......................................................... 514/473 The present invention provides a methods and compositions See application file for complete search history. for treating a host afflicted with a viral infection, particularly a Flaviviridae infection, including hepatitis C infection, com (56) References Cited prising administering an effective antiviral amount of a derivative of andrographolide alone or in combination or U.S. PATENT DOCUMENTS alternation with another antiviral compound. 6,140,063 A 10, 2000 Wheelocket al. 2002fOO16363 A1 2/2002 Nanduri et al. 2 Claims, 13 Drawing Sheets -- ANTIRALEFFECT -o- TDTICITY ews WIRLYIELD 1. 1-8 18 1-6 18-5 18-4 18-3 18-2 1e -- ANTIVIRALEFFECT --- COTOCTY awe IRALE 100 40 le--O 0. 1. ANTIVIRAL CONCENTRATIONCM) US 8,445,533 B2 Page 2 OTHER PUBLICATIONS Ebola and Respiratory Syncytlal Viruses: A Comparative Analysis With Fluorogenic Peptides.” Biochem. 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Furin, PC5 and PC7 in the Cleavage of Surface Glycoproteins of Hong Kong, * cited by examiner U.S. Patent May 21, 2013 Sheet 1 of 13 US 8.445,533 B2 -O- ANTIVIRAL EFFECT --O-- CYTOTOXCITY OVO WIRALYIELD 1e-6 1e--5 6 O 4. O le-3 1e-2 2 O 1e-1 1e--0 -O- ANTIVIRAL EFFECT --O-- CYTOTOXCITY OV a VIRALYIELD ASSAY2 1OO Oy (O V v - - - - - - - - 1e-8 1e-1 8 O 1e--6 1e--5 6 O 4. O 1e-2 2 O ANTIVIRAL CONCENTRATION (uM) Y-v-1 FIG. 1 U.S. Patent May 21, 2013 Sheet 2 of 13 US 8.445,533 B2 -O- ANTIVIRAL EFFECT --O-- CYTOTOXICITY OVO WRALYIELD 1OO le+8 le+7 8O 1e-6 s gse 60 le+5 E. 1e--4 5 40 1e +3 1e--2 2 2O es le+ 1 O 1e--0 O.OO1 0.01 0.1 1 -O- ANTIVIRAL EFFECT --O- CYTOTOXCITY o Vo VRALYIELD ASSAY 2 le+8 1OO le+7 8O le+6 s s le+5 60 as s 1e--4 5 40 1e +3 le+2 2 O e le+ 1 O.OO1 0.01 0.1 1 ANTIVIRAL CONCENTRATION (mg/ml) Y V-1 FIG. 2 U.S. Patent May 21, 2013 Sheet 3 of 13 US 8.445,533 B2 -O- BWDV-INDUCED CELLKILLING -V-- CYTOTOXICITY --V-- PROGENY WIRALYIELD 90 1e-- s 8O le+6 a 5 70 a 60 1e--5 E 50 1e--4 2 40 le+3 53 1e4-2 5 20 S. 10 1e-1 O 1e--0 1 10 (ug/ml) 1OO 1OOO -O- BWDV-INDUCED CELLKILLING --V-- CYTOTOXICITY V - PROGENY WIRALYIELD ASSAY 2 90 1e-- 8O 1e4-6 5 5 70 le+5 S 60 e+3 5 50 le+ 4 se 40 1e--3 SE s 3 1e-2 2 20 - es 10 v 1e-1 1e--0 RIBAVIRIN (ug/ml) FIG. 3B U.S. Patent May 21, 2013 Sheet 4 of 13 US 8.445,533 B2 -O- BWDW-INDUCED CELLKILLING --V-- CYTOTOXICITY V - PROGENY WIRALYIELD 1OO 1e--8 90 1e-l SQ 80 1e--6 es5 as 70 1e--5 F Sse 60 e 50 1e--4 5 3s 40 1e--3 5 30 1e +2 20 es 10 1e-1 O 1e--0 O.OOO1 O.OO1 O.O1 0.1 1 1O 1OO 1OOO INTERFERON-ALPHA (ng/ml) -O- BYDW-INDUCED CELLKILLING --V-- CYTOTOXICITY V - PROGENY WIRALYIELD 90 1e-- & 80 1e--6 Es 7060 ite is 50 1e--4 5 5CS 40 1e--3 s 30 s 1e-2 5 20 e 10 1e-1 O 1e--0 1 1O (ug/ml) 1OO 1OOO FIG. 4A U.S. Patent May 21, 2013 Sheet 5 of 13 US 8.445,533 B2 -O- BVDY-INDUCED CELLKILLING --V-- CYTOTOXICITY V - PROGENY WIRALYIELD 1OO 1e--8 e-s 90 1e-- SQ 8O 1e-6 es5 5 70 P as 60 1e--5 50 1e--4 5 gs 40 1e-3 s. 5 30 s 20 1e-2 s 10 1e-1 O 1e--0 0.01 O.1 1 10 RIBAVIRIN (ug/ml) -O- BWDW-INDUCED CELLKILLING --V-- CYTOTOXICITY V - PROGENY WIRALYIELD 100- w-O A to -O-O-O 1e--8 se 90 \ / M -va 1e-- a. 80 1e-6 S 70 F s 60 1e--5 S 50 1e--4 5 40 1e +3 3020 1e +2 3es 10 1e-1 O 1e--0 O.OOO1 O.OO1 O.O1 0.1 1 1O 1OO 1000 INTERFERON-ALPHA (ng/ml) FIG. 4C U.S. Patent May 21, 2013 Sheet 6 of 13 US 8.445,533 B2 ara 1 O O e--8 e--7 e--6 e--5 5 1e-- 4 e--3 e--2 - e-- e--O U.S. Patent May 21, 2013 Sheet 7 of 13 US 8.445,533 B2 -o- CYOTOXCITY RIBAVRIN - - -v- - - PROGENY VIRUS YELD s 1 OO 1e--8 ?? 9 O 1e--7 C 8O 1e--6 . x2 70 1e--5 h O 60 g Her 1 e-4 till 50 NY S 40 1e--5 2. JSO e--2 >- st 20 3. O 1e-- g ty o 1e--O O.O1 O.1 1 O RIBAVIRIN (ng/ml) FIG. 5B U.S. Patent May 21, 2013 Sheet 8 of 13 US 8.445,533 B2 -o- CYOTOXCITY NERFERON-ALPA - - V - - PROGENY VIRUS YED $100 1e-8 90 1e-7 f 8O e--6 le p 70 h 25 50 e-- 4 N Ya 9 40 1e--3 G sarra- 1e-2 tii> - ir- O 1e-1 gS O O e--O 1 O OO 1 OOO CYTOTOXICITY INTERFERON-ALPHA (ng/ml) FIG. 5C U.S. Patent May 21, 2013 Sheet 9 of 13 US 8.445,533 B2 -O- BWD-INDUCED CELLKILLING ---V-- CYTOTOXICITY RIBAWIRIN -O- WRALYIELDS CUMULATIVERIBAWIRIN ACTIVITY IN BWOW ASSAY 1OO 90 8O TO 60 50 40 30 2O 10 0.1 1 10 1OO RIBAVIRIN (M) FIG. 6A -O- VIRALYIELDS -O- BVD-INDUCED CELLKILLING ---V-- CYTOTOXICITY FNC. CUMULATIVE IFNC, ACTIVITY IN BVDV. ASSAY O.OOO1 O.OO1 O.O1 O.1 1 10 1OO 1000 IFNo. (ng/ml) FIG. 6B U.S. Patent May 21, 2013 Sheet 10 of 13 US 8.445,533 B2 -O- BWDW-INDUCED CELLKILLING V CYTOTOXICITYMT-3 -O- WRALYIELDS ACTIVITY OF MT-3 TEST MATERIAL 1OO 90 8O TO 60 SO 40 3O 2O 1O O.O1 0.1 1 1O 1OO MT-3 (ug/ml) FIG.