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(12) Patent Application Publication (10) Pub US 2004OO63666A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0063666 A1 Christianson et al. (43) Pub. Date: Apr. 1, 2004 (54) COMPOSITIONS FOR INHIBITING 09/545,737, filed on Apr. 10, 2000, now Pat. No. ARGINASE ACTIVITY 6,387,890, which is a continuation-in-part of appli cation No. PCT/US98/21430, filed on Oct. 9, 1998. (76) Inventors: David Christianson, Media, PA (US); Ricky Baggio, Waltham, MA (US); Publication Classification Daniel Elbaum, Newton, MA (US) (51) Int. Cl. ................................................ A61K 31/69 Correspondence Address: (52) U.S. Cl. ................................................................ 514/64 DUANE MORRIS, LLP ATTN: WILLIAM H. MURRAY ONE LIBERTY PLACE (57) ABSTRACT 1650 MARKET STREET PHILADELPHIA, PA 19103-7396 (US) Compositions and methods for inhibiting arginase activity, Appl. No.: 10/661,965 including arginase activity in a mammal, are provided. (21) Methods of making the compositions of the invention are (22) Filed: Sep. 12, 2003 also provided as are methods of using the compositions therapeutically. The compositions described herein are use Related U.S. Application Data ful for alleviating or inhibiting a variety of arinase- and NO Synthase-related disorders, including heart diseae, gas (60) Continuation of application No. 10/053,939, filed on trointestinal motility disorders, and penile erectile dysfunc Jan. 23, 2002, which is a division of application No. tion in humans. Patent Application Publication Apr. 1, 2004 Sheet 2 of 34 US 2004/0063666A1 Patent Application Publication Apr. 1, 2004 Sheet 3 of 34 US 2004/0063666A1 Patent Application Publication Apr. 1, 2004 Sheet 4 of 34 US 2004/0063666A1 Patent Application Publication Apr. 1, 2004 Sheet 5 of 34 US 2004/0063666A1 Patent Application Publication Apr. 1, 2004 Sheet 6 of 34 US 2004/0063666A1 O2 Cl (D C N1 Ó O1 2-O C2 C3 C. {) C4 C5 O -O C6 (3)c. o, -(R" -O O3 Fig. 7 Patent Application Publication Apr. 1, 2004 Sheet 7 of 34 US 2004/0063666A1 Patent Application Publication Apr. 1, 2004 Sheet 8 of 34 US 2004/0063666A1 Patent Application Publication Apr. 1, 2004 Sheet 9 of 34 US 2004/0063666A1 VII?!!! Patent Application Publication Apr. 1, 2004 Sheet 11 of 34 US 2004/0063666A1 3. 's - -2 S5 - 2N S’ S.- CN o c o C -- i. cs bO -- CN o a No C T - C 2> a.is Cn CS r c 3 3 O O C C CN Z. 2. c={ 2. qs S3 -- 9) CN Crs- t < CN wa S 5 bo + 2 Z. s t en Z. Z. 9. e Patent Application Publication Apr. 1, 2004 Sheet 14 of 34 US 2004/0063666A1 TIME (sec) O 2000 4000 6000 8000 q/t (LLCal/sec) dq/d7 (kcal/mole) 0.0 0.5 1.0 1.5 2.0 2.5 (7/Arginase Fig. 15 Patent Application Publication Apr. 1, 2004 Sheet 15 of 34 US 2004/0063666 A1 TIME (Sec) O 2000 4000 6000 8000 10000 -0.5 q/t (Lucal/sec) -1-0 dq/d15) 4 (kcal/mole) -6 -8 -10 -12 olo 0.5 1.0 15 2.0 2.5 3.0 3.5 15)/Arginase Fig. 16 Patent Application Publication Apr. 1, 2004 Sheet 16 of 34 US 2004/0063666 A1 ca. CN 2e. c O C9 t i. C N as r-ct, H gld - ve \s. bo CN e yL s ) Vco OA 1, s - en r T at S R - O < t r 3 =(ZH)SHOE Z· Oy 09 08 J_NÃORIHd TWWIXWW SWINITTWA NOISNOEL 3———————/Á001 Patent Application Publication Apr. 1, 2004 Sheet 18 of 34 US 2004/0063666A1 (~oO ~ S | O ? (ZH)SHQI . Z ..., I /// HO 09 08 NITTWH. ———————/Á001 JN30?IGHd TWWIXWW NOISNÉHJ, Patent Application Publication Apr. 1, 2004 Sheet 21 of 34 US 2004/0063666A1 (W„OIXI)HQV+VNN-TW7–V7 (W„OIX£)HAV+VNN-Iw— |OJ?u0OO—O (Wç-OIX£)VNN-T©–O S/ 07 09 08 J.NHOR{{d TWWIXWW NITITWH NOISNEJ, Patent Application Publication Apr. 1, 2004 Sheet 22 of 34 US 2004/0063666A1 TONINOOO-O (ZH)SHR 07 09 08 NITTW? SWI NOISNAL Patent Application Publication Apr. 1, 2004 Sheet 23 of 34 US 2004/0063666A1 4. (ZH)SHR ... Z 1.4. HCO 41-21HKC 07 09 08 ———————//DOI J.NGHORIHd TITWH. SWINI NOISNÉHL Patent Application Publication Apr. 1, 2004 Sheet 24 of 34 US 2004/0063666A1 W[],LORRI SWI ÇZ?IH 0008 000L 0009 000$ Patent Application Publication Apr. 1, 2004 Sheet 25 of 34 US 2004/0063666A1 SWI0–o WOJLOGINIW— NIWR|{{w– YIJAITO–O00I (Wii)SININIOIV-TAXONICI?H 9Z?IH {{SWNIORIW% ÄLIAILOW Patent Application Publication Apr. 1, 2004 Sheet 26 of 34 US 2004/0063666A1 O 3 N. > O is6, a S. 25 N A49 A t - O O < ( - 5A O <g N CN csCN spo t C cs C Co Co C C Co Cd Cd gd C C C C S. o dxed N- Wid l r er CN w f Patent Application Publication Apr. 1, 2004 Sheet 27 of 34 US 2004/0063666A1 08 09 J.NGIORÍ8d ÅLIAILOW£{SWNIORIW Patent Application Publication Apr. 1, 2004 Sheet 28 of 34 US 2004/0063666A1 08 09 Off JLNEORIHd £{SWNIORIVLOWÄLIAI Patent Application Publication Apr. 1, 2004 Sheet 29 of 34 US 2004/0063666 A1 00I 08 09 JANEIORIºd £{SWNIORIWLOWÅJLIAI Patent Application Publication Apr. 1, 2004 Sheet 30 of 34 US 2004/0063666A1 08 09 07 Patent Application Publication Apr. 1, 2004 Sheet 31 of 34 US 2004/0063666A1 Patent Application Publication Apr. 1, 2004 Sheet 32 of 34 US 2004/0063666A1 O cy o O. c CC O y - O co e Her Patent Application Publication Apr. 1, 2004 Sheet 33 of 34 US 2004/0063666A1 09||USV/ Patent Application Publication Apr. 1, 2004 Sheet 34 of 34 US 2004/0063666A1 F. Frequency (Hz) 5. 2 5 O Frequency (Hz) 5g ---A 1 2 5 O 1 mM ABH A 10 nin Pap/SNP Frequency (Hz) 1 2 5 1 O 3Oe 20 O 40 X d 60 n & 800 control o 0.5 mm ABH O 0.1 mM ABH 1 mM ABH 100 3DCF. S. | Gcontain 0.5 mM ABH q 1 mM ABH 3 400 T 9 O and () & > d5 E. Frequency (Hz) US 2004/0063666 A1 Apr. 1, 2004 COMPOSITIONS FOR INHIBITING ARGINASE 0006 To date, the X-ray crystal structure of one of the ACTIVITY enzymes of mammalian arginine catabolism, namely rat liver arginase, is available (Kanyo et al., 1996, Nature CROSS-REFERENCE TO RELATED 383:554-557). Rat liver arginase is a trimeric metalloen APPLICATIONS Zyme which contains a bi-nuclear manganese cluster in the 0001. This application is a continuation of U.S. patent active site of each Subunit. This bi-nuclear cluster is required application Ser. No. 10/053,939 (pending), which is a divi for maximal catalytic activity (Reczkowski et al., 1992, J. sional of U.S. patent application Ser. No. 09/545,737 (now Am. Chem. Soc. 114:10992-10994). U.S. Pat. No. 6,387,890), which is itself a continuation-in 0007 As noted herein, arginase catalyzes divalent cation part of International Patent Application PCT/US98/21430, dependent hydrolysis of L-arginine to form L-ornithine and published in the English language on Oct. 9, 1998, and is urea. The enzyme is currently known to Serve three impor entitled to priority pursuant to 35 U.S.C. S119(e) to U.S. tant functions: production of urea, production of ornithine, provisional patent application No. 60/061,607, filed Oct. 10, and regulation of Substrate arginine levels for nitric oxide 1997. synthase (Jenkinson et al., 1996, Comp. Biochem. Physiol. 114B:107-132; Kanyo et al., 1996, Nature 383:554-557; STATEMENT REGARDING FEDERALLY Christianson, 1997, Prog. Biophys. Molec. Biol. 67:217 SPONSORED RESEARCH OR DEVELOPMENT 252). Urea production provides a mechanism to excrete nitrogen in the form of a highly Soluble, non-toxic com 0002 This research was supported in part by U.S. Gov pound, thus avoiding the potentially dangerous conse ernment funds (U.S. National Institutes of Health grants quences of high ammonia levels. L-ornithine is a precursor number GM45614 and DK44841), and the U.S. Government for the biosynthesis of polyamines, Spermine, and Spermi may therefore have certain rights of the invention. dine, which have important roles in cell proliferation and differentiation. Finally, arginase modulates production of BACKGROUND OF THE INVENTION nitric oxide by regulating the levels of arginine present 0003. The invention relates generally to enzyme inhibi within tissues. tors, more particularly to inhibitors of the enzyme desig 0008 Since both NO synthase and arginase compete for nated arginase. the same Substrate, the possibility of reciprocal regulation of 0004. Each individual excretes roughly ten kilograms of both arginine metabolic pathways has recently been urea per year, as a result of the hydrolysis of arginine in the explored (Modelell et al., 1995, Eur. J. Immunol. 25:1101 final cytosolic step of the urea cycle (Krebs et al., 1932, 1104, Wang et al., 1995, Biochem. Biophys. Res. Commun. Hoppe-Seyler's Z. Physiol. Chem. 210:33-66). The activity 210:1009-1016). Furthermore, N'-hydroxy-L-arginine of the liver enzyme, arginase, permits disposal of nitrog (L-HO-Arg), an intermediate in the NO synthase reaction enous wastes which result from protein catabolism (Herzfeld (Pufahl et al., 1992, Biochemistry 31:6822-6828; Klau et al, et al., 1976, Biochem.J. 153:469-478). In tissues which lack 1993, J. Biol. Chem. 268: 14781-14787; Furchgom, 1995, a complete complement of the enzymes which catalyze the Annu. Rev. Pharmacol. Toxicol., 35:1-27; Yamaguchi et al., reactions of the urea cycle, arginase regulates cellular con 1992, Eur.
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