US 20090253782A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0253782 A1 Ratan et al. (43) Pub. Date: Oct. 8, 2009
(54) COMPOUNDS FOR ENHANCING ARGINASE Related U.S. Application Data ACTIVITY AND METHODS OF USE (60) Provisional application No. 60/807,661, filed on Jul. THEREOF 18, 2006. (76) Inventors: Rajiv R. Ratan, Scarsdale, NY Publication Classification (US); Marie T. Filbin, New York, (51) Int. Cl. NY (US) A63L/352 (2006.01) A6IP 25/28 (2006.01) Correspondence Address: (52) U.S. Cl...... 514/454 Irving N. Feit Hoffmann & Baron, LLP (57) ABSTRACT 6900 Jericho Turnpike The present invention relates to a method for enhancing argi Syosset, NY 11791 (US) nase activity in a damagedor injured cell. In other aspects, the invention provides a method for treating a disorder that can be (21) Appl. No.: 12/309,500 treated by enhancing arginase activity in a human in need thereof, the method comprising administering to the human (22) PCT Filed: Jul.18, 2007 an effective amount of a compound that enhances arginase activity. Such disorders include ischemia, hypoxia, neurode (86). PCT No.: PCT/US2007/016335 generative disease or condition, stroke or trauma of the ner Vous system. In yet another aspect, the invention provides S371 (c)(1), methods for promoting regeneration of a neural cell in a (2), (4) Date: Jun. 23, 2009 human in need thereof. Patent Application Publication Oct. 8, 2009 Sheet 1 of 33 US 2009/0253782 A1
Figure 1
J. An. Chen. Soc., 1940, 62:3512
J. Chem. Soc., 1959, 2679
Phytochem
istry 1968, 7:70
Phytochen istry 1972, 1:1089
Patent Application Publication Oct. 8, 2009 Sheet 2 of 33 US 2009/0253782 A1
Dehydrovariabilin CH4O4 Phytochen istry 1978, 17:417
Chrysophanol Phytochem istry 1972, 11:222 Patent Application Publication Oct. 8, 2009 Sheet 3 of 33 US 2009/0253782 A1
C17H14O4 282.3
Dehydrovariabilin C17H14O4. 282.3 OO21,0658 4'-methoxychalcone C16H14O2 238.3 OD211475 5,7-dimethoxyisoflavone C17H14O4 as 26964-35 2 Retusin 7-methyl ether C17H1405 298.3 OO24O645 oozoss 2-hydroxyxanthone C13H8O3 soos. 5,74'-trimethoxyflavone C18H16O5 Patent Application Publication Oct. 8, 2009 Sheet 4 of 33 US 2009/0253782 A1
rom
color easo as to or ento a ?ia
octor |tio orotator to to
O 1500473 also rear to a 2 f
oisoarseO 50 2 1 98 Anisindone retario is O1504044 C15H1403 is acrossmoor Teimo I as f 120-46-7
a torm to a on to eatio to 101OOOO3 Biochanin a C16H12O5 284.3 Patent Application Publication Oct. 8, 2009 Sheet 5 of 33 US 2009/0253782 A1 Patent Application Publication Oct. 8, 2009 Sheet 6 of 33 US 2009/0253782 A1
Figure 3
te RT-PCR better than control x2 activity in Tillorone primary screen AO2 O23OOOO9 x2 activity in Phenirdione primary screen . AO3 O1500477 not quite 2x data O1501139 Pramoxine in primary hydrochloride Screers x2 activity in Indoprofen primary screen Phenazopyridine x2 activity in hydrochloride primary Screen O1 50 04 73 not quite 2x data in primary Piperine SCreefs AO7 O1500B73 x2 activity in 6,3'-dimethoxyflavone primary screen AO8 O1504132 x2 activity in Anisindione primary screen AO9 O1502198. not quite 2x data in primary 5,4'-dimethoxyflavone Sctees A1 O 00211227 x2 activity in Pinosylwin primary screen OO2O1066 x2 activity in Der Tustone primary screen 1O O1401419 X x2 activity in 4,7-dimethoxyflavone primary screen O0210554 x2 activity in Daidzeir primary screen B04 O0200789 X x2 activity in
4'-methoxychalcone primary screen O02.11475 x2 activity in Tranilast primary screen B x2 activity in Biochanin a diacetate primary screen BO7 OO3OOSO1 Resveratrol 4'-methyl 7 BO8 O1504O44 Patent Application Publication Oct. 8, 2009 Sheet 7 of 33 US 2009/0253782 A1
ether primary screen x4 activity in Dertubone primary screen BO9 OO2O1650 x2 activity in
Chlorpropham primary screen OO330051 x2 activity in . primary screen O0210296 x2 activity in Dehydrovariabilin primary Screen CO2 OO210658 X x2 activity in Retusin 7-methyl ether primary screen CO3 OO24. x2 activity in Xanthone primary screen CO4 OO200523 x2 activity in Pinosylwin methyl ether primary screen COS O02O1067 x2 activity in Chrysophanol primary screen CO6 O O3005455 x2 activity in Apigenin primary screen CO7 OO020O846 not quite 2x data in primary 2-methoxyxanthone SCeeS 27 C08 O024O736 x2 activity in Apigenin triacetate primary screen C09 OO2005O2 x2 activity in Fenbendazole primary screen 10 O150.1016 x2 activity in Dibenzoylmethane primary screen C11 x2 activity in Methoxyvone primary screen O14006.66 x4 activity in
Ginkgetin, k salt primary screen OO200436 x4 activity in Methyl robustone primary screen Dolotovo not quite 2x data Liquinitigenin dimethyl in primary ether SCsees oO 1 6O O x2 activity in primary screen O14O1406 Patent Application Publication Oct. 8, 2009 Sheet 8 of 33 US 2009/0253782 A1
channan x2 activity in Biochanin a primary screen 5,7- x2 activity in dimethoxyisoflavone primary screen x2 activity in For Thononetin primary screen not quite 2x data in primary 4'-methoxyflavone SCees x2 activity in Acacetin diacetate primary screen Patent Application Publication Oct. 8, 2009 Sheet 9 of 33 US 2009/0253782 A1
Figure 4
| Patent Application Publication Oct. 8, 2009 Sheet 10 of 33 US 2009/0253782 A1
Patent Application Publication Oct. 8, 2009 Sheet 11 of 33 US 2009/0253782 A1
Figure 6
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Figure 7
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92 s S RSN to 9 S C 3. S. E SR2 53 O) h CC
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Figure 9
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Figure 10
Overcoming MAG inhibition in vitro
Post-treatment
Acetaminphen
Resveratol 4-methyl ether Chrysophanol (low dose)
7
11 Epicatechin pentaacetate
N/A Patent Application Publication Oct. 8, 2009 Sheet 16 of 33 US 2009/0253782 A1
Figure 11
se
.2 Patent Application Publication Oct. 8, 2009 Sheet 17 of 33 US 2009/0253782 A1
Figure 12 Patent Application Publication Oct. 8, 2009 Sheet 18 of 33 US 2009/0253782 A1
Figure 13
Overcoming MAG inhibition in vitro
Pretreatent Post treatment
1 Acetaminphen None
Chrysophanol (low dose) 5 None 6 methoxyvone S None 7 dehydrovariabilin 8 Phenethyl caffeate (CAPE)
11 Epicatech in pentaacetate Yes Patent Application Publication Oct. 8, 2009 Sheet 19 of 33 US 2009/0253782 A1
Figure 14
12)
10) Ev SO 3 e Co Daidzein 2. is on MAG s p up 40 2
methoxyvone or dadzeina AG tarcoprazero on A. MAG
Lanzoprazole 20ty on fi AG Neurite outgrowth assay Patent Application Publication Oct. 8, 2009 Sheet 20 of 33 US 2009/0253782 A1
Figure 15
Arginase Actin Patent Application Publication Oct. 8, 2009 Sheet 21 of 33 US 2009/0253782 A1
Figure 16
2-hydroxyxanthoneto Irm2-hydroxy- 9H in xanther- 9- one
2-methoxyxanthone 2-methoxy- 9H xanthen- 9- one
3 3-methyl-1,2- methylcholanthrene dihydrocyclopentaijtetr aphene
4,7- 7-methoxy-2-(4- dimethoxyflavone methoxyphenyl)-4H chotel-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) Patent Application Publication Oct. 8, 2009 Sheet 22 of 33 US 2009/0253782 A1
4'-methoxyflavone 2-(4-methoxyphenyl)- 4H-chromen-4-one (IUPAC),
5,4'- 5-methoxy-2-(4- dimethoxyflavone methoxyphenyl)-4H chronen-4-One (IUPAC)
5,7,4'- 5,7-dimethoxy-2-(4- trimethoxyflavone methoxyphenyl)-4H chromen-4-one (IUPAC)
5,7- 5,7-dimethoxy-3- dimethoxyisoflavon phenyl-4H-chromen-4- e one (IUPAC)
6,3'- 6-methoxy-2-(3- dimethoxyflavone methoxyphenyl)-4H chromen-4-One (IUPAC)
Acacetin diacetate 2-(4-methoxyphenyl)-4- oxo-4H-chromene-5,7- diyl diacetate Patent Application Publication Oct. 8, 2009 Sheet 23 of 33 US 2009/0253782 A1
MOLENAME ar. In Anisindione 2-(4-methoxyphenyl)- 1H-indene-1,3(2H) - dione
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 (common), C. l. natural yellow 1 (common), flavone,4',5,7- trihydroxy- (chemical), NP-OOO448 (extract), pelargidenon 1449 (common), spigenin (common), versulin (common)
Apigenin triacetate 2- (4-acetoxyphenyl)- 4-oxo- 4H- chronene 5,7-diyl diacetate Patent Application Publication Oct. 8, 2009 Sheet 24 of 33 US 2009/0253782 A1
Biochanin a 5,7-diydroxy-3-(4- methoxyphenyl)- 4H chromen- 4- one
Biochanin a 3-(4-methoxyphenyl)-4- diacetate oxo-4H-chromene-5,7- diyl diacetate
Chlorpropham isopropyl (3- chlorophenyl) Carbamate
Chrysophanol 1,8-dihydroxy-3-methyl 9,10-anthraquinone
Patent Application Publication Oct. 8, 2009 Sheet 25 of 33 US 2009/0253782 A1
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-benzopyrian-4- one (chemical), 7 hydroxy-3-(4- hydroxyphenyl)-4H chromen-4-one (IUPAC), 7-hydroxy-3- (4-hydroxyphenyl) chromen-4-one (chemical), daidzein (common, vendor, MeSH, primary common), daidzeol (common)
Dehydrovariabilin 3,9-dimethoxy-6H 1)benzofuro3,2- cchromene
Patent Application Publication Oct. 8, 2009 Sheet 27 of 33 US 2009/0253782 A1
Formononetn 7-hydroxy-3-(4- methoxyphenyl)-4-h- chromen- 4- one
36586 (NSC), 4'5,7- trihydroxyisoflavone (chemical), 4H-1-. benzopyran-4-one, 5,7- dihydroxy-3-(4- hydroxyphenyl)- (chemical), 5,7,4'- trihydroxyisoflavone (chefiltal), 5,7- dihydroxy-3-(4- hydroxyphenyl)-4- benzopyrone (chemical), 5,7- dihydroxy-3-(4- hydroxyphenyl)-4H chromer-4-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)
Patent Application Publication Oct. 8, 2009 Sheet 29 of 33 US 2009/0253782 A1
Lansoprazole 2-((3-methyl-4- (prevacid) (2.2.2- trifluoroethoxy) pyridin-2- yl)methyl)sulfinyl)-1H benzinidazole (IUPAC), 2-(S)-(3- methyl-4-(2,2,2- trifluoroethoxy) pyridin 2-yl)methyl)sulfinyl)- 1H-benzimidazole (IUPAC)
Liquiritigenin (2R)-7-methoxy-2- dimethyl ether (4-methoxyphenyl)- 2,3-dihydro- 4H chromen- 4- one) (note: liquiritigenin is also called 7,4'- dihydroxyflavanone and 5-deoxyflavone Patent Application Publication Oct. 8, 2009 Sheet 30 of 33 US 2009/0253782 A1
MOLENAME
Methoxyvone 7-methoxy-5-methyl-2- phenyl-4H-chromen-4- Cre
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)
Phenindione 2-phenyl-1H-indene 1,302H)-dione (IUPAC)
Pinosylvin 5-(E)-2- phenyvinylbenzene 1,3-diol (IUPAC)
Pinosylvin methyl 3-methoxy-5-(E)-2- ether phenylvinyl)phenol (IUPAC) Patent Application Publication Oct. 8, 2009 Sheet 31 of 33 US 2009/0253782 A1
Piperineow. In1-(2E,4E)-5-(1,3- benzodioxol-5-yl)penta 2,4-dienoyl)piperidine (IUPAC), 1-(5-(1,3- benzodioxol-5-yl)penta 2,4-dienoylpiperidine (IUPAC),
Pramoxine 25573 (NSC), 4-3-(4- hydrochloride butoxyphenoxy) propynorpholine (IUPAC),
Resveratrol 4'- 5-(E)-2-(4- methyl ether methoxyphenyl) vinyl)benzene-1,3-diol
Retusin 7-methyl 8-hydroxy-7-methoxy ether 3-(4-methoxyphenyl)- 4H-chromen-4-One (IUPAC)
Patent Application Publication Oct. 8, 2009 Sheet 33 of 33 US 2009/0253782 A1
Tranilast (aka MK- 2-((2E)-3-(3,4- 341) dimethoxyphenyl)prop 2-enoyl)amino)benzoic acid (IUPAC)
9H-xanthen-9-one (IUPAC) US 2009/0253782 A1 Oct. 8, 2009
COMPOUNDS FOR ENHANCING ARGINASE oxyflavone; 5,4'-dimethoxyflavone; 5,7,4'-trimethoxyfla ACTIVITY AND METHODS OF USE vone; 5,7-dimethoxyisoflavone; 6.3'-dimethoxyflavone: THEREOF Acacetin diacetate; Anisindione: Apigenin; Apigenin triac etate; Biochanina, Biochanin a diacetate; Chlorpropham; Chrysophanol; Daidzein, Dehydrovariabilin: Derrubone; 0001. This application asserts priority to U.S. Provisional Derrusnin; Derrustone; Dibenzoylmethane: Fenbendazole; Application No. 60/807,661, filed on Jul. 18, 2006, which is Formononetn; Genistein; Ginkgetin; Indoprofen; Iprafla hereby incorporated by reference in its entirety. vone: Liquiritigenin dimethyl ether; Methoxyvone; Methyl 0002. The invention was made with funds from New York robustone; Phenazopyridine hydrochloride; Phenindione: State Department of Health, contract number CO19772. New Pinosylvin; Pinosylvin methyl ether; Piperine; Pramoxine York State has certain rights in this invention. hydrochloride: Resveratrol 4'-methyl ether, Retusin 7-methyl ether; Robustone; Spironolactone; Tilorone; Tranilast; or BACKGROUND OF THE INVENTION Xanthone. 0003. It is reported that the adult mammalian central ner 0011. In another aspect, the invention provides a method Vous system (CNS) shows little spontaneous regeneration for enhancing arginase activity in a damaged or injured cell. after injury despite that fact that there are many molecules The method comprises administering to a human in need present which promote nerve and axonal growth. In contrast thereof an effective amount of Lansoprazole. to the CNS, the adult peripheral nervous system (PNS) is 0012. In another aspect, the invention provides a method capable of regenerating to Some extent. for treating a disorder that can be treated by enhancing argi nase activity in a human in need thereof. The method com 0004. It is believed that the lack of regeneration in the CNS prises administering to the human an effective amount of a is caused by the presence of molecules which actively prevent compound that enhances arginase activity, wherein the com or inhibit regeneration. Such molecules include Nogo (an pound is any one of the following: 2-hydroxyXanthone; antigen of the IN-1 antibody), myelin-associated glycopro 2-methoxyxanthone: 3-methylcholanthrene; 4,7-dimethoxy tein, and myelin-oligodendrocyte glycoprotein. flavone; 4'-methoxychalcone; 4'-methoxyflavone; 5,4'- 0005 Arginase is an enzyme that catalyzes the conversion dimethoxyflavone: 5,7,4'-trimethoxyflavone; 5,7- of the amino acid arginine to urea and ornithine. Arginase has dimethoxyisoflavone; 6.3'-dimethoxyflavone; Acacetin been reported to reverse the inhibition of neural regeneration diacetate; Anisindione: Apigenin; Apigenin triacetate; Bio in the central and peripheral nervous system. Thus, enhancing chanina; Biochanin a diacetate; Chlorpropham; Chrysopha arginase activity would be beneficial for reversing the inhibi nol; Daidzein: Dehydrovariabilin: Derrubone; Derrusnin; tion of neural regeneration. Derrustone; Dibenzoylmethane; Fenbendazole; Formonon 0006 Arginase I is a 35- to 38-kDa cytoplasnmic protein etn; Genistein, Ginkgetin; Indoprofen; Ipraflavone; Liquiriti that cleaves arginine into urea and ornithine. Arginine is the genin dimethyl ether, Methoxy vone; Methyl robustone: only Substrate capable of donating the guanidine group nec Phenazopyridine hydrochloride; Phenindione; Pinosylvin; essary for nitric oxide production. Nitric oxide is produced Pinosylvin methyl ether, Piperine: Pramoxine hydrochloride: from arginine by three nitric oxide synthase (NOS) isoforms. Nitric oxide production can be regulated by modulating the Resveratrol 4'-methyl ether; Retusin 7-methyl ether, Robus levels of arginine. Arginase I can limit the pool of arginine tone; Spironolactone; Tilorone; Tranilast; or Xanthone. available for nitric oxide synthase (NOS), thereby influencing 0013. In yet another aspect, the invention provides a the production of nitric oxide. method for treating a disorder that can be treated by enhanc 0007 Neuronal damage can be caused by excess levels of ing arginase activity in a human in need thereof The method nitric oxide (NO). NO is a diffusible neuronal second mes comprises administering to the human an effective amount of senger synthesized in the nervous system by three enzymes: Lansoprazole. neuronal NO synthase, endothelial NO synthase, and induc 0014. In a further aspect, the invention provides a method ible NO synthase. Excess NO generated by NO synthase is promoting regeneration of a neural cell in a human in need associated with various neurodegenerative diseases and con thereof. The method comprises administering to the human ditions, such as multiple Sclerosis, dementia, Huntington's an effective amount of a compound that enhances arginase disease, Alzheimer's disease, etc. activity, wherein the compound is any one of the following: 0008. The amino acid arginine is the only endogenous 2-hydroxyXanthone; 2-methoxyxanthone; 3-methylcholan substrate of NO synthase. It is reported that arginase can threne; 4.7dimethoxyflavone; 4'-methoxychalcone; 4'-meth reduce cell death in the nervous system by competing with oxyflavone; 5,4'-dimethoxyflavone; 5,7,4'-trimethoxyfla NO synthase for their common Substrate, arginine. vone; 5,7-dimethoxyisoflavone; 6.3'-dimethoxyflavone: 0009. Therefore, enhancing arginase activity would be Acacetin diacetate; Anisindione: Apigenin; Apigenin triac beneficial for promoting neural regeneration or reducing neu etate; Biochanina, Biochanin a diacetate; Chlorpropham; ral damage in diseases and conditions associated with neural Chrysophanol; Daidzein, Dehydrovariabilin: Derrubone; damage. Derrusnin; Derrustone; Dibenzoylmethane: Fenbendazole; Formononetn; Genistein; Ginkgetin; Indoprofen; Iprafla vone: Liquiritigenin dimethyl ether, Methoxyvone; Methyl SUMMARY OF THE INVENTION robustone; Phenazopyridine hydrochloride; Phenindione: 0010. The present invention provides a method for Pinosylvin; Pinosylvin methyl ether; Piperine; Pramoxine enhancing arginase activity in a damaged or injured cell. The hydrochloride: Resveratrol 4'-methyl ether; Retusin 7-methyl method comprises administering to a human in need thereof ether, Robustone; Spironolactone; Tilorone; Tranilast; or with an effective amount of one of the following compounds: Xanthone. 2-hydroxyXanthone; 2-methoxyxanthone; 3-methylcholan 0015. In yet a further aspect, the invention provides a threne; 4,7-dimethoxyflavone; 4'-methoxychalcone; 4'-meth method for promoting regeneration of a neural cellina human US 2009/0253782 A1 Oct. 8, 2009 in need thereof. The method comprises administering to the 0030 FIG. 15. Western blot and immunostaining results human an effective amount of Lansoprazole. for arginase I protein. Neurons were treated with, diazein (20 uM), methoxyvone (5 uM) or lanzoprazole (20 uM) for 18 BRIEF DESCRIPTION OF THE FIGURES hours and were then lysed and Subjected to gel electrophore sis, followed by western blotting and immunostaining for 0016 FIG.1. Chemical structures of compounds. Arginase I protein. As a positive control, neurons were treated 0017 FIG. 2. Table listing compounds that upregulated with 1 mM db cAMP. arginase I at least above or near 2-fold. 0031 FIG. 16. The chemical formula, structures, and ref 0018 FIG. 3. Table listing compounds that were tested in erences for a genus of compounds useful in the methods of the quantitative RT-PCR and immunoblot analysis. invention. 0019 FIG. 4. Western blot results that measured level of enhanced arginase 1 protein associated with the compounds DETAILED DESCRIPTION OF THE INVENTION listed in FIG. 3. 0020 FIG. 5. Western blot results that measured level of enhanced arginase 1 protein associated with the compounds Method for Enhancing Arginase Activity listed in FIG. 3. 0032. In one aspect, the invention provides a method for 0021 FIG. 6. Quantitative RT-PCR results presented in a enhancing arginase activity in a damaged or injured cell in a graph showing levels of arginase I messenger RNA (mRNA) human in need thereof. Any isoform of arginase can be upregulation associated with compounds 1-10 listed in FIG. enhanced by the methods of the present invention. Examples 3. of arginase isoforms include arginase I and arginase II. 0022 FIG. 7. Quantitative RT-PCR results presented in a 0033. The method comprises administering to the human graph showing levels of arginase I messenger RNA (MRNA) an effective amount of a member of the genus of compounds upregulation associated with compounds 11-20 listed in FIG. selected from: 2-hydroxyXanthone; 2-methoxyXanthone; 3. 3-methylcholanthrene ; 4,7-dimethoxyflavone; 4'-methoxy 0023 FIG. 8. Quantitative RT-PCR results presented in a chalcone; 4'-methoxyflavone; 5,4'-dimethoxyflavone; 5.7,4'- graph showing levels of arginase I messenger RNA (MRNA) trimethoxyflavone: 5,7-dimethoxyisoflavone; 6,3'- upregulation associated with compounds 21-30 listed in FIG. dimethoxyflavone; Acacetin diacetate; Anisindione; 3. Apigenin; Apigenin triacetate; Biochanin a; Biochanin a 0024 FIG. 9. Quantitative RT-PCR results presented in a diacetate; Chlorprophan; Chrysophanol; Daidzein: Dehydro graph showing levels of arginase I messenger RNA (mRNA) variabilin: Derrubone; Derrusnin; Derrustone; Dibenzoyl upregulation associated with compounds 31-40 listed in FIG. methane; Fenbendazole; Formononetn; Genistein; Ginkge 3 tin, Indoprofen; Ipraflavone; Liquiritigenin dimethyl ether; 0025 FIG. 10. Table summarizing results of compounds Methoxyvone; Methyl robustone; Phenazopyridine hydro tested for their ability to overcome myelin-associated glyco chloride; Phenindione: Pinosylvin; Pinosylvin methyl ether; protein (MAG) inhibition in P7 rat cerebellar neurons, as Piperine; Prarnoxine hydrochloride: Resveratrol 4'-methyl compared with rho kinase inhibitor. The compounds were ether; Retusin 7-methyl ether, Robustone; Spironolactone: administered after (post-treatment) the neurons were plated. Tilorone; Tranilast; or Xanthone. 0026 FIG. 11. Graph showing results from testing the 0034. The terms "enhancing arginase activity” or compounds (10 nM) listed in FIG. 10 for their ability to "enhanced arginase activity” refer to an increased level of overcome myelin-associated glycoprotein (MAG) inhibition measurable arginase activity in a given assay in the presence in P7 rat cerebellar neurons, as compared with rho kinase of a candidate compound relative to the measurable level of inhibitor. arginase activity in the absence of the candidate compound, 0027 FIG. 12. Graph showing results from testing the when tested under the same conditions. compounds (40 nM) listed in FIG. 10 for their ability to 0035 Activity is considered enhanced according to the overcome myelin-associated glycoprotein (MAG) inhibition invention if it is enhanced at least about 10% greater, prefer in P7 rat cerebellar neurons, as compared with rho kinase ably at least about 25% greater, more preferably at least about inhibitor. 50% greater, even more preferably at least about 75% greater, 0028 FIG. 13. Table summarizing results of compounds most preferably at least about 90% greater, or more than in the tested for their ability to overcome myelin-associated glyco absence of the candidate compound. protein (MAG) inhibition in P7 rat cerebellar neurons, as 0036 Arginase activity as used herein can be enhanced by compared with rho kinase inhibitor. The compounds were any mechanism. For example, arginase activity could be administered before (pretreatment) and after (post treatment) enhanced by transcriptional induction of its cognate messen the neurons were plated. ger RNA (mRNA), increased stability of its mRNA, increased 0029 FIG. 14. P5 cerebellar neurons plated on CONT and translation of mRNA into protein, increased stability of argi MAG expressing CHO cells. FIG. 14A-E are pictures repre nase protein, increased arginase activity (in the presence or sentative of BIII tubulin positive cells. Neurons are plated absence of increased protein), or any other mechanism. either with DMSO (0.1%) on control CHO monolayers (FIG. Increases in arginase activity could be realized by the ability 14A), or on substrate inhibitor (MAG) (FIG. 14B-E) and of a compound to increase the affinity of the arginase enzyme treated with DMSO (0.1%) (FIG.14B), methoxyvone (5uM) for its prototypical Substrate, arginine. (FIG. 14C), daidzein (20 uM) and Lanzoprazole (20 uM). 0037. The arginase activity is enhanced in any damaged or Graph in FIG. 14 depicts the average length of the longest injured cell that benefits from enhanced arginase or from a neurite (percentage of the longest neurite of the control). At reduction of nitric oxide or arginine. The damage or injury least 400 neurons were measured in each assay and the may be to any part of a cell. Such as to membranes, DNA, experiment was carried out at least twice. RNA, and ribosomes. US 2009/0253782 A1 Oct. 8, 2009
0038 Examples of cells that may be damaged or injured vone; 5.7,4'-trimethoxyflavone; Acacetin diacetate; include cells of the central nervous system (CNS) or periph Biochanin a; Biochanin a diacetate; Formononetn; and eral nervous system (PNS), including neurons, ganglia, Retusin 7-methyl ether. Schwann cells, astrocytes, oligodendrocytes, microglia cells, 0047. In still another embodiment, the method comprises endothelial cells, immune cells (e.g., macrophages, T cells, B administering to a human in need thereof a compound com cells, and neutrophils), etc. In one embodiment, the damaged prising a 1,3-benzodioxol Selected from a group consisting or injured cell is in a human. of Derrubone; Derrusnin, Derrustone; Methyl robustone: 0039. In one embodiment, the method comprises admin Piperine; and Robustone. istering to a human in need thereofan effective amount of any one or any combination of the following compounds: Pino 0048. In yet a further embodiment, the method comprises Sylvin; Derrustone; Methoxy vone; DehydroVariabilin; or administering to a human in need thereofan effective amount Chrysophanol. of a compound selected from any one or a combination of 0040. In another embodiment, the method comprises compounds listed in FIG. 2 and/or FIG. 3. administering to a human in need thereofan effective amount 0049. In another aspect of the invention, the method com of any one or any combination of the following compounds: prises administering to the human in need thereofan effective Resveratrol 4'-methyl ether, Derrubone; Ginkgetin; or amount of Lansoprazole. Methyl robustone. 0041. In yet another embodiment, the method comprises Method for Treating a Disorder That Can Be Treated by administering to a human in need thereofan effective amount Enhancing Arginase Activity of any one or any combination of the following compounds: 0050. In another aspect, the invention provides a method Tilorone; Phenindione: Pramoxine hydrochloride; Indopro for treating a disorder that can be treated by enhancing argi fen; Phenazopyridine hydrochloride; Piperine; 6.3'- nase activity in a human in need thereof The method includes dimethoxyflavone; Anisindione; 5,4'-dimethoxyflavone: administering to the human an effective amount of a com Pinosylvin; Derrustone; 4,7-dimethoxyflavone; Daidzein; pound that enhances arginase activity, wherein the compound 4'-methoxychalcone; Tranilast; Biochanin a diacetate; Res is any one of the following: 2-hydroxyXanthone, 2-methox Veratrol 4'-methyl ether, Derrubone; Chlorprophan; yxanthone: 3-methylcholanthrene ; 4,7-dimethoxyflavone: Genistein: Dehydrovariabilin; Retusin 7-methyl ether; Xan 4'-methoxychalcone; 4'-methoxyflavone; 5,4'-dimethoxyfla thone; PinoSylvin methyl ether, Chrysophanol: Apigenin: vone; 5,7,4'-trimethoxyflavone; 5,7-dimethoxyisoflavone: 2-methoxyxanthone: Apigenin triacetate: Fenbendazole; 6.3'-dimethoxyflavone; Acacetin diacetate; Anisindione; Dibenzoylmethane; Methoxyvone; Ginkgetin, k salt; Methyl Apigenin; Apigenin triacetate; Biochanin a; Biochanin a robustone; Liquiritigenin dimethyl ether, DerruSnin; Biocha diacetate; Chlorpropham; Chrysophanol; Daidzein: Dehy nina; 5,7-dimethoxyisoflavone; Formononetin; 4'-methoxy drovariabilin; Derrubone; Derrusnin, Derrustone; Diben flavone; or Acacetin diacetate. Zoylmethane: Fenbendazole; Formononetn; Genistein; 0042. In a further embodiment, the method comprises Ginkgetin, Indoprofen; Ipraflavone; Liquiritigenin dimethyl administering to a human in need thereofan effective amount ether; Methoxyvone; Methyl robustone; Phenazopyridine of any one or any combination of the following compounds: hydrochloride; Phenindione; Pinosylvin; Pinosylvin methyl Pinosylvin; Derrustone; Daidzein; 4'-methoxychalcone; Tra ether, Piperine; Pramoxine hydrochloride: Resveratrol 4'-me nilast; Biochanin a diacetate; Resveratrol 4'-methyl ether; thyl ether, Retusin 7-methyl ether; Robustone; Spironolac Dehydrovariabilin: Chrysophanol; or Methoxyvone. tone; Tilorone; Tranilast; or Xanthone. 0043. In yet a further embodiment, the method comprises 0051. In one embodiment, the method comprises admin administering to a human in need thereofan effective amount istering to the human an effective amount of any one or any of any one or any combination of the following compounds: combination of the following compounds: Pinosylvin; Der Daidzein or Methoxy vone. rustone; Methoxyvone; Dehydrovariabilin; or Chrysophanol. 0044. In another embodiment, the method comprises 0052. In another embodiment, the method comprises administering to a human in need thereofan effective amount administering to the human an effective amount of any one or of a compound comprising a 9H-Xanthen-9-one selected from any combination of the following compounds: Resveratrol a group consisting of 2-hydroxyXanthone, 2-methoxyxan 4'-methyl ether; Derrubone; Ginkgetin; or Methyl robustone. thone, and Xanthone. 0053. In yet another embodiment, the method comprises 0045. In yet another embodiment, the method comprises administering to the human an effective amount of any one or administering to a human in need thereofan effective amount any combination of the following compounds: Tilorone; of a compound comprising a 4H-chromen4-one selected from Phenindione; Pramoxine hydrochloride; Indoprofen; a group consisting of 4.7-dimethoxyflavone; 4'-methoxyfla Phenazopyridine hydrochloride; Piperine; 6.3'-dimethoxy vone; 5,4'-dimethoxyflavone; 5,7,4'-trimethoxyflavone; 5,7- flavone; Anisindione; 5,4'-dimethoxyflavone; Pinosylvin; dimethoxyisoflavone; 6.3'-dimethoxyflavone; Acacetin diac Derrustone; 4,7-dimethoxyflavone; Daidzein; 4'-methoxy etate; Apigenin; Apigenin triacetate; Biochanina, Biochanin chalcone; Tranilast; Biochanin a diacetate; Resveratrol a diacetate; Daidzein, Derrubone; Derrustone; Formononetn; 4'-methyl ether, Derrubone; Chlorpropham; Genistein; Genistein; Ginkgetin; Ipraflavone; Liquiritigenin dimethyl Dehydrovariabilin: Retusin 7-methyl ether, Xanthone; Pino ether; Methoxy vone; and Retusin 7-methyl ether. Sylvin methyl ether, Chrysophanol: Apigenin, 2-methoxyx 0046. In a further embodiment, the method comprises anthone; Apigenin triacetate; Fenbendazole; Dibenzoyl administering to a human in need thereofan effective amount methane; Methoxyvone; Ginkgetin, k salt; Methyl robustone; of a compound comprising a (4-methoxyphenyl)4H Liquiritigenin dimethyl ether, Derrusnin; Biochanina; 5,7- chromene-4-one selected from a group consisting of 4.7- dimethoxyisoflavone; Formononetin; 4'-methoxyflavone; or dimethoxyflavone; 4'-methoxyflavone; 5,4'-dimethoxyfla Acacetin diacetate. US 2009/0253782 A1 Oct. 8, 2009
0054. In a further embodiment, the method comprises 0065. Any mammal suffering from ischemia can be administering to the human an effective amount of any one or treated in accordance with the method of the present inven any combination of the following compounds: Pinosylvin; tion. Ischemia generally refers to a condition of decreased 0055 Derrustone; Daidzein; 4'-methoxychalcone; Tra blood flow to an organ, tissue and/or cell. The decrease in nilast; Biochanin a diacetate; Resveratrol 4'-methyl ether, blood flow can be caused by, for example, constriction (e.g., Dehydrovariabilin: Chrysophanol; or Methoxyvone. hypoxemic vasoconstriction) or obstruction (e.g., clot, ath 0056. In yet a further embodiment, the method comprises erosclerotic plaque) of a blood vessel. administering to the human an effective amount of any one or 0066 Ischemia can occur in any cell, organ, and/or tissue. any combination of the following compounds: Daidzein or Examples of cells, organs, and/or tissues which can be Sub Methoxyvone. jected to ischemia include neuronal cells (e.g., neurons, gan 0057. In another embodiment, the method comprises glia, Schwann cells, astrocytes, oligodendrocytes and micro administering to the human an effective amount of a com glia), brain, spinal cord, intestinal cells, kidney cells, heart pound comprising a 9H-Xanthen-9-one selected from a group and cardiac muscle cells such as myocytes, etc. consisting of 2-hydroxyXanthone, 2-methoxyxanthone, and Xanthone. Method for Treating Hypoxia 0058. In yet another embodiment, the method comprises 0067. In yet another embodiment, the invention provides a administering to the human an effective amount of a com method for treating hypoxia in a human in need thereof The pound comprising a 4H-chromen-4one selected from a group method includes administering to the human a compound that consisting of 4.7-dimethoxyflavone; 4'-methoxyflavone; enhances arginase activity as described above, including 5,4'-dimethoxyflavone; 5,7,4'-trimethoxyflavone; 5,7- those compounds grouped in the genera and various Sub dimethoxyisoflavone; 6.3'-dimethoxyflavone; Acacetin diac genera. etate; Apigenin; Apigenin triacetate; Biochanina, Biochanin 0068 Any mammal suffering from hypoxia can be treated a diacetate; Daidzein, Derrubone; Derrustone; Formononetn; in accordance with the method of the present invention. Genistein; Ginkgetin; Ipraflavone; Liquiritigenin dimethyl Hypoxia generally refers to a lack of oxygen to cells, organs, ether; Methoxy vone; and Retusin 7-methyl ether. and/or tissues. Hypoxia can be caused by, for example, 0059. In a further embodiment, the method comprises ischemia, anemia and chemical modification of blood, Such administering to the human an effective amount of a com as carboxyhemoglobin, etc. pound comprising a (4-methoxyphenyl)4H-chromene-4-one 0069. Hypoxia can occur in any cell, organ, and/or tissue. selected from a group consisting of 4,7-dimethoxyflavone; Examples of cells, organs, and/or tissues which can be sub 4'-methoxyflavone: 5,4'-dimethoxyflavone; 5,7,4'-tri jected to hypoxia include neuronal cells (e.g., neurons, gan methoxyflavone; Acacetin diacetate; Biochanina; Biochanin glia, Schwann cells, astrocytes, oligodendrocytes and micro a diacetate; Formononetn; and Retusin 7-methyl ether. glia), brain, spinal cord, kidney cells, intestinal cells, heart 0060. In still another embodiment, the method comprises and cardiac muscle cells such as myocytes, skin cells, etc. administering to the human an effective amount of a com pound comprising a 1,3-benzodioxol Selected from a group Method for Treating Neurodegenerative Disease or Condi consisting of Derrubone; Derrusnin; Derrustone; Methyl tion robustone; Piperine; and Robustone. 0061. In yet a further embodiment, the method comprises 0070. In still another embodiment, the invention provides administering to the human an effective amount of a com a method for treating a neurodegenerative disease or condi pound selected from any one or a combination of compounds tion in a human in need thereof The method includes admin listed in FIG. 2 and/or FIG. 3. istering to the human a compound that enhances arginase 0062. In another aspect of the invention, the method com activity as described above, including those compounds prises administering to the human an effective amount of grouped in the genera and various Sub-genera. Lansoprazole. 0071. A neurodegenerative disease or condition typically 0063 Disorders and diseases in which enhancing arginase refers to a disorder generally characterized by gradual and activity is desired for treatment include ischemia, hypoxia, progressive loss of cells, tissue and/or organ of the central or neurodegenerative disease or condition, or stroke. Additional peripheral nervous system. Examples of Such cells, tissues disorders and diseases in which enhancing arginase activity is and organs include, the brain, spinal cord, neurons, ganglia, desired for treatment traumatic disorders (including but not Schwann cells, astrocytes, oligodendrocytes and microglia. limited to spinal cord injuries, spinal cord lesions, or other 0072 Any mammal suffering from any neurodegenerative CNS pathway lesions), Surgical nerve lesions, damage sec disease or condition can be treated in accordance with the ondary to infarction, infection, exposure to toxic agents, method of the present invention. For example, the neurode malignancy, paraneoplastic syndromes, or patients with vari generative disease or condition can be an acute condition. ous types of neurodegenerative disorders of the central ner Acute conditions generally occur as a result of trauma to a cell, tissue and/or organ of the nervous system. The trauma Vous system. can, for example, partially or completely block blood flow to the cell, tissue and/or organ. Examples of acute neurodegen Method for Treating Ischemia erative conditions include head injury and brain injury. 0064. In one embodiment, the invention provides a 0073. Alternatively, the neurodegenerative disease or con method for treating ischemia in a human in need thereof. The dition can be a chronic neurodegenerative condition. method comprises administering to the human a compound Examples of chronic neurodegenerative diseases and condi that enhances arginase activity as described above, including tions include Parkinson's disease, Alzheimer's disease, Hun those compounds grouped in the various genera and Sub tington's disease and Amyotrophic Lateral Sclerosis (also genera. known as Lou Gherig's disease). US 2009/0253782 A1 Oct. 8, 2009
0074. Additional examples of neurodegenerative disor example, the injury can be a complete injury to the spinal ders and diseases that can be treated by the invention include cord. Complete injury typically refers to the lack of function but are not limited to Alexander disease, Alper's disease, (e.g., no sensation and no voluntary movement) below the site Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxia of injury. Both sides of the body are usually affected. telangiectasia, Batten disease (also known as Spielmeyer I0081 Alternatively, the injury may be an incomplete Vogt-Sjogren-Batten disease), Bovine spongiform encephal injury to the spinal cord. An incomplete injury generally opathy (BSE), Canavan disease, Cockayne syndrome, Corti refers to some function below the site of injury. For instance, cobasal degeneration, Creutzfeldt-Jakob disease, Huntington a person with an incomplete injury may be able to move one disease, HIV-associated dementia, Kennedy's disease, limb more than another, may be able to feel parts of the body Krabbe disease, Lewy body dementia, Machado-Joseph dis that cannot be moved, or may have more functioning on one ease (Spinocerebellar ataxia type 3), Multiple sclerosis, Mul side of the body than the other, etc. tiple System Atrophy, Neuroborreliosis, Parkinson disease, Pelizaeus-Merzbacher Disease, Pick's disease, Primary lat Method for Promoting Regeneration of a Neural Cell in a eral sclerosis, Prion diseases, Refsum's disease, Sandhoff Human in Need Thereof disease, Schilder's disease, Schizophrenia, Spielmeyer-Vogt I0082 In another aspect, the invention provides a method Sjogren-Batten disease (also known as Batten disease), for promoting regeneration of a neural cell in a human in need Spinocerebellar ataxia (multiple types with varying charac thereof As described in Lange, et al., J Nutr. 2004 October teristics), Spinal muscular atrophy, Steele-Richardson ;134(10 Suppl):2812S-2817S: discussion 2818S-2819S, Olszewski disease, Tabes dorsalis, and other dementias. arginase has a role in the axonal regeneration pathway. Argi nase also has neuroprotective properties. Arginase is suffi Method for Treating Stroke cient in protecting neurons against several apoptosis-induc 0075. In a further aspect, the invention provides a method ing stimuli. Moreover, arginase acts as a nitric oxide for treating stroke in a human in need thereof. The method independent inhibitor of neuronal apoptosis. includes administering to the human a compound that I0083. Several investigators have found that molecules or enhances arginase activity as described above, including drugs that prevent injury in the PNS or CNS have no effect on those compounds grouped in the genera and various Sub or worse, negatively impact the ability of the nervous system genera. to regenerate or repair. There is thus an urgent need to identify 0076 Any mammal suffering from stroke can be treated in targets whose activation would provide an environment that is accordance with the method of the present invention. Stroke simultaneously instructive for neuronal protection and repair. is a type of cardiovascular disease that generally involves the It is proposed that Such an intervention would provide greater interruption of blood flow to and/or within the brain. The latitude in the timing of initiation of treatment. interruption of blood flow can be due to, for example, a I0084 Arginine can be metabolized by nitric oxide syn blockage or rupture of an artery or vessel. The blockage thase to produce nitric oxide. It can also be degraded by typically occurs from a blood clot. As a result of the interrup arginase to produce urea and ornithine, which in turn is a tion of blood flow, the brain does not receive sufficient precursor for the synthesis of polyamines. These two path amounts of blood. ways compete for arginine. Arginase thus produces polyamines at the expense of nitric oxide. Method for Treating Trauma of the CNS or PNS I0085 Polyamines have been implicated in neuronal growth and development, axonal regeneration after injury, 0077. In still a further embodiment, the invention provides and in would healing outside of the CNS. Arginase I is a method for treating trauma of the central nervous system upregulated and polyamine synthesis increases in neurons in (CNS) or peripheral nervous system (PNS) in a human in response to either dbCAMP or BDNF. need thereof. The method includes administering to the 0086. By producing polyamines, arginase can overcome human a compound that enhances arginase activity as the effects of myelin-associated glycoprotein (MAG) and described above, including those compounds grouped in the myelin on neurite outgrowth. Arginase is an enzyme that genera and various Sub-genera. mediates repair by reducing its Substrates (L-arginine) and 0078. Any type of trauma to the nervous system may be increasing its ultimate products (polyamines), respectively. treated by the methods of the claimed invention. As described I0087 As toxic levels of nitric oxide (NO) are implicated in above, trauma of the CNS or PNS include, but are not limited acute ischemic cortical injury and motor neuron loss due to to, spinal cord injuries, spinal cord lesions, other CNS path absence of trophic factors, arginase can mediate neuroprotec way lesions, as well as injuries to the PNS, such as injuries to tion. By producing polyamines, arginase can overcome the a nerve or neuron of the PNS and axon damage resulting in effects of myelin-associated glycoprotein (MAG) and myelin demyelination of the PNS. Such trauma can arise from either on neurite outgrowth Arginase is thus a bi-functional enzyme physical injury or disease. Any mammal Suffering from a that mediates neuroprotection or repair by reducing its Sub trauma of the CNS or PNS can be treated in accordance with strates (L-arginine) and increasing its ultimate products the methods of the present invention. (polyamines), respectively. 007.9 For example, spinal cord injury refers to any dam I0088 As used herein, the phrase “neural cell includes age to the spinal cord. The damage typically results in loss of nerve cells (i.e., neurons, e.g., uni-, bi-, or mulipolar neurons) function, Such as mobility or feeling. Damage to the spinal and their precursors and glial cells (e.g., macroglia Such as cord can occur, for example, as a result or trauma (car acci astrocytes, oligodendrocytes, ependymal cells, radial glia, dent, gunshot, falls, etc.) or disease (polio, spina bifida, Frie Schwann cells, Satellite cells, and microglia) and their pre dreich's Ataxia, etc). cursors. Microglia are specialized macrophages capable of 0080. Any injury to the spinal cord can be treated in. phagocytosis that protect neurons of the central nervous sys accordance with the method of the present invention. For tem. The term “precursor refers to cells which are capable of US 2009/0253782 A1 Oct. 8, 2009
developing into a specific cell type. For example, a neural cell 0097. In yet another embodiment, the method comprises precursor is a cell which is capable of developing into a administering to the human an effective amount of a com mature neural cell (i.e., a cell having the characteristic mor pound comprising a 4H-chromen4-one selected from a group phology and function of a neural cell). consisting of 4,7-dimethoxyflavone; 4'-methoxyflavone; 0089. Accordingly, the claimed invention provides meth 5,4'-dimethoxyflavone; 5,7,4'-trimethoxyflavone; 5,7- ods for promoting regeneration of a neural cell in a human in dimethoxyisoflavone; 6.3'-dimethoxyflavone; Acacetin diac need thereof. etate; Apigenin; Apigenin triacetate; Biochanina, Biochanin 0090 The method includes administering to the human an a diacetate; Daidzein, Derrubone; Derrustone; Formononetn; effective amount of a compound that enhances arginase activ Genistein; Ginkgetin; Ipraflavone; Liquiritigenin dimethyl ity, wherein the compound is any one of the following: 2-hy ether; Methoxy vone; and Retusin 7-methyl ether. droxyXanthone; 2-methoxyxanthone... 3-methylcholan 0098. In a further embodiment, the method comprises threne; 4,7-dimethoxyflavone; 4'-methoxychalcone; administering to the human an effective amount of a com 4'-methoxyflavone: 5,4'-dimethoxyflavone; 5,7,4'-tri pound comprising a (4-methoxyphenyl)4H-chromene-4-one methoxyflavone; 5,7-dimethoxyisoflavone; 6.3'-dimethoxy selected from a group consisting of 4,7-dimethoxyflavone; flavone; Acacetin diacetate; Anisindione: Apigenin; Apigenin 4'-methoxyflavone; 5,4'-dimethoxyflavone; 5,7,4'-tri triacetate; Biochanin a Biochanin a diacetate; Chlor methoxyflavone; Acacetin diacetate; Biochanina, Biochanin propham; Chrysophanol; Daidzein: Dehydrovariabilin: Der a diacetate; Formononetn; and Retusin 7-methyl ether. rubone; Derrusnin; Derrustone; Dibenzoylmethane: Fen 0099. In still another embodiment, the method comprises bendazole; Formononetn; Genistein; Ginkgetin, Indoprofen; administering to the human an effective amount of a com Ipraflavone: Liquiritigenin dimethyl ether, Methoxy vone: pound comprising a 1,3-benzodioxol selected from a group Methyl robustone; Phenazopyridine hydrochloride; Phenin consisting of Derrubone; Derrusnin; Derrustone; Methyl dione; Pinosylvin; Pinosylvin methyl ether; Piperine; robustone; Piperine; and Robustone. Pramoxine hydrochloride; Resveratrol 4'-methyl ether; 0100. In yet a further embodiment, the method comprises Retusin 7-methyl ether, Robustone; Spironolactone; Tilor administering to the human an effective amount of a com one; Tranilast; or Xanthone. pound selected from any one or a combination of compounds 0091. In one embodiment, the method comprises admin listed in FIG. 2 and/or FIG. 3. istering to the human an effective amount of any one or any 0101. In another aspect of the invention, the method com combination of the following compounds: Pinosylvin; Der prises administering to the human an effective amount of rustone; Methoxyvone; DehydroVariabilin; or Chrysophanol. Lansoprazole. 0092. In another embodiment, the method comprises administering to the human an effective amount of any one or Methods for Protecting a Neural Cell in a Human in Need any combination of the following compounds: Resveratrol Thereof 4'-methyl ether; Derrubone; Ginkgetin; or Methyl robustone. 0093. In yet another embodiment, the method comprises 0102. In yet another aspect, the invention provides a administering to the human an effective amount of any one or method for protecting a neural cellina human in need thereof. any combination of the following compounds: Tilorone; The method includes administering to the human a compound Phenindione; Pramoxine hydrochloride; Indoprofen; that enhances arginase activity, wherein the compound com Phenazopyridine hydrochloride; Piperine; 6.3'-dimethoxy prises any one of the following compounds: 2-hydroxyXan flavone; Anisindione; 5,4'-dimethoxyflavone; Pinosylvin; thone: 2-methoxyxanthone; 3-methylcholanthrene; 4.7- Derrustone; 4,7-dimethoxyflavone; Daidzein; 4'-methoxy dimethoxyflavone; 4'-methoxychalcone; 4'-methoxyflavone: chalcone; Tranilast; Biochanin a diacetate; Resveratrol 5,4'-methoxyflavone: 5,7,4'-trimethoxyflavone; 5,7- 4'-methyl ether, Derrubone; Chlorpropham; Genistein; dimethoxyisoflavone; 6.3'-dimethoxyflavone; Acacetin diac Dehydrovariabilin: Retusin 7-methyl ether, Xanthone; Pino etate; Anisindione: Apigenin; Apigenin triacetate; Biochanin Sylvin methyl ether; Chrysophanol: Apigenin, 2-methoxyx a; Biochanin a diacetate; Chlorpropham; Chrysophanol: anthone: Apigenin triacetate; Fenbendazole; Dibenzoyl Daidzein: Dehydrovariabilin: Derrubone; Derrusnin; Derrus methane; Methoxyvone; Ginkgetin, k salt; Methyl robustone; tone; Dibenzoylmethane: Fenbendazole; Formononetn; Liquiritigenin dimethyl ether; Derrusnin; Biochanina; 5,7- Genistein; Ginkgetin; Indoprofen; Ipraflavone; Liquiritige dimethoxyisoflavone; Formononetin; 4'-methoxyflavone; or nin dimethyl ether; Methoxyvone; Methyl robustone; Acacetin diacetate. Phenazopyridine hydrochloride; Phenindione; Pinosylvin; 0094. In a further embodiment, the method comprises Pinosylvin methyl ether, Piperine: Pramoxine hydrochloride: administering to the human an effective amount of any one or Resveratrol 4'-methyl ether, Retusin 7-methyl ether; Robus any combination of the following compounds: Pinosylvin; tone; Spironolactone; Tilorone; Tranilast; or Xanthone. Derrustone; Daidzein; 4'-methoxychalcone; Tranilast; Bio 0103) In another aspect of the invention, the method com chanin a diacetate; Resveratrol 4'-methyl ether, Dehydrova prises administering to the human an effective amount of riabilin; Chrysophanol; or Methoxyvone. Lansoprazole. The method includes administering to the 0095. In yet a further embodiment, the method comprises human any one or a combination of compounds that enhances administering to the human a an effective amount of n effec arginase activity included in the genera and Sub-genera of tive amount of any one or any combination of the following compounds described above. compounds: Daidzein or Methoxyvone. Compounds 0096. In another embodiment, the method comprises administering to the human an effective amount of a com 0104 Compounds useful in the methods of the present pound comprising a 9H-Xanthen-9-one selected from a group invention include 2-hydroxyXanthone: 2-methoxyxanthone; consisting of 2-hydroxyXanthone, 2-methoxyxanthone, and 3-methylcholanthrene; 4,7-dimethoxyflavone; 4'-methoxy Xanthone. chalcone; 4'-methoxyflavone; 5,4'-dimethoxyflavone; 5.7,4'- US 2009/0253782 A1 Oct. 8, 2009 trimethoxyflavone: 5,7-dimethoxyisoflavone; 6,3'- conditions suitable for enhancing arginase activity. Suitable dimethoxyflavone; Acacetin diacetate; Anisindione; incubation conditions can be readily determined by those Apigenin; Apigenin triacetate; Biochanin a; Biochanin a skilled in the art. diacetate; Chlorpropham; Chrysophanol; Daidzein: Dehy 0113 Ex vivo methods typically include cells, organs or drovariabilin; Derrubone; Derrusnin, Derrustone; Diben tissues removed from a mammal, such as a human. The cells, Zoylmethane: Fenbendazole; Formononetn; Genistein; organs or tissues can, for example, be incubated with the Ginkgetin, Indoprofen; Ipraflavone; Liquiritigenin dimethyl compound under appropriate conditions. The contacted cells, ether, Methoxyvone; Methyl robustone; Phenazopyridine organs or tissues are normally returned to the donor, placed in hydrochloride; Phenindione; Pinosylvin; Pinosylvin methyl a recipient, or stored for future use. Thus, the compound is ether; Piperine; Pramoxine hydrochloride; Resveratrol generally in a pharmaceutically acceptable carrier. 4'-methyl ether; Retusin 7-methyl ether; Robustone; Spirono 0114. In vivo methods are typically limited to the admin lactone; Tilorone; Tranilast; or Xanthone. istration of a compound, Such as those described above, to a mammal, preferably a human. The compounds useful in the 0105. Another compound useful in the methods of the methods of the present invention are administered to a mam present invention includes Lansoprazole. mal in an amount effective in enhancing arginase activity or 0106 Further examples compounds useful in the methods treating the mammal. The effective amount is determined of the present invention include any one or a combination of during pre-clinical trials and clinical trials by methods famil compounds listed in FIG. 2 and/or FIG. 3. iar to physicians and clinicians. 0107 These compounds are known in the art. The chemi 0.115. An effective amount of a compound useful in the cal formula, structures, and references for a genus of com methods of the present invention, preferably in a pharmaceu pounds described above are shown in FIG. 16. tical composition, may be administered to a mammal in need 0108. The chemical formula, structures, and references for thereof by any of a number of well-known methods for Pinosylvin, Derrustone, Methoxyvone, Dehydrovariabilin, administering pharmaceutical compounds. The compound and Chrysophanol are shown in FIG. 1. may be administered systemically or locally. 0109 The compounds can be in the form of a pharmaceu 0116 For example, the compound may be administered tically acceptable salt The term “pharmaceutically acceptable orally, intravenously, intranasally, intramuscularly, Subcuta salt” refers to a well-tolerated, nontoxic salt prepared from neously, or transdermally. Other routes of administration any basic or acidic compound mentioned above, and an acid include intracerebroventricularly or intrathecally. Intracere or base, respectively The acids may be inorganic or organic broventiculatly refers to administration into the ventricular acids of any one of the compounds mentioned above. system of the brain. Intrathecally refers to administration into Examples of inorganic acids include hydrochloric, hydrobro the space under the arachnoid membrane of the spinal cord. mic, nitric hydroiodic, Sulfuric, and phosphoric acids. Thus intracerebroventricular or intrathecal administration Examples of organic acids include carboxylic and Sulfonic may be preferred for those diseases and conditions which acids. The radical of the organic acids may be aliphatic or affect the organs or tissues of the central nervous system. aromatic. Some examples of organic acids include formic, 0117 The compounds useful in the methods of the inven acetic, phenylacetic, propionic, succinic, glycolic, glucu tion may also be administered to mammals by Sustained ronic, maleic, furoic, glutamic, benzoic, anthranilic, Salicylic, release, as is known in the art. Sustained release administra phenylacetic, mandelic, embonic (pamoic), methanesulfonic, tion is a method of drug delivery to achieve a certain level of ethanesulfonic, panthenoic, benzenesulfonic, Stearic, Sulfa the drug over a particular period of time. The level typically is nilic, alginic, tartaric, citric, gluconic, gulonic, arylsulfonic, measured by serum or plasma concentration. and galacturonic acids. Appropriate organic bases may be 0118. A description of methods for delivering a compound selected, for example, from N,N-dibenzylethylenediamine, by controlled release can be found in international PCT chloroprocaine, choline, diethanolamine, ethylenediamine, Application No. WO 02/083106. The PCT application is meglumine (N-methylglucamine), and procaine. incorporated herein by reference in its entirety. Other con 0110. Throughout this specification, parameters are trolled release agents are described, for example, in U.S. Pat. defined by maximum and minimum amounts. Each minimum Nos. 5,567,439; 6,838,094; 6,863,902; and 6,905,708. The amount can be combined with each maximum amount to controlled release agents and methods for making them in define a range. these patents are incorporated herein by reference. 0119) Any formulation known in the art of pharmacy is Administration suitable for administration of the compounds useful in the methods of the present invention. For oral administration, 0111. The compounds are administered to a human. The liquid or solid formulations may be used. Some examples of compound is administered to the human in an amount effec formulations include tablets, gelatin capsules, pills, troches, tive in achieving its purpose. The effective amount of the elixirs, Suspensions, syrups, wafers, chewing gum and the compound to be administered can be readily determined by like. The compounds can be mixed with a suitable pharma those skilled in the art during pre-clinical trials and clinical ceutical carrier (vehicle) or excipient as understood by prac trials by methods familiar to physicians and clinicians. Typi titioners in the art. Examples of carriers and excipients cal daily doses include approximately 1 mg to 1000 mg. include starch, milk, Sugar, certain types of clay, gelatin, 0112 Any method known to those in the art for contacting lactic acid, Stearic acid or salts thereof, including magnesium a cell, organ or tissue with a compound may be employed. or calcium Stearate, talc, vegetable fats or oils, gums and Suitable methods include invitro, ex vivo, or in vivo methods. glycols. In vitro methods typically include cultured samples. For I0120 For systemic, intracerebroventricular, intrathecal, example, a cell can be placed in a reservoir (e.g., tissue culture topical, intranasal, Subcutaneous, or transdermal administra plate), and incubated with a compound under appropriate tion, formulations of the compounds useful in the methods of US 2009/0253782 A1 Oct. 8, 2009
the present inventions may utilize conventional diluents, car activity. Compounds and their respective Chem ID numbers riers, or excipients etc.. Such as those known in the art to that upregulated arginase 1 above or near 2 fold were identi deliver the compounds. For example, the formulations may fied. See FIG. 2. The protocol that was used to screen for the comprise one or more of the following: a stabilizer, a Surfac Arginase I upregulators is described in detail below. tant, preferably a nonionic Surfactant, and optionally a salt and/or a buffering agent. The compound may be delivered in Materials the form of an aqueous solution, or in a lyophilized form. I0127. T75 flask, Corning 430641 vented 0121 The stabilizer may, for example, be an amino acid, I0128 96 well plate (for tissue culture), Mictotest Primaria, Such as for instance, glycine; or an oligosaccharide, such as flat bottom, #35-3872 for example. Sucrose, tetralose, lactose or a dextran. Alterna I0129 96 well plate (for chemical compound), MictotestTM tively, the stabilizer may be a Sugar alcohol. Such as for 96, flat bottom, #35-3072 instance, mannitol; or a combination thereof Preferably the stabilizer or combination of stabilizers constitutes from about I0130. Dulbecco's Phosphate Buffered Saline (1xPBS), 0.1% to about 10% weight for weight of the compound. The from Gibco #14190-144 Surfactant is preferably a nonionic Surfactant, such as a I0131 0.25% Trypsin-EDTA solution, from Sigma polysorbate. Some examples of Suitable surfactants include HT4049 Tween20, Tween80; a polyethylene glycol or a polyoxyeth 0.132. DMEM (Dulbecco's Modified Eagle's Medium), ylene polyoxypropylene glycol, such as Pluronic F-68 at from from Gibco H1 1995-040 & H 11965-092 about 0.001% (w/v) to about 10% (w/v). I0133) Fetal Bovine Serum (FCS), from Gibco #10082-147 0122) The salt or buffering agent may be any salt or buff I0134) Penicillin 10,000 IU/ml & Streptomycin 10,000 ering agent, such as for example, sodium chloride, or Sodium/ ug/ml (P/S) from Cellgro #30-002-C1 potassium phosphate, respectively. Preferably, the buffering 0.135 Puromycin 1 mg/ml stock agent maintains the pH of the pharmaceutical composition in 0.136 10 mM stock of chemical compounds in DMSO, the range of about 5.5 to about 7.5. The salt and/or buffering The Spectrum CollectionTM from MicroSource Discovery agent is also useful to maintain the osmolality at a level System. Inc.; stored at -80°C. suitable for administration to a human or an animal. Prefer I0137 Lysis Reagent (5x), from Promega #E153A ably the salt or buffering agent is presentata roughly isotonic 0.138 Luciferase Assay Substrate, from Promega i E151A concentration of about 150 mM to about 300 mM. I0139 Luciferase Assay Buffer, from Promega #E 152A 0123. The formulations of the compounds useful in the methods of the present invention may additionally contain 0140 LimaxII from Molecular Device one or more conventional additive. Some examples of Such 0141 70% Ethanol additives include a solubilizer Such as, for example, glycerol; 0142 96-well NUNC 236107 white plate (Luciferase an antioxidant Such as for example, benzalkonium chloride (a Assay) mixture of quaternary ammonium compounds, known as 0143 UV Plate for plate reader (Protein Assay) “quats'), benzyl alcohol, chloretone or chlorobutanol; anaes thetic agent Such as for example a morphine derivative; or an Reagents isotonic agent etc.. Such as described above. As a further precaution against oxidation or other spoilage, the pharma 0144) ceutical compositions may be stored under nitrogen gas in vials sealed with impermeable stoppers. Ingredient Stock Final conc. Volume EXAMPLES DMEM for HT22 - 4.8Kb Arg-Puro Example 1 DMEM (#11965-092) 445 ml Screening for Arginase I Upregulators FCS 10% 50 ml PS 3 ml 0.124. A 2000 compound library was tested to identify Puromycin 1 mg/ml 4 g/ml 2 ml arginase-1 upregulators. The library tested was The Spectrum Total 500 ml CollectionTM from MicroSource Discovery System, Inc. Lysis Reagent (10 ml per plate) (Groton, Conn.). The 2000 compounds in the library are primarily Food and Drug Administration (FDA)-approved Lysis Reagent Sx 1X 8 m compounds or natural products. An alphabetical list of the ddH2O 32 m compounds is available at the MicroSource Discovery web site at www.msdiscovery.com/spect.html. The compounds Total 40 ml are supplied as 10 mM solutions in dimethyl sulfoxide (DMSO). Day 1. Harvest cell and seeding 0.125. The library was screened using murine hippocampal HT22 cells transfected with a luciferase-arginase I construct Materials: on 96 well plates. Total protein was also measured to use in normalization. The luciferase assay result for each compound (0145 is normalized to protein content and is expressed as luciferasefug protein. The fold increase was measured rela tive to untreated control, represented as sample #1 in each 12x 96 well plates (#35-3872) Two types of DMEM medium HT22 Arg plate. 8x T75 Flasks Trypsin solution 0126 Ratios of the results from the “luciferase assay” and 2x Glass sterile pipette Timer “protein assay” were tabulated. A comparison of these nor Sterile pipette (25 ml, 10 ml, 5 ml) malized responses indicates amount of enhanced arginase US 2009/0253782 A1 Oct. 8, 2009
(0167 7. Use repeat pipette to add 2 ul/well 2.5 mMcAMP -continued stock (positive control—fic. 50 uM DFO) to columni 12 for all plates; columni 1 is negative control (Blank). 6x 50 ml centrifuge tubes Repeat pipette & tips (0168 8. Label and incubate at 37° C. incubator for 24 hours 0169. 9. Wrap and store two secondary stock plates at 4 Steps C. refrigerator 0170 10. Leave original chemical plates at basement-80° 0146 1. Remove old medium from T4s flask by suction C. freezer 0147 2. Rinse once with 5 ml 1xPBS: discard 1XPBS 0148 3. Add3 ml Trypsin to treat cells and incubate at 37° Day 3. Lysis and Read C. incubator for 3 min 0149 4. Observe under microscope to see if all cells Materials: detached 0150 5.Stop trypsin reaction by adding 10 ml DMEM and 0171 then transfer the solution into a 50 ml centrifuge tube 0151. 6. Centrifuge at 980 rpm/4 min 0152 7. Prepare three T75 by adding 10 ml fresh medium 2x Glass sterile pipette 1x Lysis Reagent (10 ml for each plate) to each flask 12x 96-well NUNC white plate Luciferase Assay Substrate & Buffer 12x 96-well UV plate (1 set per plate) 0153 8. Prepare two 50 ml centrifuge tubes by adding 30 10 ultips (24x regular) Repeat pipette & tips ml fresh medium to each tube Timer 0154) 9. Discard supernatant by suction and add 8ml fresh DMEM into the tube to resuspend the cell pellet 0155 10. Mix and Titrate cell suspension and add 2 ml cell suspension into three T75 flasks (total 12 ml) and 1 ml to each Steps 50 ml centrifuge tube. 0172 1. After 24 hrs incubation, remove all medium by 0156 11. Use repeat pipette and sterile tips to dispense suction and add 100 ul/well 1xLysis Reagent
0193 11. Save and calculate data polyacrylamide gels. Proteins were transferred to a nitrocel lulose membrane (Bio-Rad) by electroblotting. Nonspecific binding was inhibited by incubation in Tris-buffered saline Chemical plate 1 (duplicate) with Tween 20 (TBST: 50 mM Tris-HCl, pH 8.0, 0.9% NaCl, Chemical plate 2 (duplicate) and 0.1% Tween 20) containing 5% nonfat milk for at least 1.5h. Primary antibodies against arginase, a-tubulin (Sigma), Chemical plate 3 (duplicate) were diluted in TBST containing 5% milk overnight at 4°C. followed by incubation with respective horseradish peroxi dase-conjugated secondary antibodies (Bio-Rad) for 2 hours Example 2 at room temperature. Immunoreactive proteins were detected according to the enhanced chemiluminescent protocol (Am Quantitative RT-PCR and Immunoblot Analysis ersham Biosciences, Sunnyvale, Calif.). See FIGS. 4 and 5. 0194 In another analysis, 40 compounds were assayed in HT22 cells on a custom plate ordered from MicroSource Example 3 Discovery System, Inc. (Groton, Conn.). (0195 FIG. 3 lists the forty compounds that were tested, MAG Inhibition Analysis; Comparison with Rho along with their Microsource Discovery System, Inc. Kinase Inhibitor ChemID numbers, their respective plate locations, and their “compound number for the purpose of these experiments. 0201 MAG is a well-characterized protein of the central An “X” mark in FIG. 3 indicates whether the compound nervous system (CNS) and the peripheral nervous system enhanced arginase activity in the respective assay. (PNS). MAO has been identified as an inhibitor of axonal (0196. Western blots were performed to measure the level regeneration and neurite outgrowth. A consequence of of enhanced arginase activity, i.e., arginase I expression, and elevated cAMP is the synthesis of polyamines, resulting from MRNA levels of arginase I. In addition, B-actin messages an up-regulation of Arginase I, a key enzyme in their synthe were measured. Western blot results were repeated, and the sis. Inhibiting polyamine synthesis blocks the cAMP effect results of the two experiments are shown in FIGS. 4 and 5. on regeneration. Either over-expression of Arginase I or exog The sample numbers listed on the top of each blot of FIGS. 4 enous polyamines can overcome inhibition by MAG and by and 5 correspond to the sample number and compound listed myelin in general, as described by Cai, et al., Neuron. 2002 in FIG, 3. Aug. 15:35(4):711-9. (0197). In FIGS. 6 through9, the levels of Arginase I mes 0202 Compounds were tested for their ability to over senger RNA (mRNA) upregulated by each of the forty com come myelin-associated glycoprotein (MAG) inhibition in pounds, normalized to B-actin, are presented. A comparison P7 rat cerebellar neurons. of the levels of Arginase IMRNA upregulated by each of the 0203. In experiments performed in the Roman Giger labo forty compounds normalized to actin indicates amount of ratory at the University of Rochester School of Medicine and enhanced arginase activity. Phosphate buffered saline (PBS) Dentistry, twelve compounds were tested, using the materials was used as a negative control, and cAMP was used as a and methods described by Venkatesh, et al., J Neurosci 2005 positive control. The sample numbers (e.g., c1, c2) listed on Jan. 26:25(4):808-22. The compounds and their respective the bottom of each graph of FIGS. 6 through 9 correspond to post-treatment results are listed in FIG. 10. the sample number and compound listed in FIG. 3. 0204 The twelve compounds tested were Acetaminphen; (0198 The protocols used for the quantitative RT-PCR and Pinosylvin; Resveratol 4-methyl ether, Chrysophanol (low Immunoblot analyses are described in detail below. dose); Daidzein; Anisomycin; Methoxy vone; DehydroVari (0199 Quantitative RT-PCR'Total RNA was prepared abilin; Phenethyl caffeate (cape); Fenbendazole; Derrustone: from primary mixed cortical neurons using TriZOL (Invitro Epicatechin pentaacetate; Lansoprazole. gen) and cDNA generating using a SuperScript III First 0205 The twelve compounds were tested in two different Strand Synthesis System for RT-PCR kit (Invitrogen), dilutions (10 nM and 40 nM final concentration). FIG. 11 according to the manufacturer's protocol. Real time PCRs shows the assay results for the compounds at 10 nM and FIG. were performed as a duplex reaction using arginase gene 12 shows the assay results for the compounds at 40 nM. The expression assay which uses a FAM-labeled probe, and P-ac compound sample numbers listed on the bottom of FIGS. 11 tin gene expression assay which uses a VIC-labeled probe and 12 correspond to the compound sample numbers listed in (Applied Biosystems, Foster City, Calif.) so that arginase FIG 10. amplification could be normalized to B-actin. Real time PCRs (0206 MAG-overexpressing CHO cells and control CHO were performed using a 7500 Real Time PCR System (Ap feeder layers were used in the assay. All experiments were plied Biosystems) using standard PCR protocol and amplifi done with Percoll purified P7 rat cerebellar neurons. The cation conditions. See FIGS. 6 through 9. twelve compounds were tested in a “post-treatment manner. 0200 Immunoblot Analysis Cell lysates were obtained Post-treatment refers to administering the compounds after by rinsing neurons with cold PBS followed by lysis in NP40 the neurons were plated. lysis buffer (Boston Bioproducts, Worcester, Mass.). Protein 0207. After 24 h in culture, cells were fixed and stained concentrations in lysates were quantified by Bradford assay with Tulj 1 (cells with neurites longer than 1 cell body diam (Bio-Rad, Hercules, Calif.). Nuclear and cytoplasmic protein eter were quantified). Untreated cells (first column of FIGS. extractions were obtained using NE-PERNuclear and Cyto 11 and 12) and DMSO only (second column of FIGS. 11 and plasmic Extraction Reagents (Pierce Biotechnology, Rock 12) were used as a negative controls. RHO-kinase inhibitor ford, Ill.) according to the manufacturer's protocol. Samples Y27632 (15 microM) (third column of FIGS. 11 and 12) was were boiled in Laemmli buffer and electrophoresed under used as a positive control. All experiments have been repeated reducing conditions on 12% (or 7.5% for pRb immunoblots) 3-5 times independently. US 2009/0253782 A1 Oct. 8, 2009
0208. In FIGS. 11 and 12, there are two bars for each 0218. Next, the compounds were tested for their ability to compound tested. One bar represents the results on the CHO overcome inhibition by MAG when the neurons were MAG cells (labeled “MAG”), and the other bar (labeled “r2) exposed to the compounds prior to being exposed to MAG-a is the result on control CHO cells. procedure termed, “priming.” Neurons were primed with the 0209. The following compounds were found to have no individual compounds at various concentrations ranging effect on releasing MAG inhibition: #1 (Acetaminphen), #3 from 2-20 uM overnight and were then plated onto either the (Resveratol 4-methyl ether), #5 (Daidzein), #7 (Methox MAG-expressing or control CHO cell monolayers. Of the 12 yvone), #9 CAPE, #1 (Derrustone), and #12 (epicatechin compounds tested, three were able to overcome inhibition by pentaacetate). MAG completely: Daidzein (20 uM), Lanzoprazole (20 uM) 0210. The experiment was repeated three times with the and Methoxyvone (5uM). See FIGS. 13 and 14. most promising compounds, which were compound #6 (Ani 0219. In FIG. 14, P5 cerebellar neurons were plated on somycin), #8 (DehydroVariabilin), #10 (Fenbendazole), #2 CONT and MAG expressing CHO cells. See FIG. 14A-E, (Pinosylvin) and #4 (Chrysophanol at low doses). which are pictures representative offIII tubulin positive cells. Neurons are plated either with DMSO (0.1%) on control Example 4 CHO monolayers (FIG. 14A), or on substrate inhibitor (MAG) (FIGS. 14B-E) and treated with DMSO (0.1%) (FIG. MAG Inhibition Analysis 14B), methoxy vone (5uM) (FIG.14C), daidzein (20M) and 0211 MAG has been identified as an inhibitor of axonal Lanzoprazole (20 uM). Graph in FIG. 14 depicts the average regeneration and neurite outgrowth. A consequence of length of the longest neurite (percentage of the longest neurite elevated cAMP is the synthesis of polyamines, resulting from of the control). At least 400 neurons were measured in each an up-regulation of Arginase I, a key enzyme in their synthe assay and the experiment was carried out at least twice. sis. Inhibiting polyamine synthesis blocks the cAMP effect 0220. The other compounds did not show as strong of an on regeneration. Either over-expression of Arginase I or exog effect in overcoming MAG inhibition as the compounds enous polyamines can overcome inhibition by MAG and by Daidzein (20 uM), Lanzoprazole (20 uM) and Methoxyvone myelin in general, as described by Cai, et al., Neuron. 2002 (5 uM). See FIG. 13. The overcoming of MAG inhibition Aug. 15:35(4):71 1-9. occurred when the cells were “preconditioned.” Precondi 0212 Compounds were tested for their ability to over tioning treatment means that neurons were first treated over come myelin-associated glycoprotein (MAG) inhibition in night with the compound and then trypsinized, plated, and primary rat neurons. allowed to grow for neurite extension for 16-18h on the top of 0213 Inhibiting polyamine synthesis blocks the cAMP the CHO monolayers. Neurons that were directly treated with effect on regeneration. Either over-expression of Arginase I or the different compounds on the top of the monolayers did not exogenous polyamines can overcome inhibition by MAG and show any overcoming of MAG inhibition. by myelin in general. 0221. In a second experiment, the twelve compounds were 0214. In experiments performed in the Marie Filbin labo tested for their ability to upregulate arginase I protein. Neu ratory at Hunter College on p5 rat cerebellar neurons, twelve rons were treated with, diazein (20 uM), methoxyvone (5uM) or lanzoprazole (20 uM) for 18 hours and were then lysed and compounds were tested, using the materials and methods subjected to gel electrophoresis, followed by western blotting described by Mukhopadhyay, et a, Neuron. 1994 Sep: 13(3): and immunostaining for Arginase I protein. See FIG. 15. As a 757-67. The compounds and their respective post-treatment positive control, neurons were treated with 1 mM db cAMP results are listed in FIG. 13. (FIG. 15). Db-cAMP was previously shown to increased 0215. The twelve compounds were Acetaminphen; Pino Arginase I protein 3-fold as described by Cai, et a, Neuron. Sylvin; Resveratol 4-methyl ether: Chrysophanol (low dose): 2002 Aug. 15:35(4):71 1-9. Daidzein; Anisomycin; Methoxy vone; DehydroVariabilin; 0222. As was shown previously (Cai et al., 2002), Argin Phenethyl caffeate (cape); Fenbendazole; Derrustone: Epi ase I is up-regulated in response to treatment with dbcAMP. catechin pentaacetate; Lansoprazole. Two experiments were As shown presently, Arginase I protein level is also up-regu done. lated after the treatment with diadzein (20 uM), methoxyvone 0216. In a first experiment, the twelve compounds were (5uM) or Lanzoprazole (20 uM). tested for their ability to overcome inhibition by MAG in culture. The compounds were tested for their ability to inhibit What is claimed is: neurite outgrowth of MAG-expressing CHO cells. Cerebellar 1. A method for enhancing arginase activity in a damaged neurons were cultured on MAG-expressing CHO cells and or injured cell in a human in need thereof, the method com neurite length is compared to neurons growing on control prising administering to the human an effective amount of one of the following compounds: 2-hydroxyXanthone; CHO cells, not expressing MAG. 2-methoxyxanthone: 3-methylcholanthrene; 4,7-dimethoxy 0217. The cells were assessed for their ability to overcome flavone; 4'-methoxychalcone; 4'-methoxyflavone; 5,4'- MAG inhibition when added directly to the cultures at a range dimethoxyflavone: 5,7,4'-trimethoxyflavone; 5,7- of concentrations from 2 - 20 uM. Neurons were plated at a dimethoxyisoflavone; 6.3'-dimethoxyflavone; Acacetin density of about 10,000 neurons per well of an 8-well culture diacetate; Anisindione: Apigenin; Apigenin triacetate; Bio dish containing a monolayer of either MAG-expressing or chanina, Biochanin a diacetate; Chlorpropham; Chrysopha control CHO cells. The co-cultures were, incubated for 16-18 nol; Daidzein: Dehydrovariabilin: Derrubone; Derrusnin; hand then immunostained for BIII tubulin. See FIG. 14 A-E. Derrustone; Dibenzoylmethane; Fenbendazole; Formonon Neurite outgrowth was quantified as previously described etn; Genistein, Ginkgetin; Indoprofen; Ipraflavone; Liquiriti (Mukhopadhyay et al., 1994). When added directly to the genin dimethyl ether; Methoxy vone; Methyl robustone: co-cultures, none of the compounds had any effect on inhibi Phenazopyridine hydrochloride; Phenindione; Pinosylvin; tion by MAG. MAG inhibited neurite outgrowth as potently Pinosylvin methyl ether, Piperine: Pramoxine hydrochloride: as when the compound was absent Resveratrol 4'-methyl ether, Retusin 7-methyl ether; Robus US 2009/0253782 A1 Oct. 8, 2009
tone; Spironolactone; Tilorone: Tranilast; or Xanthone; or a Alexander disease, Alper's disease, Alzheimer's disease, pharmaceutically acceptable salt of any such compound. Amyotrophic lateral Sclerosis, Ataxia telangiectasia, Batten 2. A method according to claim 1, wherein the compound disease (also known as Spielmeyer-Vogt-Sjogren-Batten dis is Pinosylvin; Derrustone; Methoxyvone; DehydroVariabilin; ease), Bovine spongiform encephalopathy (BSE), Canavan or Chrysophanol; or a pharmaceutically acceptable salt of any disease, Cockayne syndrome, Corticobasal degeneration, Such compound. Creutzfeldt-Jakob disease, Huntington disease, HIV-associ 3. A method according to claim 1, wherein the compound ated dementia, Kennedy's disease, Krabbe disease, Lewy comprises a 9H-Xanthen-9-one selected from a group consist body dementia, Machado-Joseph disease (Spinocerebellar ing of 2-hydroxyXanthone, 2-methoxyxanthone, and Xan ataxia type 3), Multiple sclerosis, Multiple System Atrophy, thone; or a pharmaceutically acceptable salt of any such com Neuroborreliosis, Parkinson disease, Pelizaeus-Merzbacher pound. Disease, Pick's disease, Primary lateral sclerosis, Prion dis 4. A method according to claim 1, wherein the compound eases, Refsum's disease, Sandhoff disease, Schilder's dis comprises a 4H-chromen-4-one selected from a group con ease, Schizophrenia, Spielmeyer-Vogt-Sjogren-Batten dis sisting of 4,7-dimethoxyflavone; 4'-methoxyflavone; 5,4'- ease (also known as Batten disease), Spinocerebellar ataxia dimethoxyflavone: 5,7,4'-trimethoxyflavone; 5,7- (multiple types with varying characteristics), Spinal muscu dimethoxyisoflavone; 6.3'-dimethoxyflavone; Acacetin laratrophy, Steele-Richardson-Olszewski disease, Tabes dor diacetate; Apigenin; Apigenin triacetate; Biochanina, Bio salis, or other dimentias. chanin a diacetate; Daidzein; Derrubone; Derrustone; For 13. A method according to claim 8, wherein the disorder is mononetn; Genistein, Ginkgetin; Ipraflavone; Liquiritigenin stroke. dimethyl ether, Methoxyvone; and Retusin 7-methyl ether, or 14. A method according to claim 8, wherein the disorder is a pharmaceutically acceptable salt of any such compound. trauma to the central nervous system or peripheral nervous 5. A method according to claim 4, wherein the compound system. comprises a (4-methoxyphenyl)-4H-chromene-4-one 15. A method according to claim 8, wherein the compound selected from a group consisting of 4.7-dimethoxyflavone; is Pinosylvin; Derrustone; Methoxyvone; DehydroVariabilin; 4'-methoxyflavone: 5,4'-dimethoxyflavone; 5,7,4'-tri or Chrysophanol, or a pharmaceutically acceptable Salt of any methoxyflavone; Acacetin diacetate; Biochanina; Biochanin Such compound. a diacetate; Formononetn; and Retusin 7-methyl ether; or a 16. A method for treating a disorder that can be treated by pharmaceutically acceptable salt of any such compound. enhancing arginase activity in a human in need thereof, the 6. A method according to claim 1, wherein the compound method comprising administering to the human an effective comprises a 1,3-benzodioxol Selected from a group consist amount of Lansoprazole; or a pharmaceutically acceptable ing of Derrubone; Derrusnin, Derrustone; Methyl robustone: Piperine; and Robustone; or a pharmaceutically acceptable salt of Such compound. salt of any Such compound. 17. A method for promoting regeneration of a neural cell in 7. A method for enhancing arginase activity in a damaged a human in need thereof, the method comprising administer or injured cell in a human in need thereof, the method com ing to the human an effective amount of a compound that prising administering to the human an effective amount of enhances arginase activity, wherein the compound is any one Lansoprazole; or a pharmaceutically acceptable salt of Such of the following: 2-hydroxyXanthone; 2-methoxyxanthone; compound. 3-methylcholanthrene ; 4,7-dimethoxyflavone; 4'-methoxy 8. A method for treating a disorder that can be treated by chalcone; 4'-methoxyflavone; 5,4'-dimethoxyflavone; 5.7,4'- enhancing arginase activity in a human in need thereof, the trimethoxyflavone: 5,7-dimethoxyisoflavone; 6,3'- method comprising administering to the human an effective dimethoxyflavone; Acacetin diacetate; Anisindione; amount of a compound that enhances arginase activity, Apigenin; Apigenin triacetate; Biochanin a; Biochanin a wherein the compound is any one of the following: 2-hydrox diacetate; Chlorpropham; Chrysophanol; Daidzein: Dehy yXanthone; 2-methoxyxanthone; 3-methylcholanthrene; 4.7- drovariabilin; Derrubone; Derrusnin, Derrustone; Diben dimethoxyflavone; 4'-methoxychalcone; 4'-methoxyflavone: Zoylmethane: Fenbendazole; Formononetn; Genistein; 5,4'-dimethoxyflavone; 7,4'-trimethoxyflavone; 5.7-meth Ginkgetin, Indoprofen; Ipraflavone; Liquiritigenin dimethyl oxyisoflavone; 6.3'-dimethoxyflavone: Acacetin diacetate; ether; Methoxyvone; Methyl robustone; Phenazopyridine Anisindione: Apigenin; Apigenin triacetate; Biochanin a; hydrochloride; Phenindione; Pinosylvin; Pinosylvin methyl Biochanin a diacetate; Chlorpropham; Chrysophanol; Daid ether, Piperine; Pramoxine hydrochloride: Resveratrol 4'-me Zein: Dehydrovariabilin: Derrubone; Derrusnin; Derrustone: thyl ether; Retusin 7-methyl ether; Robustone; Spironolac Dibenzoylmethane: Fenbendazole; Formononetn; Genistein; tone; Tilorone; Tranilast; or Xanthone; or a pharmaceutically Ginkgetin, Indoprofen; Ipraflavone; Liquiritigenin dimethyl acceptable salt of any Such compound. ether; Methoxyvone; Methyl robustone; Phenazopyridine 18. A method according to claim 17, wherein the com hydrochloride; Phenindione; Pinosylvin; Pinosylvin methyl pound is Pinosylvin; Derrustone; Methoxyvone; Dehydrova ether, Piperine; Pramoxine hydrochloride: Resveratrol 4'-me riabilin; or Chrysophanol; or a pharmaceutically acceptable thyl ether, Retusin 7-methyl ether, Robustone; Spironolac salt of any Such compound. tone; Tilorone; Tranilast; or Xanthone; or a pharmaceutically 19. A method for promoting regeneration of a neural cell in acceptable salt of any such compound. a human in need thereof, the method comprising administer 9. A method according to claim 8, wherein the disorder is ing to the human an effective amount of Lansoprazole; or a ischemia. pharmaceutically acceptable salt of Such compound. 10. A method according to claim 8, wherein the disorder is 20. A method according to claim 19, wherein the neural cell hypoxia is a neuron. 11. A method according to claim 8, wherein the disorder is 21. A method according to claim 19, wherein the neural cell a neurodegenerative disease or condition. is a glial cell. 12. A method according to claim 11, wherein the neurode generative disease or condition is any one of the following: