USOO9345694B2
(12) United States Patent (10) Patent No.: US 9,345,694 B2 Ratan et al. (45) Date of Patent: May 24, 2016
(54) COMPOUNDS FOR ENHANCING ARGINASE (56) References Cited ACTIVITY AND METHODS OF USING SAME U.S. PATENT DOCUMENTS (75) Inventors: Rajiv R. Ratan, Scarsdale, NY (US); 3,867,531 A 2f1975 Shemano Marie T. Filbin, New York, NY (US) 4,966,918 A 10, 1990 Watanabe et al. 6,972,195 B2 12/2005 Xu (73) Assignees: Cornell Research Foundation, Inc., 8,206,741 B2 * 6/2012 Plachetka ...... 424,472 Ithaca, NY (US): The Research 2004/0121004 A1* 6/2004 Taneja ...... 424/465 Foundation of the City University of 2006/0142241 A1* 6/2006 Yoo ...... 514/59 New York, New York, NY (US) 2006/0276393 A1 12/2006 Milburn et al. (*) Notice: Subject to any disclaimer, the term of this FOREIGN PATENT DOCUMENTS patent is extended or adjusted under 35 WO O3066595 A2 8, 2003 U.S.C. 154(b) by 0 days. OTHER PUBLICATIONS (21) Appl. No.: 13/416,885 Colin Howden (Am J Med. 2004; 117(5A):44S-48S.).* (22) Filed: Mar. 9, 2012 Schaller et al. (Am. J. Gastroenterol. Jul. 2006; 101(7): 1655-1665).* Badiola et al. (PLOS ONE. Mar. 2013; 8(3): e58837, pp. 1-8).* (65) Prior Publication Data Ishige et al., “Flavonoids Protect Neuronal Cells from Oxidative Stress by Three Distinct Mechanisms”. Free Radical Biology & US 2013/0245070 A1 Sep. 19, 2013 Medicine, vol. 30, No. 4, pp. 433-446; 2001. Bastianetto et al., “Natural Antioxidants and Neurodegenerative Dis Related U.S. Application Data eases”. Frontiers in Bioscience, vol. 9, pp. 3447-3452; 2004. (63) Continuation of application No. 12/309,500, filed as Uwabe et al., “HU0622: A Small Molecule Promoting GAP-43 Acti application No. PCT/US2007/016335 on Jul. 18, vation and Neurotrophic Effects'. Neuropharmacology, vol. 51, pp. 2007, now abandoned. 727-736; 2006. (60) Provisional application No. 60/807,661, filed on Jul. * cited by examiner 18, 2006. (51) Int. Cl. Primary Examiner — Scott Long A6 IK3I/4439 (2006.01) (74) Attorney, Agent, or Firm — Hoffman & Baron, LLP A6 IK3I/05 (2006.01) A6 IK3I/085 (2006.01) (57) ABSTRACT A6 IK3I/22 (2006.01) A6 IK3I/352 (2006.01) The present invention relates to a method for enhancing argi A6 IK3I/484 (2006.01) nase activity in a damagedor injured cell. In other aspects, the A6 IK3I/36 (2006.01) invention provides a method for treating a disorder that can be A6 IK3I/7048 (2006.01) treated by enhancing arginase activity in a human in need thereof, the method comprising administering to the human (52) U.S. Cl. an effective amount of a compound that enhances arginase CPC ...... A6 IK3I/4439 (2013.01); A61 K3I/05 activity. Such disorders include ischemia, hypoxia, neurode (2013.01); A61 K3I/085 (2013.01); A61 K generative disease or condition, stroke or trauma of the ner 31/122 (2013.01); A61 K3I/352 (2013.01); Vous system. In yet another aspect, the invention provides A6 IK3I/36 (2013.01); A61 K3I/4184 methods for promoting regeneration of a neural cell in a (2013.01); A61K3I/7048 (2013.01) human in need thereof. (58) Field of Classification Search CPC ...... A61K 31/4439; A61 K31/7048 See application file for complete search history. 1 Claim, 37 Drawing Sheets U.S. Patent May 24, 2016 Sheet 1 of 37 US 9,345,694 B2
Figure 1A
Name Reference
Pinosylwin J. Ann. Chen. Soc., 1940, 62:3512
J. Chen. Soc., 1959, 2679 Phytochen istry 1968, 7:701 Phytochen istry 1972, O89 Methoxyvone C7H14O3 U.S. Patent May 24, 2016 Sheet 2 of 37 US 9,345,694 B2
Figure 1B
Dehydrovariabilin C17H140 Phytochem istry 1978, 7:1417
Chrysophanol C15H10O4 Phytochem istry 1972, 11:2122
U.S. Patent May 24, 2016 Sheet 4 of 37 US 9,345,694 B2
Figure 2B E: formula assis "assig
33. Crystphati |colso Si.
36 Eisairs discstate T C20H16O7awryxxsrs -xxxxxxxxxxxxaasaxsaxSara
33 3-rriethylchoiartyrere C-1s
oss Corptophar cis-12CNO2 i-im---- i 24 2- EiEEE graf: wife 8-33 faki S É. ESS; ethickycine F-83 : 4. ficity reas:ore 8-33S. 3,3 fix 2 ties erstir 93. S. fold ---28 iša ::1 Estie r 838 2- 45.835 incioscief CF33 old2- . 9578 hydric:Perazopyridine (iii.2CNS" "2497 3. 5. a 1500.477 eitsife C15H10O2 s : ------rrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrronw5. 5:53. Spiritreactive 32CS Fixxxmain33 5. oisoto18 easti xxxaswax 53-32S
34 g oisoales fissions iii.23
35 told 81504044''
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of 5053t 4. ristic A.33, 8-Fs 5505333
: versit is: ------28-383
"Sisi 25 s s U.S. Patent May 24, 2016 Sheet 5 Of 37 US 9,345,694 B2
Figure 3A
Western Blot RT-PCR better than Chern Narine control x2 activity in Tillorone primary screen 02300009
x2 activity in primary screen D1500477 not quite 2x data 15O139 Pramoxine in primary hydrochloride ScreenS x2 activity in Indoprofen primary screen O1500351 Phenazopyridine x2 activity in hydrochloride primary screen AO6 O1500473 not quite 2x data in primary Piperine SCS x2 activity in 6,3'-dimethoxyflavone primary screen x2 activity in Anisindione primary screen e A09 O1502198. not quite 2x data in primary 5,4'-dimethoxyflavone screers . A10 00211227 x2 activity in Pinosylwin primary screen A11 002O1066 x2 activity in Derrustone primary screen 302 O401419 x2 activity in 4,7-dimethoxyflavone primary screen BO3 O0210554 x2 activity in Daidzein primary screen 002OO789 x2 activity in 4'-methoxychalcone primary screen O0211475 x2 activity in Tranilast primary screen 15 1505.333 x2 activity in Biochanin a diacetate primary Screen 16 OO3OO601 O1504044 X Resveratrol 4'-meth U.S. Patent May 24, 2016 Sheet 6 of 37 US 9,345,694 B2
Figure 3B
Western Blot RT-PCR better tar control chemether name primary screen
x4 activity in Derrubone primary screen BO9 OD2O65 x2 activity in
Chlorpropham primary screen 9 B 10 OO330051 x2 activity in Genistein primary screen 2O B11 OO2O296 x2 activity in Dehydrovariabilin primary Screen 21 CO2 00210658 X x2 activity in Retusin 7-methyl ether primary screen 22 CO3 OO24O645 x2 activity in Xanthone primary screen 23 CO4 OO200523 x2 activity in Pinosylvin methyl ether primary screen 24 CO5 OO20067 x2 activity in Chrysophanol primary screen 2 5 COS OO300545 X x2 activity in Apigenin primary screen 26 CO7 OO2ODEas not quite 2x data
in primary 2-methoxyxanthone screers 27 CO8 OO24O736 x2 activity in Apigenin triacetate primary screen 28 CO9 OO2O)5O2 x2 activity in Fenbendazole primary screen 29 C10 O150016
x2 activity in Dibenzoylmethane primary screen C11 O1505311 x2 activity in Methoxyvone primary screen 31 OO2 40O8ESS X X X x4 activity in Ginkgetin, k salt primary screen 32 OO3 OO2004.36 x4 activity in Methyl robustone primary Screen 33 O4 O4O1401 not quite 2x data Liquiritigenin dimethyl in primary ether screes 34 D05 O16OO56 x2 activity in
Deusnin primary screen O14O1406 U.S. Patent May 24, 2016 Sheet 7 Of 37 US 9,345,694 B2
Figure 3C
Western Blot RT-PCR better than control
x2 activity in primary screen x2 activity in For Toroneti primary screen not quite 2x data in primary 4'-methoxyflavone Sctees x2 activity in Acacetin diacetate primary screen U.S. Patent May 24, 2016 Sheet 8 of 37
U.S. Patent May 24, 2016 Sheet 9 Of 37 US 9,345,694 B2
U.S. Patent May 24, 2016 Sheet 10 Of 37 US 9,345,694 B2
Figure 6
COmound 1.10 U.S. Patent May 24, 2016 Sheet 11 of 37 US 9,345,694 B2
Figure 7
Arg-Screen/Custom Plate Compound #11-20
4.
3
2
O c wis ah- na sh wn A so se s a c r o O U.S. Patent May 24, 2016 Sheet 12 of 37 US 9,345,694 B2
Figure 8
Argl-Screen/Custom Plate Compound #21-30
U.S. Patent May 24, 2016 Sheet 13 of 37 US 9,345,694 B2
Figure 9
Arg-ScreenlCustom Plate Compound #31-40
3. U 2 A. U.S. Patent May 24, 2016 Sheet 14 of 37 US 9,345,694 B2
Figure 10
Overcoming MAG inhibition in vitro
Post-Teatment Effect Acetaminphen None 2 None Reveral methylene None cryoprano (owdo None
7 dehydrovariabilin
CAPE
1 1 deriustone None 1 2 Epicatechin pentaacetate
Lansoprazole N/A U.S. Patent May 24, 2016 Sheet 15 Of 37 US 9,345,694 B2
s 3 U.S. Patent May 24, 2016 Sheet 16 of 37 US 9,345,694 B2
U.S. Patent May 24, 2016 Sheet 17 Of 37 US 9,345,694 B2
Figure 13
Overcoming MAG inhibition in vitro
Pretreatment Post treatment
Crnod Name 1 Acetamin phen None
None Resveratol 4-methyl ether Chrysophanol (low dose)
5 daidzein S Yes None 7 dehydrovariabilin Phenethyl Caffeate (CAPE) fenbendazole
Epicatechin pentaacetate
Yes U.S. Patent May 24, 2016 Sheet 18 Of 37 US 9,345,694 B2
CON Figure 14A U.S. Patent May 24, 2016 Sheet 19 Of 37 US 9,345,694 B2
MAG Figure 14B U.S. Patent May 24, 2016 Sheet 20 Of 37 US 9,345,694 B2
Methoxyzone 5puM on MAG Figure 14C U.S. Patent May 24, 2016 Sheet 21 Of 37 US 9,345,694 B2
WW - Y W. W. W. W. W. W. W. W. W. W. W. W. W. W. W. W. aidzein 20M on MAG Figure 14D U.S. Patent May 24, 2016 Sheet 22 Of 37 US 9,345,694 B2
anzoprazole 20M on MAG Figure 14E
U.S. Patent May 24, 2016 Sheet 24 of 37 US 9,345,694 B2
Arginase
Acti U.S. Patent May 24, 2016 Sheet 25 Of 37 US 9,345,694 B2
Figure 16A
MOLENAE At armes Structure
2-hydroxyxanthone 2-hydroxy- 9H xanthen- 9- one
2-methoxycanthone 2-methoxy- 9H xanther- 9- one (GIGIGl 3-methyl-1,2- methylcholanthrene dihydrocyclopenta?itetr aphene
4,7- 7-methoxy-2-(4- dimethoxyflavone methoxyphenyl)-4H H chronel-4-one (IUPAC)
4'- (2E)-1-(4- methoxychalcone methoxyphenyl)-3- phenylprop-2-en-1-one (IUPAC), 1-(4- methoxyphenyl)-3- phenylprop-2-en-1-one (IUPAC) U.S. Patent May 24, 2016 Sheet 26 of 37 US 9,345,694 B2
Figure 16B
MOLENAME At nates '-methoxyflavone 2-(4-methoxyphenyl)- 4H-chromen-a-one (IUPAC),
5,4'- 5-methoxy-2-(4- dimethoxyflavone methoxyphenyl)-4H chromen-4-one (IUPAC)
5,74'. 5,7-dimethoxy-2-(4- trimethoxyflavone Tethoxyphenyl)-4H corter-4-ore (IUPAC)
5,7- 5,7-dimethoxy-3- dimethoxyisoflavon phenyl-4H-chromen-4- e one (IUPAC)
6,3'- 6-methoxy-2-(3- dimethoxyflavone methoxyphenyl)-4H chroner-4-one (IUPAC)
Acacetin diacetate 2-(4-methoxyphenyl)-4- oxo-4H-chronene-5,7- diyl diacetate U.S. Patent May 24, 2016 Sheet 27 Of 37 US 9,345,694 B2
Figure 16C
Anisindione 2-(4-methoxyphenyl)- 1H-indene-1,3(2H) - diore
Apigenin 4,5,7-trihydroxyflavone (chemical), 4H-1- benzopyran-4- one, 5,7-dihydroxy-2- (4-hydroxyphenyl)- (chemical), 5,7,4'- trihydroxy- flavone (chemical), 5,7- dihydroxy-2-(4- hydroxyphenyl)-4H chromen- 4- one (IUPAC), 83244 (NSC), apigenin (common, vendor, MeSH, primary common), apigenine (common), apigenol (cornrnon), C. l. natural yellow t (common), flavone,4',5,7- trihydroxy- (chemical), NP-000448 (extract), pelargidenon 1449 (common), spigenin (common), versulin (common)
Apigenin triacetate 2-(4-acetoxyphenyl)- 4-oxo- 4H- chromene 5,7-diyi diacetate U.S. Patent May 24, 2016 Sheet 28 of 37 US 9,345,694 B2
Figure 16D
Biochanin a 5,7-diydroxy-3-(4- methoxyphenyl)- 4H chromen- 4- one
Biocharina 3-(4-methoxyphenyl)-4- diacetate oxo-4H-chromene-5,7- diyl diacetate
Chlorpropham isopropy (3- chlorophenyl) carbamate
1,8-dihydroxy-3-methyl lar 9,10-anthraquinone U.S. Patent May 24, 2016 Sheet 29 Of 37 US 9,345,694 B2
Figure 16E
MOLENAME At names Structure
Daidzein 4,7-dihydroxy-iso flavone (chemical), 4',7- dihydroxyisoflavone (chemical), 4H-1- benzopyran-4-one,7- hydroxy-3-(4- hydroxyphenyl)- (chemical), 7,4'- dihydroxyisoflavone (chemical), 7-hydroxy 3-(4-hydroxyphenyl)-4- benzopyrone (chemical), 7-hydroxy 3-(4-hydroxyphenyl)- 4H-1-benzopyran-4- one (chemical), 7 hydroxy-3-(4- hydroxyphenyl)-4H chromen-4-one (IUPAC), 7-hydroxy-3- (4-hydroxyphenyl) chronen-4-one (chemical), daidZein (common, vendor. MeSH, primary common), daidzeol (common)
Dehydrovariabilin 3,9-dimethoxy-6H 1)benzofuro3,2- cchromene
U.S. Patent May 24, 2016 Sheet 31 of 37 US 9,345,694 B2
Figure 16G
MOLENAME Ait names Structure
Formononet 7-hydroxy-3-(4- methoxyphenyl)-4H chromen- 4- one
Genistein 36586 (NSC), 4',5,7- trihydroxyisoflavone (chemical), 4H-1- . benzopyran-4-one, 5,7- dihydroxy-3-(4- hydroxyphenyl)- (chemical), 5,7,4'- trihydroxyisoflavone (chemical), 5,7- dihydroxy-3-(4- hydroxyphenyl)-4- benzopyrone (chemical), 5,7- dihydroxy-3-(4- hydroxyphenyl)-4H chomer-1-one (IUPAC), differenol A (common), genistein (common, MeSH, primary-common), genisteol (common), genisterin (common), isoflavone,4,5,7- trihydroxy-(chemical), NP-001561 (extract), prunetol (common), sophoricol (common)
U.S. Patent May 24, 2016 Sheet 34 of 37 US 9,345,694 B2
Figure 16J
MOLENAE Structure
7-methoxy-5-methyl-2- phenyl-4H-chromen-4- e
Methyl robustone 7-(1,3-benzodioxol-5- yl)-5-methoxy-2,2- dimethyl-2H,6H pyrano,3,2-gchromen 6-one
Phenazopyridine 3-(phenyldiazenyl) hydrochloride pyridine-2,6-diamine (IUPAC)
Phenindice 2-phenyl-1H-indene 1,3(2H)-dione (IUPAC)
Pinosylwin 5-(E)-2- phenylvinyl)benzene 1,3-diol (IUPAC)
Pinosylwin methyl 3-methoxy-5-(E)-2- ether phenylwinyl)phenol (IUPAC) U.S. Patent May 24, 2016 Sheet 35 of 37 US 9,345,694 B2
Figure 16K
MOLENAME At 1ames Structure
Piperine 1-(2E,4E)-5-(1,3- benzodioxol-5-yl)penta- Cs k 2,4-dienoyl)piperidine H (IUPAC),benzodioxol-5-yl)penta- 1-(5-(1,3- / \ N 2,4-dienoylpiperidine h (IUPAC),
Pranoxie 25573 (NSC), 4-3-(4-
hydrochloride butoxyphenoxy) propylimorpholine (IUPAC),
Resveratrol 4'- 5-(E)-2-(4- methyl ether methoxyphenyl) vinylbenzene-1,3-diol
Retusin 7-methyl 8-hydroxy-7-methoxy ether 3-(4-methoxyphenyl)- 4H-chromen-4-one (IUPAC)
U.S. Patent May 24, 2016 Sheet 37 Of 37 US 9,345,694 B2
Figure 16M
MOLENAME Structure
Tranilast (aka MK- 2-(2E)-3-(3,4- 341) dimethoxyphenyl)prop 2-enoyl)amino)benzoic acid (IUPAC)
Xanthone 9-xanthen-9-one (IUPAC) US 9,345,694 B2 1. 2 COMPOUNDS FOR ENHANCING ARGINASE droxyXanthone, 2-methoxyxanthone: 3-methylcholanthrene; ACTIVITY AND METHODS OF USING SAME 4,7-dimethoxyflavone; 4'-methoxychalcone; 4'-methoxyfla vone; 5,4'-dimethoxyflavone; 5,7,4'-trimethoxyflavone; 5,7- CROSS REFERENCE TO RELATED dimethoxyisoflavone; 6.3'-dimethoxyflavone; Acacetin diac APPLICATIONS etate; Anisindione: Apigenin; Apigenin triacetate; Biochanin a; Biochanin a diacetate; Chlorpropham; Chrysophanol: This application is a continuation of U.S. Ser. No. 12/309, Daidzein: Dehydrovariabilin: Derrubone; Derrusnin; Derrus 500, filed on Jun. 23, 2009, abandoned, which is a 371 appli tone; Dibenzoylmethane: Fenbendazole; Formononetn; cation of PCT/US2007/016335, filed Jul. 18, 2007, which Genistein; Ginkgetin; Indoprofen; Ipraflavone; Liquiritige claims the benefit of U.S. Provisional Application Ser. No. 10 nin dimethyl ether; Methoxyvone; Methyl robustone; 60/807,661, filed on Jul.18, 2006. The contents of the afore Phenazopyridine hydrochloride; Phenindione; Pinosylvin; mentioned prior applications are incorporated herein by ref Pinosylvin methyl ether, Piperine: Pramoxine hydrochloride: erence in their entireties. Resveratrol 4'-methyl ether; Retusin 7-methyl ether; Robus The invention was made with funds from New York State tone; Spironolactone; Tilorone; Tranilast; or Xanthone. Department of Health, contract number CO19772. New York 15 In another aspect, the invention provides a method for State has certain rights in this invention. enhancing arginase activity in a damaged or injured cell. The method comprises administering to a human in need thereof BACKGROUND OF THE INVENTION an effective amount of Lansoprazole. In another aspect, the invention provides a method for It is reported that the adult mammalian central nervous treating a disorder that can be treated by enhancing arginase system (CNS) shows little spontaneous regeneration after activity in a human in need thereof. The method comprises injury despite that fact that there are many molecules present administering to the human an effective amount of a com which promote nerve and axonal growth. In contrast to the pound that enhances arginase activity, wherein the compound CNS, the adult peripheral nervous system (PNS) is capable of is any one of the following: 2-hydroxyXanthone, 2-methox regenerating to some extent. 25 yxanthone: 3-methylcholanthrene; 4,7-dimethoxyflavone: It is believed that the lack of regeneration in the CNS is 4'-methoxychalcone; 4'-methoxyflavone; 5,4'-dimethoxyfla caused by the presence of molecules which actively prevent vone; 5,7,4'-trimethoxyflavone; 5,7-dimethoxyisoflavone: or inhibit regeneration. Such molecules include Nogo (an 6.3'-dimethoxyflavone; Acacetin diacetate; Anisindione; antigen of the IN-1 antibody), myelin-associated glycopro Apigenin; Apigenin triacetate; Biochanin a; Biochanin a tein, and myelin-oligodendrocyte glycoprotein. 30 diacetate; Chlorpropham; Chrysophanol; Daidzein: Dehy Arginase is an enzyme that catalyzes the conversion of the drovariabilin; Derrubone; Derrusnin, Derrustone; Diben amino acid arginine to urea and ornithine. Arginase has been Zoylmethane: Fenbendazole; Formononetn; Genistein; reported to reverse the inhibition of neural regeneration in the Ginkgetin, Indoprofen; Ipraflavone; Liquiritigenin dimethyl central and peripheral nervous system. Thus, enhancing argi ether; Methoxyvone; Methyl robustone; Phenazopyridine nase activity would be beneficial for reversing the inhibition 35 hydrochloride; Phenindione; Pinosylvin; Pinosylvin methyl of neural regeneration. ether; Piperine; Pramoxine hydrochloride; Resveratrol Arginase I is a 35- to 38-kDa cytoplasmic protein that 4'-methyl ether; Retusin 7-methyl ether; Robustone; Spirono cleaves arginine into urea and ornithine. Arginine is the only lactone; Tilorone; Tranilast; or Xanthone. Substrate capable of donating the guanidine group necessary In yet another aspect, the invention provides a method for for nitric oxide production. Nitric oxide is produced from 40 treating a disorder that can be treated by enhancing arginase arginine by three nitric oxide synthase (NOS) isoforms. Nitric activity in a human in need thereof. The method comprises oxide production can be regulated by modulating the levels of administering to the human an effective amount of Lansopra arginine. Arginase I can limit the pool of arginine available for Zole. nitric oxide synthase (NOS), thereby influencing the produc In a further aspect, the invention provides a method pro tion of nitric oxide. 45 moting regeneration of a neural cell in a human in need Neuronal damage can be caused by excess levels of nitric thereof. The method comprises administering to the human oxide (NO). NO is a diffusible neuronal second messenger an effective amount of a compound that enhances arginase synthesized in the nervous system by three enzymes: neu activity, wherein the compound is any one of the following: ronal NO synthase, endothelial NO synthase, and inducible 2-hydroxyXanthone; 2-methoxyxanthone; 3-methylcholan NO synthase. Excess NO generated by NO synthase is asso 50 threne; 4,7-dimethoxyflavone; 4'-methoxychalcone; 4'-meth ciated with various neurodegenerative diseases and condi oxyflavone; 5,4'-dimethoxyflavone; 5,7,4'-trimethoxyfla tions, such as multiple Sclerosis, dementia, Huntington's dis vone; 5,7-dimethoxyisoflavone; 6.3'-dimethoxyflavone: ease, Alzheimer's disease, etc. Acacetin diacetate; Anisindione: Apigenin; Apigenin triac The amino acid arginine is the only endogenous Substrate etate; Biochanina, Biochanin a diacetate; Chlorpropham; of NO synthase. It is reported that arginase can reduce cell 55 Chrysophanol; Daidzein, Dehydrovariabilin: Derrubone; death in the nervous system by competing with NO synthase Derrusnin; Derrustone; Dibenzoylmethane: Fenbendazole; for their common Substrate, arginine. Formononetn; Genistein; Ginkgetin; Indoprofen; Iprafla Therefore, enhancing arginase activity would be beneficial vone: Liquiritigenin dimethyl ether; Methoxyvone; Methyl for promoting neural regeneration or reducing neural damage robustone; Phenazopyridine hydrochloride; Phenindione: in diseases and conditions associated with neural damage. 60 Pinosylvin; Pinosylvin methyl ether; Piperine; Pramoxine hydrochloride: Resveratrol 4'-methyl ether; Retusin 7-methyl SUMMARY OF THE INVENTION ether; Robustone; Spironolactone; Tilorone; Tranilast; or Xanthone. The present invention provides a method for enhancing In yet a further aspect, the invention provides a method for arginase activity in a damaged or injured cell. The method 65 promoting regeneration of a neural cell in a human in need comprises administering to a human in need thereof with an thereof. The method comprises administering to the human effective amount of one of the following compounds: 2-hy an effective amount of Lansoprazole. US 9,345,694 B2 3 4 BRIEF DESCRIPTION OF THE FIGURES FIG. 14C. P5 cerebellar neurons were plated on substrate inhibitor MAG expressing CHO cells and treated with meth FIG. 1A. Chemical structures of compounds pinosylvin, oxy vone (5 uM). Picture is representative of BIII tubulin derrustone and methoxy vone. positive cells. FIG. 1B. Chemical structures of compounds dehydroVari FIG. 14D. P5 cerebellar neurons were plated on substrate abilin and chrysophanol. inhibitor MAG expressing CHO cells and treated with daid FIG. 2A. Table listing compounds that upregulated argin Zein (20 uM). Picture is representative of BIII tubulin positive ase I at least above or near 2-fold. cells. FIG. 2B. Table listing compounds that upregulated argin FIG. 14E. P5 cerebellar neurons were plated on substrate 10 inhibitor MAG expressing CHO cells and treated with Lan ase I at least above or near 2-fold. Zoprazole (20 uM). Picture is representative of BIII tubulin FIG.3A. Table listing compounds that were tested in quan positive cells. titative RT-PCR and immunoblot analysis. FIG. 14F. P5 cerebellar neurons were plated on CONT and FIG.3B. Table listing compounds that were tested in quan substrate inhibitor (MAG) expressing CHO cells. Neurons titative RT-PCR and immunoblot analysis. 15 were plated either with DMSO(0.1%) on control CHO mono FIG.3C. Table listing compounds that were tested in quan layers, or on MAG and treated with DMSO (0.1%), methox titative RT-PCR and immunoblot analysis. yvone (5uM), daidzein (20 uM) and Lanzoprazole (20 uM). FIG. 4. Western blot results that measured level of Graph depicts the average length of the longest neurite (per enhanced arginase 1 protein associated with the compounds centage of the longest neurite of the control). At least 400 listed in FIG. 3. neurons were measured in each assay and the experiment was FIG. S. Western blot results that measured level of carried out at least twice. enhanced arginase 1 protein associated with the compounds FIG. 15. Western blot and immunostaining results for argi listed in FIG. 3. nase I protein. Neurons were treated with, daidzein (20 uM), FIG. 6. Quantitative RT-PCR results presented in a graph methoxy vone (5 uM) or lanzoprazole (20 uM) for 18 hours showing levels of arginase I messenger RNA (mRNA) 25 and were then lysed and Subjected to gel electrophoresis, upregulation associated with compounds 1-10 listed in FIG. followed by western blotting and immunostaining for Argin 3. ase I protein. As a positive control, neurons were treated with FIG. 7. Quantitative RT-PCR results presented in a graph 1 mM db cAMP. showing levels of arginase I messenger RNA (mRNA) FIG. 16A. The chemical formula, structures, and refer upregulation associated with compounds II-20 listed in FIG. 30 ences for a genus of compounds useful in the methods of the 3. invention. FIG. 16B. The chemical formula, structures, and refer FIG. 8. Quantitative RT-PCR results presented in a graph ences for a genus of compounds useful in the methods of the showing levels of arginase I messenger RNA (mRNA) invention. upregulation associated with compounds 21-30 listed in FIG. 35 FIG. 16C. The chemical formula, structures, and refer 3. ences for a genus of compounds useful in the methods of the FIG. 9. Quantitative RT-PCR results presented in a graph invention. showing levels of arginase I messenger RNA (mRNA) FIG. 16D. The chemical formula, structures, and refer upregulation associated with compounds 31-40 listed in FIG. ences for a genus of compounds useful in the methods of the 3. 40 invention. FIG. 10. Table summarizing results of compounds tested FIG. 16E. The chemical formula, structures, and refer for their ability to overcome myelin-associated glycoprotein ences for a genus of compounds useful in the methods of the (MAG) inhibition in P7 rat cerebellar neurons, as compared invention. with rho kinase inhibitor. The compounds were administered FIG.16F. The chemical formula, structures, and references after (post-treatment) the neurons were plated. 45 for a genus of compounds useful in the methods of the inven FIG. 11. Graph showing results from testing the com tion. pounds (10 nM) listed in FIG. 10 for their ability to overcome FIG. 16G. The chemical formula, structures, and refer myelin-associated glycoprotein (MAG) inhibition in P7 rat ences for a genus of compounds useful in the methods of the cerebellar neurons, as compared with rho kinase inhibitor. invention. FIG. 12. Graph showing results from testing the com 50 FIG. 16H. The chemical formula, structures, and refer pounds (40 nM) listed in FIG. 10 for their ability to overcome ences for a genus of compounds useful in the methods of the myelin-associated glycoprotein (MAG) inhibition in P7 rat invention. FIG.16I. The chemical formula, structures, and references cerebellar neurons, as compared with rho kinase inhibitor. for a genus of compounds useful in the methods of the inven FIG. 13. Table summarizing results of compounds tested 55 tion. for their ability to overcome myelin-associated glycoprotein FIG.16.J. The chemical formula, structures, and references (MAG) inhibition in P7 rat cerebellar neurons, as compared for a genus of compounds useful in the methods of the inven with rho kinase inhibitor. The compounds were administered tion. before (pretreatment) and after (post treatment) the neurons FIG. 16K. The chemical formula, structures, and refer were plated. 60 ences for a genus of compounds useful in the methods of the FIG. 14A. P5 cerebellar neurons were plated on CONT invention. expressing CHO cells with DMSO (0.1%) on control CHO FIG. 16L. The chemical formula, structures, and refer monolayers. Picture is representative of BIII tubulin positive ences for a genus of compounds useful in the methods of the cells. invention. FIG. 14B. P5 cerebellar neurons were plated on substrate 65 FIG. 16M. The chemical formula, structures, and refer inhibitor MAG expressing CHOcells and treated with DMSO ences for a genus of compounds useful in the methods of the (0.1%). Picture is representative of III tubulin positive cells. invention. US 9,345,694 B2 5 6 DETAILED DESCRIPTION OF THE INVENTION or any combination of the following compounds: Resveratrol 4'-methyl ether; Derrubone; Ginkgetin; or Methyl robustone. Method for Enhancing Arginase Activity In yet another embodiment, the method comprises admin In one aspect, the invention provides a method for enhanc istering to a human in need thereofan effective amount of any ing arginase activity in a damaged or injured cell in a human one or any combination of the following compounds: Tilor in need thereof. Any isoform of arginase can be enhanced by one; Phenindione; Pramoxine hydrochloride; Indoprofen; the methods of the present invention. Examples of arginase Phenazopyridine hydrochloride; Piperine; 6.3'-dimethoxy isoforms include arginase I and arginase II. flavone; Anisindione; 5,4'-dimethoxyflavone; Pinosylvin; The method comprises administering to the human an Derrustone; 4,7-dimethoxyflavone; Daidzein; 4'-methoxy 10 chalcone; Tranilast; Biochanin a diacetate; Resveratrol effective amount of a member of the genus of compounds 4'-methyl ether; Derrubone; Chlorpropham; Genistein; selected from: 2-hydroxyXanthone; 2-methoxyXanthone; Dehydrovariabilin: Retusin 7-methyl ether, Xanthone; Pino 3-methylcholanthrene; 4,7-dimethoxyflavone; 4'-methoxy Sylvin methyl ether; Chrysophanol: Apigenin, 2-methoxyx chalcone; 4'-methoxyflavone; 5,4'-dimethoxyflavone; 5.7,4'- anthone; Apigenin triacetate; Fenbendazole; Dibenzoyl trimethoxyflavone: 5,7-dimethoxyisoflavone; 6,3'- 15 methane; Methoxyvone; Ginkgetin, k salt; Methyl robustone; dimethoxyflavone; Acacetin diacetate; Anisindione; Liquiritigenin dimethyl ether; Derrusnin; Biochanina; 5,7- Apigenin; Apigenin triacetate; Biochanin a; Biochanin a dimethoxyisoflavone; Formononetin; 4'-methoxyflavone; or diacetate; Chlorpropham; Chrysophanol; Daidzein: Dehy Acacetin diacetate. drovariabilin; Derrubone; Derrusnin, Derrustone; Diben In a further embodiment, the method comprises adminis Zoylmethane: Fenbendazole; Formononetn; Genistein; tering to a human in need thereof an effective amount of any Ginkgetin, Indoprofen; Ipraflavone; Liquiritigenin dimethyl one or any combination of the following compounds: Pino ether; Methoxyvone; Methyl robustone; Phenazopyridine Sylvin; Derrustone; Daidzein; 4'-methoxychalcone; Tra hydrochloride; Phenindione; Pinosylvin; Pinosylvin methyl nilast; Biochanin a diacetate; Resveratrol 4'-methyl ether; ether; Piperine; Pramoxine hydrochloride; Resveratrol Dehydrovariabilin: Chrysophanol; or Methoxyvone. 4'-methyl ether; Retusin 7-methyl ether; Robustone; Spirono 25 In yet a further embodiment, the method comprises admin lactone; Tilorone; Tranilast; or Xanthone. istering to a human in need thereofan effective amount of any The terms "enhancing arginase activity” or 'enhanced one or any combination of the following compounds: Daid arginase activity” refer to an increased level of measurable Zein or Methoxyvone. arginase activity in a given assay in the presence of a candi In another embodiment, the method comprises administer date compound relative to the measurable level of arginase 30 ing to a human in need thereof an effective amount of a activity in the absence of the candidate compound, when compound comprising a 9H-Xanthen-9-one selected from a tested under the same conditions. group consisting of: 2-hydroxyxanthone, 2-methoxyxan Activity is considered enhanced according to the invention thone, and Xanthone. if it is enhanced at least about 10% greater, preferably at least In yet another embodiment, the method comprises admin about 25% greater, more preferably at least about 50% 35 istering to a human in need thereof an effective amount of a greater, even more preferably at least about 75% greater, most compound comprising a 4H-chromen-4-one selected from a preferably at least about 90% greater, or more than in the group consisting of 4.7-dimethoxyflavone; 4'-methoxyfla absence of the candidate compound. vone; 5,4'-dimethoxyflavone; 5,7,4'-trimethoxyflavone; 5,7- Arginase activity as used herein can be enhanced by any dimethoxyisoflavone; 6.3'-dimethoxyflavone; Acacetin diac mechanism. For example, arginase activity could be 40 etate; Apigenin; Apigenin triacetate; Biochanina, Biochanin enhanced by transcriptional induction of its cognate messen a diacetate; Daidzein, Derrubone; Derrustone; Formononetn; ger RNA (mRNA), increased stability of its mRNA, increased Genistein; Ginkgetin; Ipraflavone; Liquiritigenin dimethyl translation of mRNA into protein, increased stability of argi ether; Methoxy vone; and Retusin 7-methyl ether. nase protein, increased arginase activity (in the presence or In a further embodiment, the method comprises adminis absence of increased protein), or any other mechanism. 45 tering to a human in need thereof an effective amount of a Increases in arginase activity could be realized by the ability compound comprising a (4-methoxyphenyl)-4H-chromene of a compound to increase the affinity of the arginase enzyme 4-one selected from a group consisting of 4.7-dimethoxyfla for its prototypical Substrate, arginine. vone; 4'-methoxyflavone; 5,4'-dimethoxyflavone; 5.7,4'-tri The arginase activity is enhanced in any damaged or methoxyflavone; Acacetin diacetate; Biochanina, Biochanin injured cell that benefits from enhanced arginase or from a 50 a diacetate; Formononetn; and Retusin 7-methyl ether. reduction of nitric oxide or arginine. The damage or injury In still another embodiment, the method comprises admin may be to any part of a cell. Such as to membranes, DNA, istering to a human in need thereof a compound comprising a RNA, and ribosomes. 1,3-benzodioxol Selected from a group consisting of Derru Examples of cells that may be damaged or injured include bone; Derrusnin; Derrustone; Methyl robustone; Piperine; cells of the central nervous system (CNS) or peripheral ner 55 and Robustone. Vous system (PNS), including neurons, ganglia, Schwann In yet a further embodiment, the method comprises admin cells, astrocytes, oligodendrocytes, microglia cells, endothe istering to a human in need thereof an effective amount of a lial cells, immune cells (e.g., macrophages, T cells, B cells, compound selected from any one or a combination of com and neutrophils), etc. In one embodiment, the damaged or pounds listed in FIG. 2 and/or FIG. 3. injured cell is in a human. 60 In another aspect of the invention, the method comprises In one embodiment, the method comprises administering administering to the human in need thereof an effective to a human in need thereof an effective amount of any one or amount of Lansoprazole. any combination of the following compounds: Pinosylvin; Method for Treating a Disorder that can be Treated by Derrustone; Methoxyvone: Dehydrovariabilin; O Enhancing Arginase Activity Chrysophanol. 65 In another aspect, the invention provides a method for In another embodiment, the method comprises administer treating a disorder that can be treated by enhancing arginase ing to a human in need thereofan effective amount of any one activity in a human in need thereof. The method includes US 9,345,694 B2 7 8 administering to the human an effective amount of a com from a group consisting of 4,7-dimethoxyflavone; 4'-meth pound that enhances arginase activity, wherein the compound oxyflavone; 5,4'-dimethoxyflavone; 5,7,4'-trimethoxyfla is any one of the following: 2-hydroxyXanthone, 2-methox Vone; Acacetin diacetate; Biochanina, Biochanina diacetate; yxanthone: 3-methylcholanthrene; 4,7-dimethoxyflavone: Formononetn; and Retusin 7-methyl ether. 4'-methoxychalcone; 4'-methoxyflavone; 5,4'-dimethoxyfla In still another embodiment, the method comprises admin vone; 5,7,4'-trimethoxyflavone; 5,7-dimethoxyisoflavone: istering to the human an effective amount of a compound 6.3'-dimethoxyflavone; Acacetin diacetate; Anisindione; comprising a 1,3-benzodioxol Selected from a group consist Apigenin; Apigenin triacetate; Biochanin a; Biochanin a ing of Derrubone; Derrusnin; Derrustone; Methyl robustone: diacetate; Chlorpropham; Chrysophanol; Daidzein: Dehy Piperine; and Robustone. drovariabilin; Derrubone; Derrusnin, Derrustone; Diben 10 Zoylmethane: Fenbendazole; Formononetn; Genistein; In yet a further embodiment, the method comprises admin Ginkgetin, Indoprofen; Ipraflavone; Liquiritigenin dimethyl istering to the human an effective amount of a compound ether; Methoxyvone; Methyl robustone; Phenazopyridine selected from any one or a combination of compounds listed hydrochloride; Phenindione; Pinosylvin; Pinosylvin methyl in FIG. 2 and/or FIG. 3. ether; Piperine; Pramoxine hydrochloride; Resveratrol 15 In another aspect of the invention, the method comprises 4'-methyl ether; Retusin 7-methyl ether; Robustone; Spirono administering to the human an effective amount of Lansopra lactone; Tilorone; Tranilast; or Xanthone. Zole. In one embodiment, the method comprises administering Disorders and diseases in which enhancing arginase activ to the human an effective amount of any one or any combi ity is desired for treatment include ischemia, hypoxia, neuro nation of the following compounds: Pinosylvin; Derrustone; degenerative disease or condition, or stroke. Additional dis Methoxyvone; DehydroVariabilin; or Chrysophanol. orders and diseases in which enhancing arginase activity is In another embodiment, the method comprises administer desired for treatment traumatic disorders (including but not ing to the human an effective amount of any one or any limited to spinal cord injuries, spinal cord lesions, or other combination of the following compounds: Resveratrol 4'-me CNS pathway lesions), Surgical nerve lesions, damage sec thyl ether; Derrubone; Ginkgetin; or Methyl robustone. 25 ondary to infarction, infection, exposure to toxic agents, In yet another embodiment, the method comprises admin malignancy, paraneoplastic syndromes, or patients with vari istering to the human an effective amount of any one or any ous types of neurodegenerative disorders of the central ner combination of the following compounds: Tilorone; Phenin Vous system. dione; Pramoxine hydrochloride; Indoprofen; Phenazopyri Method for Treating Ischemia dine hydrochloride; Piperine; 6.3'-dimethoxyflavone: Anis 30 In one embodiment, the invention provides a method for indione; 5,4'-dimethoxyflavone; Pinosylvin; Derrustone; 4.7- treating ischemia in a human in need thereof. The method dimethoxyflavone; Daidzein; 4'-methoxychalcone; Tranilast; comprises administering to the human a compound that Biochanin a diacetate; Resveratrol 4'-methyl ether; Derru enhances arginase activity as described above, including bone; Chlorpropham; Genistein: DehydroVariabilin; Retusin those compounds grouped in the various genera and Sub 7-methyl ether, Xanthone; Pinosylvin methyl ether; 35 genera. Chrysophanol: Apigenin, 2-methoxyxanthone; Apigenin tri Any mammal Suffering from ischemia can be treated in acetate: Fenbendazole; Dibenzoylmethane; Methoxy vone: accordance with the method of the present invention. Ginkgetin, k salt; Methyl robustone; Liquiritigenin dimethyl Ischemia generally refers to a condition of decreased blood ether; Derrusnin; Biochanin a 5,7-dimethoxyisoflavone: flow to an organ, tissue and/or cell. The decrease in blood flow Formononetin; 4'-methoxyflavone; or Acacetin diacetate. 40 can be caused by, for example, constriction (e.g., hypoxemic In a further embodiment, the method comprises adminis vasoconstriction) or obstruction (e.g., clot, atherosclerotic tering to the human an effective amount of any one or any plaque) of a blood vessel. combination of the following compounds: Pinosylvin; Der Ischemia can occur in any cell, organ, and/or tissue. rustone; Daidzein; 4'-methoxychalcone; Tranilast; Biochanin Examples of cells, organs, and/or tissues which can be Sub a diacetate; Resveratrol 4'-methyl ether; DehydroVariabilin; 45 jected to ischemia include neuronal cells (e.g., neurons, gan Chrysophanol; or Methoxyvone. glia, Schwann cells, astrocytes, oligodendrocytes and micro In yet a further embodiment, the method comprises admin glia), brain, spinal cord, intestinal cells, kidney cells, heart istering to the human an effective amount of any one or any and cardiac muscle cells such as myocytes, etc. combination of the following compounds: Daidzein or Meth Method for Treating Hypoxia oxyvone. 50 In yet another embodiment, the invention provides a In another embodiment, the method comprises administer method for treating hypoxia in a human in need thereof. The ing to the human an effective amount of a compound com method includes administering to the human a compound that prising a 9H-Xanthen-9-one selected from a group consisting enhances arginase activity as described above, including of 2-hydroxyXanthone, 2-methoxyxanthone, and Xanthone. those compounds grouped in the genera and various Sub In yet another embodiment, the method comprises admin 55 genera. istering to the human an effective amount of a compound Any mammal Suffering from hypoxia can be treated in comprising a 4H-chromen-4-one selected from a group con accordance with the method of the present invention. sisting of 4,7-dimethoxyflavone; 4'-methoxyflavone; 5,4'- Hypoxia generally refers to a lack of oxygen to cells, organs, dimethoxyflavone: 5,7,4'-trimethoxyflavone; 5,7- and/or tissues. Hypoxia can be caused by, for example, dimethoxyisoflavone; 6.3'-dimethoxyflavone; Acacetin 60 ischemia, anemia and chemical modification of blood, Such diacetate; Apigenin; Apigenin triacetate; Biochanina, Bio as carboxyhemoglobin, etc. chanin a diacetate; Daidzein; Derrubone; Derrustone; For Hypoxia can occur in any cell, organ, and/or tissue. mononetn; Genistein, Ginkgetin; Ipraflavone; Liquiritigenin Examples of cells, organs, and/or tissues which can be Sub dimethyl ether; Methoxyvone; and Retusin 7-methyl ether. jected to hypoxia include neuronal cells (e.g., neurons, gan In a further embodiment, the method comprises adminis 65 glia, Schwann cells, astrocytes, oligodendrocytes and micro tering to the human an effective amount of a compound com glia), brain, spinal cord, kidney cells, intestinal cells, heart prising a (4-methoxyphenyl)-4H-chromene-4-one selected and cardiac muscle cells such as myocytes, skin cells, etc. US 9,345,694 B2 9 10 Method for Treating Neurodegenerative Disease or Condi (CNS) or peripheral nervous system (PNS) in a human in tion need thereof. The method includes administering to the In still another embodiment, the invention provides a human a compound that enhances arginase activity as method for treating a neurodegenerative disease or condition described above, including those compounds grouped in the in a human in need thereof. The method includes administer 5 genera and various Sub-genera. ing to the human a compound that enhances arginase activity Any type of trauma to the nervous system may be treated by as described above, including those compounds grouped in the methods of the claimed invention. As described above, the genera and various Sub-genera. trauma of the CNS or PNS include, but are not limited to, A neurodegenerative disease or condition typically refers spinal cord injuries, spinal cord lesions, other CNS pathway to a disorder generally characterized by gradual and progres 10 lesions, as well as injuries to the PNS, such as injuries to a sive loss of cells, tissue and/or organ of the central or periph nerve or neuron of the PNS and axon damage resulting in eral nervous system. Examples of Such cells, tissues and demyelination of the PNS. Such trauma can arise from either organs include, the brain, spinal cord, neurons, ganglia, physical injury or disease. Any mammal Suffering from a Schwann cells, astrocytes, oligodendrocytes and microglia. trauma of the CNS or PNS can be treated in accordance with Any mammal Suffering from any neurodegenerative dis 15 the methods of the present invention. ease or condition can be treated in accordance with the For example, spinal cord injury refers to any damage to the method of the present invention. For example, the neurode spinal cord. The damage typically results in loss of function, generative disease or condition can be an acute condition. Such as mobility or feeling. Damage to the spinal cord can Acute conditions generally occur as a result of trauma to a occur, for example, as a result or trauma (car accident, gun cell, tissue and/or organ of the nervous system. The trauma shot, falls, etc.) or disease (polio, spina bifida, Friedreich's can, for example, partially or completely block blood flow to Ataxia, etc). the cell, tissue and/or organ. Examples of acute neurodegen Any injury to the spinal cord can be treated in accordance erative conditions include head injury and brain injury. with the method of the present invention. For example, the Alternatively, the neurodegenerative disease or condition injury can be a complete injury to the spinal cord. Complete can be a chronic neurodegenerative condition. Examples of 25 injury typically refers to the lack of function (e.g., no sensa chronic neurodegenerative diseases and conditions include tion and no voluntary movement) below the site of injury. Parkinson's disease, Alzheimer's disease, Huntington's dis Both sides of the body are usually affected. ease and Amyotrophic Lateral Sclerosis (also known as Lou Alternatively, the injury may be an incomplete injury to the Gehrig's disease). spinal cord. An incomplete injury generally refers to some Additional examples of neurodegenerative disorders and 30 function below the site of injury. For instance, a person with diseases that can be treated by the invention include but are an incomplete injury may be able to move one limb more than not limited to Alexander disease, Alper's disease, Alzhe another, may be able to feel parts of the body that cannot be imer's disease, Amyotrophic lateral sclerosis, Ataxia telang moved, or may have more functioning on one side of the body iectasia, Batten disease (also known as Spielmeyer-Vogt than the other, etc. Sjogren-Batten disease), Bovine spongiform encephalopathy 35 Method for Promoting Regeneration of a Neural Cell in a (BSE), Canavan disease, Cockayne syndrome, Corticobasal Human in Need Thereof degeneration, Creutzfeldt-Jakob disease, Huntington disease, In another aspect, the invention provides a method for HIV-associated dementia, Kennedy's disease, Krabbe dis promoting regeneration of a neural cell in a human in need ease, Lewy body dementia, Machado-Joseph disease thereof. As described in Lange, et al., J. Nutr 2004 October; (Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple 40 134(10 Suppl):28125-28175; discussion 2818S-2819S, argi System Atrophy, Neuroborreliosis, Parkinson disease, Pel nase has a role in the axonal regeneration pathway. Arginase izaeus-Merzbacher Disease, Pick's disease, Primary lateral also has neuroprotective properties. Arginase is Sufficient in sclerosis, Prion diseases, Refsum’s disease, Sandhoff dis protecting neurons against several apoptosis-inducing ease, Schilder's disease, Schizophrenia, Spielmeyer-Vogt stimuli. Moreover, arginase acts as a nitric oxide-independent Sjogren-Batten disease (also known as Batten disease), 45 inhibitor of neuronal apoptosis. Spinocerebellar ataxia (multiple types with varying charac Several investigators have found that molecules or drugs teristics), Spinal muscular atrophy, Steele-Richardson that prevent injury in the PNS or CNS have no effect on or Olszewski disease, Tabes dorsalis, and other dementias. worse, negatively impact the ability of the nervous system to Method for Treating Stroke regenerate or repair. There is thus an urgent need to identify In a further aspect, the invention provides a method for 50 targets whose activation would provide an environment that is treating stroke in a human in need thereof. The method simultaneously instructive for neuronal protection and repair. includes administering to the human a compound that It is proposed that Such an intervention would provide greater enhances arginase activity as described above, including latitude in the timing of initiation of treatment. those compounds grouped in the genera and various Sub Arginine can be metabolized by nitric oxide synthase to genera. 55 produce nitric oxide. It can also be degraded by arginase to Any mammal Suffering from stroke can be treated in accor produce urea and ornithine, which in turn is a precursor for the dance with the method of the present invention. Stroke is a synthesis of polyamines. These two pathways compete for type of cardiovascular disease that generally involves the arginine. Arginase thus produces polyamines at the expense interruption of blood flow to and/or within the brain. The of nitric oxide. interruption of blood flow can be due to, for example, a 60 Polyamines have been implicated in neuronal growth and blockage or rupture of an artery or vessel. The blockage development, axonal regeneration after injury, and in would typically occurs from a blood clot. As a result of the interrup healing outside of the CNS. Arginase I is upregulated and tion of blood flow, the brain does not receive sufficient polyamine synthesis increases in neurons in response to amounts of blood. either dbCAMP or BDNF. Method for Treating Trauma of the CNS or PNS 65 By producing polyamines, arginase can overcome the In still a further embodiment, the invention provides a effects of myelin-associated glycoprotein (MAG) and myelin method for treating trauma of the central nervous system on neurite outgrowth. Arginase is an enzyme that mediates US 9,345,694 B2 11 12 repair by reducing its Substrates (L-arginine) and increasing In a further embodiment, the method comprises adminis its ultimate products (polyamines), respectively. tering to the human an effective amount of any one or any As toxic levels of nitric oxide (NO) are implicated in acute combination of the following compounds: Pinosylvin; Der ischemic cortical injury and motor neuron loss due to absence rustone; Daidzein; 4'-methoxychalcone; Tranilast; Biochanin of trophic factors, arginase can mediate neuroprotection. By 5 a diacetate; Resveratrol 4'-methyl ether, DehydroVariabilin; producing polyamines, arginase can overcome the effects of Chrysophanol; or Methoxyvone. myelin-associated glycoprotein (MAG) and myelin on neu In yet a further embodiment, the method comprises admin rite outgrowth. Arginase is thus a bi-functional enzyme that istering to the human a an effective amount of n effective mediates neuroprotection or repair by reducing its Substrates amount of any one or any combination of the following com (L-arginine) and increasing its ultimate products 10 pounds: Daidzein or Methoxy vone. (polyamines), respectively. In another embodiment, the method comprises administer ing to the human an effective amount of a compound com As used herein, the phrase “neural cell includes nerve prising a 9H-Xanthen-9-one selected from a group consisting cells (i.e., neurons, e.g., uni-, bi-, or mulipolar neurons) and of 2-hydroxy Xanthone, 2-methoxyxanthone, and Xanthone. their precursors and glial cells (e.g., macroglia Such as astro 15 In yet another embodiment, the method comprises admin cytes, oligodendrocytes, ependymal cells, radial glia, istering to the human an effective amount of a compound Schwann cells, Satellite cells, and microglia) and their pre comprising a 4H-chromen-4-one selected from a group con cursors. Microglia are specialized macrophages capable of sisting of 4,7-dimethoxyflavone; 4'-methoxyflavone; 5,4'- phagocytosis that protect neurons of the central nervous sys dimethoxyflavone: 5,7,4'-trimethoxyflavone; 5,7- tem. The term “precursor refers to cells which are capable of dimethoxyisoflavone; 6.3'-dimethoxyflavone; Acacetin developing into a specific cell type. For example, a neural cell diacetate; Apigenin; Apigenin triacetate; Biochanina, Bio precursor is a cell which is capable of developing into a chanin a diacetate; Daidzein, Derrubone; Derrustone; For mature neural cell (i.e., a cell having the characteristic mor mononetn; Genistein, Ginkgetin; Ipraflavone; Liquiritigenin phology and function of a neural cell). dimethyl ether; Methoxyvone; and Retusin 7-methyl ether. Accordingly, the claimed invention provides methods for 25 In a further embodiment, the method comprises adminis promoting regeneration of a neural cell in a human in need tering to the human an effective amount of a compound com thereof. prising a (4-methoxyphenyl)-4H-chromene-4-one selected The method includes administering to the human an effec from a group consisting of 4,7-dimethoxyflavone; 4'-meth tive amount of a compound that enhances arginase activity, oxyflavone; 5,4'-dimethoxyflavone; 5,7,4'-trimethoxyfla wherein the compound is any one of the following: 2-hydrox 30 Vone; Acacetin diacetate; Biochanina, Biochanina diacetate; yXanthone; 2-methoxyxanthone: 3-methylcholanthrene; 4.7- Formononetn; and Retusin 7-methyl ether. dimethoxyflavone; 4'-methoxychalcone; 4'-methoxyflavone: In still another embodiment, the method comprises admin 5,4'-dimethoxyflavone; 5,7,4'-trimethoxyflavone; 5,7- istering to the human an effective amount of a compound dimethoxyisoflavone; 6.3'-dimethoxyflavone; Acacetin diac comprising a 1,3-benzodioxol Selected from a group consist etate; Anisindione: Apigenin; Apigenin triacetate; Biochanin 35 ing of Derrubone; Derrusnin; Derrustone; Methyl robustone: a; Biochanin a diacetate; Chlorpropham; Chrysophanol: Piperine; and Robustone. Daidzein: Dehydrovariabilin: Derrubone; Derrusnin; Derrus In yet a further embodiment, the method comprises admin tone; Dibenzoylmethane: Fenbendazole; Formononetn; istering to the human an effective amount of a compound Genistein; Ginkgetin; Indoprofen; Ipraflavone; Liquiritige selected from any one or a combination of compounds listed nin dimethyl ether; Methoxyvone; Methyl robustone; 40 in FIG. 2 and/or FIG. 3. Phenazopyridine hydrochloride; Phenindione; Pinosylvin; In another aspect of the invention, the method comprises Pinosylvin methyl ether; Piperine: Pramoxine hydrochloride: administering to the human an effective amount of Lansopra Resveratrol 4'-methyl ether; Retusin 7-methyl ether; Robus Zole. tone; Spironolactone; Tilorone; Tranilast; or Xanthone. Methods for Protecting a Neural Cell in a Human in Need In one embodiment, the method comprises administering 45 Thereof to the human an effective amount of any one or any combi In yet another aspect, the invention provides a method for nation of the following compounds: Pinosylvin; Derrustone; protecting a neural cell in a human in need thereof. The Methoxyvone; DehydroVariabilin; or Chrysophanol. method includes administering to the human a compound that In another embodiment, the method comprises administer enhances arginase activity, wherein the compound comprises ing to the human an effective amount of any one or any 50 any one of the following compounds: 2-hydroxyXanthone; combination of the following compounds: Resveratrol 4'-me 2-methoxyxanthone: 3-methylcholanthrene; 4,7-dimethoxy thyl ether; Derrubone; Ginkgetin; or Methyl robustone. flavone; 4'-methoxychalcone; 4'-methoxyflavone; 5,4'- In yet another embodiment, the method comprises admin dimethoxyflavone: 5,7,4'-trimethoxyflavone; 5,7- istering to the human an effective amount of any one or any dimethoxyisoflavone; 6.3'-dimethoxyflavone; Acacetin combination of the following compounds: Tilorone; Phenin 55 diacetate; Anisindione: Apigenin; Apigenin triacetate; Bio dione; Pramoxine hydrochloride; Indoprofen; Phenazopyri chanina, Biochanin a diacetate; Chlorpropham; Chrysopha dine hydrochloride; Piperine; 6.3'-dimethoxyflavone: Anis nol; Daidzein: Dehydrovariabilin: Derrubone; Derrusnin; indione; 5,4'-dimethoxyflavone; Pinosylvin; Derrustone; 4.7- Derrustone; Dibenzoylmethane; Fenbendazole; Formonon dimethoxyflavone; Daidzein; 4'-methoxychalcone; Tranilast; etn; Genistein, Ginkgetin; Indoprofen; Ipraflavone; Liquiriti Biochanin a diacetate; Resveratrol 4'-methyl ether; Derru 60 genin dimethyl ether; Methoxy vone; Methyl robustone: bone; Chlorpropham; Genistein: DehydroVariabilin; Retusin Phenazopyridine hydrochloride; Phenindione; Pinosylvin; 7-methyl ether, Xanthone; Pinosylvin methyl ether; Pinosylvin methyl ether, Piperine: Pramoxine hydrochloride: Chrysophanol: Apigenin, 2-methoxyxanthone; Apigenin tri Resveratrol 4'-methyl ether; Retusin 7-methyl ether; Robus acetate: Fenbendazole; Dibenzoylmethane; Methoxy vone: tone; Spironolactone; Tilorone; Tranilast; or Xanthone. Ginkgetin, k salt; Methyl robustone; Liquiritigenin dimethyl 65 In another aspect of the invention, the method comprises ether; Derrusnin; Biochanin a 5,7-dimethoxyisoflavone: administering to the human an effective amount of Lansopra Formononetin; 4'-methoxyflavone; or Acacetin diacetate. Zole. The method includes administering to the human any US 9,345,694 B2 13 14 one or a combination of compounds that enhances arginase cell can be placed in a reservoir (e.g., tissue culture plate), and activity included in the genera and Sub-genera of compounds incubated with a compound under appropriate conditions described above. Suitable for enhancing arginase activity. Suitable incubation Compounds conditions can be readily determined by those skilled in the Compounds useful in the methods of the present invention 5 art. include 2-hydroxyXanthone, 2-methoxyxanthone; 3-methyl Ex vivo methods typically include cells, organs or tissues cholanthrene; 4,7-dimethoxyflavone; 4'-methoxychalcone; removed from a mammal. Such as a human. The cells, organs 4'-methoxyflavone: 5,4'-dimethoxyflavone; 5,7,4'-tri or tissues can, for example, be incubated with the compound methoxyflavone; 5,7-dimethoxyisoflavone; 6.3'-dimethoxy under appropriate conditions. The contacted cells, organs or flavone; Acacetin diacetate; Anisindione: Apigenin; Apigenin 10 tissues are normally returned to the donor, placed in a recipi triacetate; Biochanin a Biochanin a diacetate; Chlor ent, or stored for future use. Thus, the compound is generally propham; Chrysophanol; Daidzein: Dehydrovariabilin: Der in a pharmaceutically acceptable carrier. rubone; Derrusnin; Derrustone; Dibenzoylmethane: Fen In vivo methods are typically limited to the administration bendazole; Formononetn; Genistein; Ginkgetin, Indoprofen; of a compound, such as those described above, to a mammal, Ipraflavone: Liquiritigenin dimethyl ether; Methoxy vone: 15 preferably a human. The compounds useful in the methods of Methyl robustone; Phenazopyridine hydrochloride; Phenin the present invention are administered to a mammal in an dione; Pinosylvin; Pinosylvin methyl ether; Piperine; amount effective in enhancing arginase activity or treating the Pramoxine hydrochloride; Resveratrol 4'-methyl ether; mammal. The effective amount is determined during pre Retusin 7-methyl ether; Robustone; Spironolactone; Tilor clinical trials and clinical trials by methods familiar to phy one; Tranilast; or Xanthone. sicians and clinicians. Another compound useful in the methods of the present An effective amount of a compound useful in the methods invention includes Lansoprazole. of the present invention, preferably in a pharmaceutical com Further examples compounds useful in the methods of the position, may be administered to a mammal in need thereof present invention include any one or a combination of com by any of a number of well-known methods for administering pounds listed in FIG. 2 and/or FIG. 3. 25 pharmaceutical compounds. The compound may be admin These compounds are known in the art. The chemical for istered systemically or locally. mula, structures, and references for a genus of compounds For example, the compound may be administered orally, described above are shown in FIG. 16. intravenously, intranasally, intramuscularly, Subcutaneously, The chemical formula, structures, and references for Pino or transdermally. Other routes of administration include Sylvin, Derrustone, Methoxyvone, DehydroVariabilin, and 30 intracerebroventricularly or intrathecally. Intracerebroven Chrysophanol are shown in FIG. 1. ticulatly refers to administration into the ventricular system The compounds can be in the form of a pharmaceutically of the brain. Intrathecally refers to administration into the acceptable salt. The term “pharmaceutically acceptable salt space under the arachnoid membrane of the spinal cord. Thus refers to a well-tolerated, nontoxic salt prepared from any intracerebroventricular or intrathecal administration may be basic or acidic compound mentioned above, and an acid or 35 preferred for those diseases and conditions which affect the base, respectively. The acids may be inorganic or organic organs or tissues of the central nervous system. acids of any one of the compounds mentioned above. The compounds useful in the methods of the invention may Examples of inorganic acids include hydrochloric, hydrobro also be administered to mammals by Sustained release, as is mic, nitric hydroiodic, Sulfuric, and phosphoric acids. known in the art. Sustained release administration is a method Examples of organic acids include carboxylic and Sulfonic 40 of drug delivery to achieve a certain level of the drug over a acids. The radical of the organic acids may be aliphatic or particular period of time. The level typically is measured by aromatic. Some examples of organic acids include formic, serum or plasma concentration. acetic, phenylacetic, propionic, succinic, glycolic, glucu A description of methods for delivering a compound by ronic, maleic, furoic, glutamic, benzoic, anthranilic, Salicylic, controlled release can be found in international PCT Appli phenylacetic, mandelic, embonic (pamoic), methanesulfonic, 45 cation No. WO 02/083106. The PCT application is incorpo ethanesulfonic, panthenoic, benzenesulfonic, Stearic, Sulfa rated herein by reference in its entirety. Other controlled nilic, alginic, tartaric, citric, gluconic, gulonic, arylsulfonic, release agents are described, for example, in U.S. Pat. Nos. and galacturonic acids. Appropriate organic bases may be 5,567,439; 6,838,094; 6,863,902; and 6,905,708. The con selected, for example, from N,N-dibenzylethylenediamine, trolled release agents and methods for making them in these chloroprocaine, choline, diethanolamine, ethylenediamine, 50 patents are incorporated herein by reference. meglumine (N-methylglucamine), and procaine. Any formulation known in the art of pharmacy is Suitable Throughout this specification, parameters are defined by for administration of the compounds useful in the methods of maximum and minimum amounts. Each minimum amount the present invention. For oral administration, liquid or Solid can be combined with each maximum amount to define a formulations may be used. Some examples of formulations range. 55 include tablets, gelatin capsules, pills, troches, elixirs, Sus Administration pensions, syrups, wafers, chewing gum and the like. The The compounds are administered to a human. The com compounds can be mixed with a suitable pharmaceutical pound is administered to the human in an amount effective in carrier (vehicle) or excipient as understood by practitioners in achieving its purpose. The effective amount of the compound the art. Examples of carriers and excipients include starch, to be administered can be readily determined by those skilled 60 milk, Sugar, certain types of clay, gelatin, lactic acid, Stearic in the art during pre-clinical trials and clinical trials by meth acid or salts thereof, including magnesium or calcium Stear ods familiar to physicians and clinicians. Typical daily doses ate, talc, vegetable fats or oils, gums and glycols. include approximately 1 mg to 1000 mg. For systemic, intracerebroVentricular, intrathecal, topical, Any method known to those in the art for contacting a cell, intranasal, Subcutaneous, or transdermal administration, for organ or tissue with a compound may be employed. Suitable 65 mulations of the compounds useful in the methods of the methods include in vitro, ex vivo, or in vivo methods. In vitro present inventions may utilize conventional diluents, carriers, methods typically include cultured samples. For example, a or excipients etc., such as those known in the art to deliver the US 9,345,694 B2 15 16 compounds. For example, the formulations may comprise See FIG. 2. The protocol that was used to screen for the one or more of the following: a stabilizer, a surfactant, pref Arginase I upregulators is described in detail below. erably a nonionic Surfactant, and optionally a salt and/or a Materials buffering agent. The compound may be delivered in the form T75 flask, Corning 430641 vented of an aqueous solution, or in a lyophilized form. 96 well plate (for tissue culture), Mictotest Primaria, flat The stabilizer may, for example, be an amino acid, Such as bottom, #35-3872 for instance, glycine; or an oligosaccharide. Such as for 96 well plate (for chemical compound), MictotestTM 96, flat example, Sucrose, tetralose, lactose or a dextran. Alterna bottom, #35-3072 tively, the stabilizer may be a Sugar alcohol. Such as for Dulbecco's Phosphate Buffered Saline (1xPBS), from Gibco instance, mannitol; or a combination thereof. Preferably the 10 H 14190-144 stabilizer or combination of stabilizers constitutes from about 0.25% Trypsin-EDTA solution, from Sigma #T4049 0.1% to about 10% weight for weight of the compound. The DMEM (Dulbecco's Modified Eagle's Medium), from Gibco Surfactant is preferably a nonionic Surfactant, such as a H 11995-040 & #11965-092 polysorbate. Some examples of Suitable surfactants include Fetal Bovine Serum (FCS), from Gibco #10082-147 Tween20, Tween80; a polyethylene glycol or a polyoxyeth 15 Penicillin 10,000 IU/ml & Streptomycin 10,000 ug/ml (P/S) ylene polyoxypropylene glycol, such as Pluronic F-68 at from from Cellgro #30-002-C1 about 0.001% (w/v) to about 10% (w/v). Puromycin 1 mg/ml stock The salt or buffering agent may be any salt or buffering 10 mM stock of chemical compounds in DMSO, The Spec agent, such as for example, Sodium chloride, or Sodium/po trum CollectionTM from MicroSource Discovery System. Inc: tassium phosphate, respectively. Preferably, the buffering Stored at -80° C. agent maintains the pH of the pharmaceutical composition in Lysis Reagent (5x), from Promega i E153A the range of about 5.5 to about 7.5. The salt and/or buffering Luciferase Assay Substrate, from Promega i E151A agent is also useful to maintain the osmolality at a level Luciferase Assay Buffer, from Promega i E152A suitable for administration to a human or an animal. Prefer LimaxII from Molecular Device ably the salt or buffering agent is presentata roughly isotonic 25 70% Ethanol concentration of about 150 mM to about 300 mM. 96-well NUNC 236107 white plate (Luciferase Assay) The formulations of the compounds useful in the methods UV Plate for plate reader (Protein Assay) of the present invention may additionally contain one or more Reagents conventional additive. Some examples of such additives DMEM for HT22 4.8Kb Arg-Puro include a solubilizer Such as, for example, glycerol; an anti 30 oxidant such as for example, benzalkonium chloride (a mix ture of quaternary ammonium compounds, known as Ingredient Stock Final conc. Volume “quats'), benzyl alcohol, chloretone or chlorobutanol; anaes DMEM (#11965-092) 445 ml thetic agent Such as for example a morphine derivative; or an FCS 10% 50 ml isotonic agent etc.. Such as described above. As a further 35 PS 3 ml precaution against oxidation or other spoilage, the pharma Puromycin 1 mg/ml 4 g/ml 2 ml ceutical compositions may be stored under nitrogen gas in Total 500 ml vials sealed with impermeable stoppers. Lysis Reagent (10 ml per plate)
EXAMPLES 40 Lysis Reagent Sx 1X 8 mil did H2O 32 m Example 1 Total 40 ml Screening for Arginase I Upregulators 45 Day 1. Harvest Cell and Seeding A 2000 compound library was tested to identify arginase-1 Materials: upregulators. The library tested was The Spectrum Collec tionTM from MicroSource Discovery System, Inc. (Groton, 12x 96 well plates (#35-3872) Two types of DMEM medium Conn.). The 2000 compounds in the library are primarily 8x T75 Flasks Trypsin solution Food and Drug Administration (FDA)-approved compounds 50 2x Glass sterile pipette Timer or natural products. An alphabetical list of the compounds is Sterile pipette (25 ml, 10 ml, 5 ml) available at the MicroSource Discovery website at www.ms 6x 50 ml centrifuge tubes discovery.com/spect.html. The compounds are Supplied as 10 Repeat pipette & tips mM solutions in dimethyl sulfoxide (DMSO). The library was screened using murine hippocampal HT22 55 Steps cells transfected with a luciferase-arginase 1 construct on 96 1. Remove old medium from T.s flask by suction well plates. Total protein was also measured to use in normal 2. Rinse once with 5 ml 1xPBS: discard 1xRBS ization. The luciferase assay result for each compound is 3. Add 3 ml Trypsin to treat cells and incubate at 37° C. normalized to protein content and is expressed as luciferase? incubator for 3 min mg protein. The fold increase was measured relative to 60 4. Observe under microscope to see if all cells detached untreated control, represented as sample #1 in each HT22 Arg 5. Stop trypsin reaction by adding 10 ml DMEM and then plate. transfer the solution into a 50 ml centrifuge tube Ratios of the results from the “luciferase assay” and “pro 6. Centrifuge at 980 rpm/4 min teinassay” were tabulated. A comparison of these normalized 7. Prepare three T75 by adding 10 ml fresh medium to each responses indicates amount of enhanced arginase activity. 65 flask Compounds and their respective Chem ID numbers that 8. Prepare two 50 ml centrifuge tubes by adding 30 ml fresh upregulated arginase 1 above or near 2 fold were identified. medium to each tube US 9,345,694 B2 17 18 9. Discard supernatant by suction and add 8 ml fresh 5. Prepare Substrate solution by mixing Luciferase Assay DMEM into the tube to resuspend the cell pellet Substrate (keep in freezer) with 10 ml Luciferase Assay 10. Mix and Titrate cell suspension and add 2 ml cell Buffer (thaw at room temperature) suspension into three T75 flasks (total 12 ml) and 1 ml to 6. Use multiple channel pipette to transfer 10 ul/well cell each 50 ml centrifuge tube. lysate/supernatant to a 96-well NUNC white plate for 11. Use repeat pipette and sterile tips to dispense 100 reading with Luciferase Assay ul/well to six 96-well plates (for duplicate three chemi 7. Use multiple channel pipette to transfer 2.5 ul/well cell cal plate) from two 50 ml centrifuge tubes lysate/supernatant to a 96-well UV plate for reading 12. Repeat step 1-9 for another cell line (total is twelve with Protein Assay 96-well plates) 10 8. Stored the plate in 4°C. refrigerator or basement freezer 13. Label all plates and T.s flask and incubate at 37° C. 9. Set up and run LimaxII ATP Assay: incubator for 24 hours or until at least 50% confluence