USOO638789OB1 (12) United States Patent (10) Patent No.: US 6,387,890 B1 Christianson et al. (45) Date of Patent: May 14, 2002 (54) COMPOSITIONS AND METHODS FOR CA:87:6321 abs of Diss Abstr Int B 37(8) pp. 3927-8 by INHIBITING ARGINASE ACTIVITY Hartz. Albina et al., 1995, J. Immunol. 155:4391-4396. (75) Inventors: David Christianson, Media, PA (US); Bacon et al., 1988, J. Mol. Graph. 6:219–200. Ricky Baggio, Waltham; Daniel Baker et al., 1980, Fed. Proc., Fed. Am. Soc. Exp. Biol. Elbaum, Newton, both of MA (US) 39:1686. Baker et al., 1983, Biochemistry 22:2098-2103. (73) Assignee: Trustees of the University of Bergeron et al., 1987, J. Org. Chem. 52:1700–1703. Pennsylvania, Philadelphia, PA (US) Biancani et al., 1985, Gastroenterology 89:867-874. - 0 Boden, 1975, Synthesis 784. (*) Notice: Subject to any disclaimer, the term of this Boucher et al., 1994, Biochem. Biophys. Res. Commun. patent is extended or adjusted under 35 2O3:1614-1621. U.S.C. 154(b) by 0 days. 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Chem. 259: 15106–15114. * cited by examiner U.S. Patent MaV 14, 2002 Sheet 1 of 34 U.S. Patent May 14, 2002 Sheet 2 of 34 US 6,387,890 B1 U.S. Patent May 14, 2002 Sheet 3 of 34 US 6,387,890 B1 U.S. Patent May 14, 2002 Sheet 4 of 34 US 6,387,890 B1 U.S. Patent May 14, 2002 Sheet S of 34 US 6,387,890 B1 U.S. Patent May 14, 2002 Sheet 6 of 34 US 6,387,890 B1 () C1 () C N1 Ó O1 2-O C2 C3 C (C) C4 C5 O -O C6 (3) Cl o, -(r" -O O3 Fig. 7 U.S. Patent May 14, 2002 Sheet 7 of 34 US 6,387,890 B1 LLZ-TlIO ~%...•**?EIN U.S. Patent May 14, 2002 Sheet 8 of 34 US 6,387,890 B1 U.S. Patent May 14, 2002 Sheet 9 of 34 US 6,387,890 B1 VII?!!! U.S. Patent May 14, 2002 Sheet 10 of 34 US 6,387,890 B1 LLZ(110 U.S. Patent May 14, 2002 Sheet 12 of 34 US 6,387,890 B1 U.S. Patent US 6,387,890 B1 U.S. Patent May 14, 2002 Sheet 14 of 34 US 6,387,890 B1 TIME (sec) O 2000 4000 6000 8000 q/t (LLcal/sec) -2 O -2 -4 -6 dqld17 (kcal/mole) 8 -10 -12 -14 0.0 0.5 1.0 1.5 2.0 2.5 7/Arginase Fig. 15 U.S. Patent May 14, 2002 Sheet 15 of 34 US 6,387,890 B1 TIME (sec) O 2000 4000 6000 8000 10000 0.0 -0.5 q/t (Llcal/sec) -1-0 dq/d15 -4 (kcal/mole) 6 -8 -10 -12 0.0 0.5 1.0 15 2.0 2.5 3.0 3.5 15)/Arginase Fig. 16 U.S. Patent May 14, 2002 Sheet 17 of 34 US 6,387,890 B1 07 09 08 J.NGIORIHd TWWIXWW SVINITTWH NOISNHL U.S. Patent May 14, 2002 Sheet 18 of 34 US 6,387,890 B1 09 08 JÄNHORÉId TWWIXWW NITITVSH NOISNÉHJ, U.S. Patent May 14, 2002 Sheet 20 of 34 US 6,387,890 B1 09 08 JANEIO?[Hd TWWIXWW. NITTIVA NOISNÉHJ, U.S. Patent May 14, 2002 Sheet 21 of 34 US 6,387,890 B1 S/ *~~~~. OZ 07 09 08 J_NÃORIHd TWWIXWW NITTIVH. NOISNEJ, U.S. Patent May 14, 2002 Sheet 22 of 34 US 6,387,890 B1 Off 09 08 SWI JLNÉHORIGHd NITTWH. NOISNÉHJ, U.S. Patent May 14, 2002 Sheet 23 of 34 US 6,387,890 B1 ... 1.4. 41-21 09 08 JLNHORIHdH TTWH SVINI NOISNÉHJ, U.S. Patent >? ?S Sheet 24 of 34 US 6,387,890 B1 NIWR|{{W[\[OERISWIRIGHAITI SZ?IH 0008 000L 0009 st0009 U.S. Patent May 14, 2002 Sheet 27 of 34 US 6,387,890 B1 08 09 07 U.S. Patent May 14, 2002 Sheet 29 of 34 US 6,387,890 B1 08 09 07 JLNHORIH? ÄLIAILOW£{SWNIORIV U.S. Patent May 14, 2002 Sheet 30 0f 34 US 6,387,890 B1 XXX &OX XX& C CXOXOXOXO SXMY YNXXXXX 08 09 07 XLIAILOVASWNIÐHV U.S. Patent May 14, 2002 Sheet 31 of 34 US 6,387,890 B1 U.S. Patent May 14, 2002 Sheet 32 of 34 US 6,387,890 B1 U.S. Patent May 14, 2002 Sheet 34 of 34 US 6,387,890 B1 Frequency (Hz) 1 2 5 O Frequency (Hz) 5g --- yi1 2 5 10 At 1 mM ABH A 10 min Pap/SNP Frequency (Hz) 1 2 O iii. t O 20 9 40 X GS 60 i r s 80+0 Control O 0.5 mM ABH O 0.1 mM ABH 1 mM ABH 100 | T T-I- 600 - 2.