U.S. patent application number 12/309500 was filed with the patent office on 2009-10-08 for compounds for enhancing arginase activity and methods of use thereof.
Invention is credited to Marie T. Filbin, Rajiv R. Ratan.
Application Number | 20090253782 12/309500 |
Document ID | / |
Family ID | 38957361 |
Filed Date | 2009-10-08 |
United States Patent
Application |
20090253782 |
Kind Code |
A1 |
Ratan; Rajiv R. ; et
al. |
October 8, 2009 |
Compounds for Enhancing Arginase Activity and Methods of USe
Thereof
Abstract
The present invention relates to a method for enhancing arginase
activity in a damaged or injured cell. In other aspects, the
invention provides a method for treating a disorder that can be
treated by enhancing arginase activity in a human in need thereof,
the method comprising administering to the human an effective
amount of a compound that enhances arginase activity. Such
disorders include ischemia, hypoxia, neurodegenerative disease or
condition, stroke or trauma of the nervous system. In yet another
aspect, the invention provides methods for promoting regeneration
of a neural cell in a human in need thereof.
Inventors: |
Ratan; Rajiv R.; (Scarsdale,
NY) ; Filbin; Marie T.; (New York, NY) |
Correspondence
Address: |
Irving N. Feit;Hoffmann & Baron, LLP
6900 Jericho Turnpike
Syosset
NY
11791
US
|
Family ID: |
38957361 |
Appl. No.: |
12/309500 |
Filed: |
July 18, 2007 |
PCT Filed: |
July 18, 2007 |
PCT NO: |
PCT/US2007/016335 |
371 Date: |
June 23, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60807661 |
Jul 18, 2006 |
|
|
|
Current U.S.
Class: |
514/454 |
Current CPC
Class: |
A61K 31/122 20130101;
A61P 25/28 20180101; A61K 31/085 20130101; A61K 31/05 20130101;
A61K 31/4439 20130101; A61K 31/4184 20130101; A61K 31/7048
20130101; A61K 31/352 20130101; A61K 31/36 20130101 |
Class at
Publication: |
514/454 |
International
Class: |
A61K 31/352 20060101
A61K031/352; A61P 25/28 20060101 A61P025/28 |
Claims
1. A method for enhancing arginase activity in a damaged or injured
cell in a human in need thereof, the method comprising
administering to the human an effective amount of one of the
following compounds: 2-hydroxyxanthone; 2-methoxyxanthone;
3-methylcholanthrene; 4,7-dimethoxyflavone; 4'-methoxychalcone;
4'-methoxyflavone; 5,4'-dimethoxyflavone; 5,7,4'-trimethoxyflavone;
5,7-dimethoxyisoflavone; 6,3'-dimethoxyflavone; Acacetin diacetate;
Anisindione; Apigenin; Apigenin triacetate; Biochanin a; Biochanin
a diacetate; Chlorpropham; Chrysophanol; Daidzein;
Dehydrovariabilin; Derrubone; Derrusnin; Derrustone;
Dibenzoylmethane; Fenbendazole; Formononetn; Genistein; Ginkgetin;
Indoprofen; Ipraflavone; Liquiritigenin dimethyl ether;
Methoxyvone; Methyl robustone; Phenazopyridine hydrochloride;
Phenindione; Pinosylvin; Pinosylvin methyl ether; Piperine;
Pramoxine hydrochloride; Resveratrol 4'-methyl ether, Retusin
7-methyl ether; Robustone; Spironolactone; Tilorone; Tranilast; or
Xanthone; or a pharmaceutically acceptable salt of any such
compound.
2. A method according to claim 1, wherein the compound is
Pinosylvin; Derrustone; Methoxyvone; Dehydrovariabilin; or
Chrysophanol; or a pharmaceutically acceptable salt of any such
compound.
3. A method according to claim 1, wherein the compound comprises a
9H-xanthen-9-one selected from a group consisting of:
2-hydroxyxanthone, 2-methoxyxanthone, and xanthone; or a
pharmaceutically acceptable salt of any such compound.
4. A method according to claim 1, wherein the compound comprises a
4H-chromen-4-one selected from a group consisting of:
4,7-dimethoxyflavone; 4'-methoxyflavone; 5,4'-dimethoxyflavone;
5,7,4'-trimethoxyflavone; 5,7-dimethoxyisoflavone;
6,3'-dimethoxyflavone; Acacetin diacetate; Apigenin; Apigenin
triacetate; Biochanin a; Biochanin a diacetate; Daidzein;
Derrubone; Derrustone; Formononetn; Genistein; Ginkgetin;
Ipraflavone; Liquiritigenin dimethyl ether, Methoxyvone; and
Retusin 7-methyl ether, or a pharmaceutically acceptable salt of
any such compound.
5. A method according to claim 4, wherein the compound comprises a
(4-methoxyphenyl)-4H-chromene-4-one selected from a group
consisting of: 4,7-dimethoxyflavone; 4'-methoxyflavone;
5,4'-dimethoxyflavone; 5,7,4'-trimethoxyflavone; Acacetin
diacetate; Biochanin a; Biochanin a diacetate; Formononetn; and
Retusin 7-methyl ether; or a pharmaceutically acceptable salt of
any such compound.
6. A method according to claim 1, wherein the compound comprises a
1,3-benzodioxol selected from a group consisting of: Derrubone;
Derrusnin; Derrustone; Methyl robustone; Piperine; and Robustone;
or a pharmaceutically acceptable salt of any such compound.
7. A method for enhancing arginase activity in a damaged or injured
cell in a human in need thereof, the method comprising
administering to the human an effective amount of Lansoprazole; or
a pharmaceutically acceptable salt of such compound.
8. A method for treating a disorder that can be treated by
enhancing arginase activity in a human in need thereof, the method
comprising administering to the human an effective amount of a
compound that enhances arginase activity, wherein the compound is
any one of the following: 2-hydroxyxanthone; 2-methoxyxanthone;
3-methylcholanthrene ; 4,7-dimethoxyflavone; 4'-methoxychalcone;
4'-methoxyflavone; 5,4'-dimethoxyflavone; 7,4'-trimethoxyflavone;
5,7-methoxyisoflavone; 6,3'-dimethoxyflavone; Acacetin diacetate;
Anisindione; Apigenin; Apigenin triacetate; Biochanin a; Biochanin
a diacetate; Chlorpropham; Chrysophanol; Daidzein;
Dehydrovariabilin; Derrubone; Derrusnin; Derrustone;
Dibenzoylmethane; Fenbendazole; Formononetn; Genistein; Ginkgetin;
Indoprofen; Ipraflavone; Liquiritigenin dimethyl ether;
Methoxyvone; Methyl robustone; Phenazopyridine hydrochloride;
Phenindione; Pinosylvin; Pinosylvin methyl ether, Piperine;
Pramoxine hydrochloride; Resveratrol 4'-methyl ether, Retusin
7-methyl ether, Robustone; Spironolactone; Tilorone; Tranilast; or
Xanthone; or a pharmaceutically acceptable salt of any such
compound.
9. A method according to claim 8, wherein the disorder is
ischemia.
10. A method according to claim 8, wherein the disorder is
hypoxia
11. A method according to claim 8, wherein the disorder is a
neurodegenerative disease or condition.
12. A method according to claim 11, wherein the neurodegenerative
disease or condition is any one of the following: Alexander
disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral
sclerosis, Ataxia telangiectasia, Batten disease (also known as
Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform
encephalopathy (BSE), Canavan disease, Cockayne syndrome,
Corticobasal degeneration, Creutzfeldt-Jakob disease, Huntington
disease, HIV-associated dementia, Kennedy's disease, Krabbe
disease, Lewy body dementia, Machado-Joseph disease
(Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple
System Atrophy, Neuroborreliosis, Parkinson disease,
Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral
sclerosis, Prion diseases, Refsum's disease, Sandhoff disease,
Schilder's disease, Schizophrenia, Spielmeyer-Vogt-Sjogren-Batten
disease (also known as Batten disease), Spinocerebellar ataxia
(multiple types with varying characteristics), Spinal muscular
atrophy, Steele-Richardson-Olszewski disease, Tabes dorsalis, or
other dimentias.
13. A method according to claim 8, wherein the disorder is
stroke.
14. A method according to claim 8, wherein the disorder is trauma
to the central nervous system or peripheral nervous system.
15. A method according to claim 8, wherein the compound is
Pinosylvin; Derrustone; Methoxyvone; Dehydrovariabilin; or
Chrysophanol, or a pharmaceutically acceptable salt of any such
compound.
16. A method for treating a disorder that can be treated by
enhancing arginase activity in a human in need thereof, the method
comprising administering to the human an effective amount of
Lansoprazole; or a pharmaceutically acceptable salt of such
compound.
17. A method for promoting regeneration of a neural cell in a human
in need thereof, the method comprising administering to the human
an effective amount of a compound that enhances arginase activity,
wherein the compound is any one of the following:
2-hydroxyxanthone; 2-methoxyxanthone; 3-methylcholanthrene ;
4,7-dimethoxyflavone; 4'-methoxychalcone; 4'-methoxyflavone;
5,4'-dimethoxyflavone; 5,7,4'-trimethoxyflavone;
5,7-dimethoxyisoflavone; 6,3'-dimethoxyflavone; Acacetin diacetate;
Anisindione; Apigenin; Apigenin triacetate; Biochanin a; Biochanin
a diacetate; Chlorpropham; Chrysophanol; Daidzein;
Dehydrovariabilin; Derrubone; Derrusnin; Derrustone;
Dibenzoylmethane; Fenbendazole; Formononetn; Genistein; Ginkgetin;
Indoprofen; Ipraflavone; Liquiritigenin dimethyl ether;
Methoxyvone; Methyl robustone; Phenazopyridine hydrochloride;
Phenindione; Pinosylvin; Pinosylvin methyl ether, Piperine;
Pramoxine hydrochloride; Resveratrol 4'-methyl ether; Retusin
7-methyl ether; Robustone; Spironolactone; Tilorone; Tranilast; or
Xanthone; or a pharmaceutically acceptable salt of any such
compound.
18. A method according to claim 17, wherein the compound is
Pinosylvin; Derrustone; Methoxyvone; Dehydrovariabilin; or
Chrysophanol; or a pharmaceutically acceptable salt of any such
compound.
19. A method for promoting regeneration of a neural cell in a human
in need thereof, the method comprising administering to the human
an effective amount of Lansoprazole; or a pharmaceutically
acceptable salt of such compound.
20. A method according to claim 19, wherein the neural cell is a
neuron.
21. A method according to claim 19, wherein the neural cell is a
glial cell.
Description
[0001] This application asserts priority to U.S. Provisional
Application No. 60/807,661, filed on Jul. 18, 2006, which is hereby
incorporated by reference in its entirety.
[0002] The invention was made with funds from New York State
Department of Health, contract number CO19772. New York State has
certain rights in this invention.
BACKGROUND OF THE INVENTION
[0003] It is reported that the adult mammalian central nervous
system (CNS) shows little spontaneous regeneration after injury
despite that fact that there are many molecules present which
promote nerve and axonal growth. In contrast to the CNS, the adult
peripheral nervous system (PNS) is capable of regenerating to some
extent.
[0004] It is believed that the lack of regeneration in the CNS is
caused by the presence of molecules which actively prevent or
inhibit regeneration. Such molecules include Nogo (an antigen of
the IN-1 antibody), myelin-associated glycoprotein, and
myelin-oligodendrocyte glycoprotein.
[0005] Arginase is an enzyme that catalyzes the conversion of the
amino acid arginine to urea and ornithine. Arginase has been
reported to reverse the inhibition of neural regeneration in the
central and peripheral nervous system. Thus, enhancing arginase
activity would be beneficial for reversing the inhibition of neural
regeneration.
[0006] Arginase I is a 35- to 38-kDa cytoplasnmic protein that
cleaves arginine into urea and ornithine. Arginine is the only
substrate capable of donating the guanidine group necessary for
nitric oxide production. Nitric oxide is produced from arginine by
three nitric oxide synthase (NOS) isoforms. Nitric oxide production
can be regulated by modulating the levels of arginine. Arginase I
can limit the pool of arginine available for nitric oxide synthase
(NOS), thereby influencing the production of nitric oxide.
[0007] Neuronal damage can be caused by excess levels of nitric
oxide (NO). NO is a diffusible neuronal second messenger
synthesized in the nervous system by three enzymes: neuronal NO
synthase, endothelial NO synthase, and inducible NO synthase.
Excess NO generated by NO synthase is associated with various
neurodegenerative diseases and conditions, such as multiple
sclerosis, dementia, Huntington's disease, Alzheimer's disease,
etc.
[0008] The amino acid arginine is the only endogenous substrate of
NO synthase. It is reported that arginase can reduce cell death in
the nervous system by competing with NO synthase for their common
substrate, arginine.
[0009] Therefore, enhancing arginase activity would be beneficial
for promoting neural regeneration or reducing neural damage in
diseases and conditions associated with neural damage.
SUMMARY OF THE INVENTION
[0010] The present invention provides a method for enhancing
arginase activity in a damaged or injured cell. The method
comprises administering to a human in need thereof with an
effective amount of one of the following compounds:
2-hydroxyxanthone; 2-methoxyxanthone; 3-methylcholanthrene;
4,7-dimethoxyflavone; 4'-methoxychalcone; 4'-methoxyflavone;
5,4'-dimethoxyflavone; 5,7,4'-trimethoxyflavone;
5,7-dimethoxyisoflavone; 6,3'-dimethoxyflavone; Acacetin diacetate;
Anisindione; Apigenin; Apigenin triacetate; Biochanin a; Biochanin
a diacetate; Chlorpropham; Chrysophanol; Daidzein;
Dehydrovariabilin; Derrubone; Derrusnin; Derrustone;
Dibenzoylmethane; Fenbendazole; Formononetn; Genistein; Ginkgetin;
Indoprofen; Ipraflavone; Liquiritigenin dimethyl ether;
Methoxyvone; Methyl robustone; Phenazopyridine hydrochloride;
Phenindione; Pinosylvin; Pinosylvin methyl ether; Piperine;
Pramoxine hydrochloride; Resveratrol 4'-methyl ether, Retusin
7-methyl ether; Robustone; Spironolactone; Tilorone; Tranilast; or
Xanthone.
[0011] In another aspect, the invention provides a method for
enhancing arginase activity in a damaged or injured cell. The
method comprises administering to a human in need thereof an
effective amount of Lansoprazole.
[0012] In another aspect, the invention provides a method for
treating a disorder that can be treated by enhancing arginase
activity in a human in need thereof. The method comprises
administering to the human an effective amount of a compound that
enhances arginase activity, wherein the compound is any one of the
following: 2-hydroxyxanthone; 2-methoxyxanthone;
3-methylcholanthrene; 4,7-dimethoxyflavone; 4'-methoxychalcone;
4'-methoxyflavone; 5,4'-dimethoxyflavone; 5,7,4'-trimethoxyflavone;
5,7-dimethoxyisoflavone; 6,3'-dimethoxyflavone; Acacetin diacetate;
Anisindione; Apigenin; Apigenin triacetate; Biochanin a; Biochanin
a diacetate; Chlorpropham; Chrysophanol; Daidzein;
Dehydrovariabilin; Derrubone; Derrusnin; Derrustone;
Dibenzoylmethane; Fenbendazole; Formononetn; Genistein; Ginkgetin;
Indoprofen; Ipraflavone; Liquiritigenin dimethyl ether,
Methoxyvone; Methyl robustone; Phenazopyridine hydrochloride;
Phenindione; Pinosylvin; Pinosylvin methyl ether; Piperine;
Pramoxine hydrochloride; Resveratrol 4'-methyl ether; Retusin
7-methyl ether, Robustone; Spironolactone; Tilorone; Tranilast; or
Xanthone.
[0013] In yet another aspect, the invention provides a method for
treating a disorder that can be treated by enhancing arginase
activity in a human in need thereof The method comprises
administering to the human an effective amount of Lansoprazole.
[0014] In a further aspect, the invention provides a method
promoting regeneration of a neural cell in a human in need thereof.
The method comprises administering to the human an effective amount
of a compound that enhances arginase activity, wherein the compound
is any one of the following: 2-hydroxyxanthone; 2-methoxyxanthone;
3-methylcholanthrene; 4,7dimethoxyflavone; 4'-methoxychalcone;
4'-methoxyflavone; 5,4'-dimethoxyflavone; 5,7,4'-trimethoxyflavone;
5,7-dimethoxyisoflavone; 6,3'-dimethoxyflavone; Acacetin diacetate;
Anisindione; Apigenin; Apigenin triacetate; Biochanin a; Biochanin
a diacetate; Chlorpropham; Chrysophanol; Daidzein;
Dehydrovariabilin; Derrubone; Derrusnin; Derrustone;
Dibenzoylmethane; Fenbendazole; Formononetn; Genistein; Ginkgetin;
Indoprofen; Ipraflavone; Liquiritigenin dimethyl ether,
Methoxyvone; Methyl robustone; Phenazopyridine hydrochloride;
Phenindione; Pinosylvin; Pinosylvin methyl ether; Piperine;
Pramoxine hydrochloride; Resveratrol 4'-methyl ether; Retusin
7-methyl ether, Robustone; Spironolactone; Tilorone; Tranilast; or
Xanthone.
[0015] In yet a further aspect, the invention provides a method for
promoting regeneration of a neural cell in a human in need thereof.
The method comprises administering to the human an effective amount
of Lansoprazole.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1. Chemical structures of compounds.
[0017] FIG. 2. Table listing compounds that upregulated arginase I
at least above or near 2-fold.
[0018] FIG. 3. Table listing compounds that were tested in
quantitative RT-PCR and immunoblot analysis.
[0019] FIG. 4. Western blot results that measured level of enhanced
arginase 1 protein associated with the compounds listed in FIG.
3.
[0020] FIG. 5. Western blot results that measured level of enhanced
arginase 1 protein associated with the compounds listed in FIG.
3.
[0021] FIG. 6. Quantitative RT-PCR results presented in a graph
showing levels of arginase I messenger RNA (mRNA) upregulation
associated with compounds 1-10 listed in FIG. 3.
[0022] FIG. 7. Quantitative RT-PCR results presented in a graph
showing levels of arginase I messenger RNA (MRNA) upregulation
associated with compounds 11-20 listed in FIG. 3.
[0023] FIG. 8. Quantitative RT-PCR results presented in a graph
showing levels of arginase I messenger RNA (MRNA) upregulation
associated with compounds 21-30 listed in FIG. 3.
[0024] FIG. 9. Quantitative RT-PCR results presented in a graph
showing levels of arginase I messenger RNA (mRNA) upregulation
associated with compounds 31-40 listed in FIG. 3.
[0025] FIG. 10. Table summarizing results of compounds tested for
their ability to overcome myelin-associated glycoprotein (MAG)
inhibition in P7 rat cerebellar neurons, as compared with rho
kinase inhibitor. The compounds were administered after
(post-treatment) the neurons were plated.
[0026] FIG. 11. Graph showing results from testing the compounds
(10 nM) listed in FIG. 10 for their ability to overcome
myelin-associated glycoprotein (MAG) inhibition in P7 rat
cerebellar neurons, as compared with rho kinase inhibitor.
[0027] FIG. 12. Graph showing results from testing the compounds
(40 nM) listed in FIG. 10 for their ability to overcome
myelin-associated glycoprotein (MAG) inhibition in P7 rat
cerebellar neurons, as compared with rho kinase inhibitor.
[0028] FIG. 13. Table summarizing results of compounds tested for
their ability to overcome myelin-associated glycoprotein (MAG)
inhibition in P7 rat cerebellar neurons, as compared with rho
kinase inhibitor. The compounds were administered before
(pretreatment) and after (post treatment) the neurons were
plated.
[0029] FIG. 14. P5 cerebellar neurons plated on CONT and MAG
expressing CHO cells. FIG. 14A-E are pictures representative of
.beta.III tubulin positive cells. Neurons are plated either with
DMSO (0.1%) on control CHO monolayers (FIG. 14A), or on substrate
inhibitor (MAG) (FIG. 14B-E) and treated with DMSO (0.1%) (FIG.
14B), methoxyvone (5 .mu.M) (FIG. 14C), daidzein (20 .mu.M) and
Lanzoprazole (20 .mu.M). Graph in FIG. 14 depicts the average
length of the longest neurite (percentage of the longest neurite of
the control). At least 400 neurons were measured in each assay and
the experiment was carried out at least twice.
[0030] FIG. 15. Western blot and immunostaining results for
arginase I protein. Neurons were treated with, diazein (20 uM),
methoxyvone (5 uM) or lanzoprazole (20 uM) for 18 hours and were
then lysed and subjected to gel electrophoresis, followed by
western blotting and immunostaining for Arginase I protein. As a
positive control, neurons were treated with 1 mM db cAMP.
[0031] FIG. 16. The chemical formula, structures, and references
for a genus of compounds useful in the methods of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
Method for Enhancing Arginase Activity
[0032] In one aspect, the invention provides a method for enhancing
arginase activity in a damaged or injured cell in a human in need
thereof. Any isoform of arginase can be enhanced by the methods of
the present invention. Examples of arginase isoforms include
arginase I and arginase II.
[0033] The method comprises administering to the human an effective
amount of a member of the genus of compounds selected from:
2-hydroxyxanthone; 2-methoxyxanthone; 3-methylcholanthrene ;
4,7-dimethoxyflavone; 4'-methoxychalcone; 4'-methoxyflavone;
5,4'-dimethoxyflavone; 5,7,4'-trimethoxyflavone;
5,7-dimethoxyisoflavone; 6,3'-dimethoxyflavone; Acacetin diacetate;
Anisindione; Apigenin; Apigenin triacetate; Biochanin a; Biochanin
a diacetate; Chlorprophan; Chrysophanol; Daidzein;
Dehydrovariabilin; Derrubone; Derrusnin; Derrustone;
Dibenzoylmethane; Fenbendazole; Formononetn; Genistein; Ginkgetin;
Indoprofen; Ipraflavone; Liquiritigenin dimethyl ether;
Methoxyvone; Methyl robustone; Phenazopyridine hydrochloride;
Phenindione; Pinosylvin; Pinosylvin methyl ether; Piperine;
Prarnoxine hydrochloride; Resveratrol 4'-methyl ether; Retusin
7-methyl ether, Robustone; Spironolactone; Tilorone; Tranilast; or
Xanthone.
[0034] The terms "enhancing arginase activity" or "enhanced
arginase activity" refer to an increased level of measurable
arginase activity in a given assay in the presence of a candidate
compound relative to the measurable level of arginase activity in
the absence of the candidate compound, when tested under the same
conditions.
[0035] Activity is considered enhanced according to the invention
if it is enhanced at least about 10% greater, preferably at least
about 25% greater, more preferably at least about 50% greater, even
more preferably at least about 75% greater, most preferably at
least about 90% greater, or more than in the absence of the
candidate compound.
[0036] Arginase activity as used herein can be enhanced by any
mechanism. For example, arginase activity could be enhanced by
transcriptional induction of its cognate messenger RNA (mRNA),
increased stability of its mRNA, increased translation of mRNA into
protein, increased stability of arginase protein, increased
arginase activity (in the presence or absence of increased
protein), or any other mechanism. Increases in arginase activity
could be realized by the ability of a compound to increase the
affinity of the arginase enzyme for its prototypical substrate,
arginine.
[0037] The arginase activity is enhanced in any damaged or injured
cell that benefits from enhanced arginase or from a reduction of
nitric oxide or arginine. The damage or injury may be to any part
of a cell, such as to membranes, DNA, RNA, and ribosomes.
[0038] Examples of cells that may be damaged or injured include
cells of the central nervous system (CNS) or peripheral nervous
system (PNS), including neurons, ganglia, Schwann cells,
astrocytes, oligodendrocytes, microglia cells, endothelial cells,
immune cells (e.g., macrophages, T cells, B cells, and
neutrophils), etc. In one embodiment, the damaged or injured cell
is in a human.
[0039] In one embodiment, the method comprises administering to a
human in need thereof an effective amount of any one or any
combination of the following compounds: Pinosylvin; Derrustone;
Methoxyvone; Dehydrovariabilin; or Chrysophanol.
[0040] In another embodiment, the method comprises administering to
a human in need thereof an effective amount of any one or any
combination of the following compounds: Resveratrol 4'-methyl
ether, Derrubone; Ginkgetin; or Methyl robustone.
[0041] In yet another embodiment, the method comprises
administering to a human in need thereof an effective amount of any
one or any combination of the following compounds: Tilorone;
Phenindione; Pramoxine hydrochloride; Indoprofen; Phenazopyridine
hydrochloride; Piperine; 6,3'-dimethoxyflavone; Anisindione;
5,4'-dimethoxyflavone; Pinosylvin; Derrustone;
4,7-dimethoxyflavone; Daidzein; 4'-methoxychalcone; Tranilast;
Biochanin a diacetate; Resveratrol 4'-methyl ether, Derrubone;
Chlorprophan; Genistein; Dehydrovariabilin; Retusin 7-methyl ether;
Xanthone; Pinosylvin methyl ether, Chrysophanol; Apigenin;
2-methoxyxanthone; Apigenin triacetate; Fenbendazole;
Dibenzoylmethane; Methoxyvone; Ginkgetin, k salt; Methyl robustone;
Liquiritigenin dimethyl ether, Derrusnin; Biochanin a;
5,7-dimethoxyisoflavone; Formononetin; 4'-methoxyflavone; or
Acacetin diacetate.
[0042] In a further embodiment, the method comprises administering
to a human in need thereof an effective amount of any one or any
combination of the following compounds: Pinosylvin; Derrustone;
Daidzein; 4'-methoxychalcone; Tranilast; Biochanin a diacetate;
Resveratrol 4'-methyl ether; Dehydrovariabilin; Chrysophanol; or
Methoxyvone.
[0043] In yet a further embodiment, the method comprises
administering to a human in need thereof an effective amount of any
one or any combination of the following compounds: Daidzein or
Methoxyvone.
[0044] In another embodiment, the method comprises administering to
a human in need thereof an effective amount of a compound
comprising a 9H-xanthen-9-one selected from a group consisting of:
2-hydroxyxanthone, 2-methoxyxanthone, and Xanthone.
[0045] In yet another embodiment, the method comprises
administering to a human in need thereof an effective amount of a
compound comprising a 4H-chromen4-one selected from a group
consisting of: 4,7-dimethoxyflavone; 4'-methoxyflavone;
5,4'-dimethoxyflavone; 5,7,4'-trimethoxyflavone;
5,7-dimethoxyisoflavone; 6,3'-dimethoxyflavone; Acacetin diacetate;
Apigenin; Apigenin triacetate; Biochanin a; Biochanin a diacetate;
Daidzein; Derrubone; Derrustone; Formononetn; Genistein; Ginkgetin;
Ipraflavone; Liquiritigenin dimethyl ether; Methoxyvone; and
Retusin 7-methyl ether.
[0046] In a further embodiment, the method comprises administering
to a human in need thereof an effective amount of a compound
comprising a (4-methoxyphenyl)4H-chromene-4-one selected from a
group consisting of: 4,7-dimethoxyflavone; 4'-methoxyflavone;
5,4'-dimethoxyflavone; 5,7,4'-trimethoxyflavone; Acacetin
diacetate; Biochanin a; Biochanin a diacetate; Fornononetn; and
Retusin 7-methyl ether.
[0047] In still another embodiment, the method comprises
administering to a human in need thereof a compound comprising a
1,3-benzodioxol selected from a group consisting of: Derrubone;
Derrusnin; Derrustone ; Methyl robustone; Piperine; and
Robustone.
[0048] In yet a further embodiment, the method comprises
administering to a human in need thereof an effective amount of a
compound selected from any one or a combination of compounds listed
in FIG. 2 and/or FIG. 3.
[0049] In another aspect of the invention, the method comprises
administering to the human in need thereof an effective amount of
Lansoprazole.
Method for Treating a Disorder That Can Be Treated by Enhancing
Arginase Activity
[0050] In another aspect, the invention provides a method for
treating a disorder that can be treated by enhancing arginase
activity in a human in need thereof The method includes
administering to the human an effective amount of a compound that
enhances arginase activity, wherein the compound is any one of the
following: 2-hydroxyxanthone; 2-methoxyxanthone;
3-methylcholanthrene ; 4,7-dimethoxyflavone; 4'-methoxychalcone;
4'-methoxyflavone; 5,4'-dimethoxyflavone; 5,7,4'-trimethoxyflavone;
5,7-dimethoxyisoflavone; 6,3'-dimethoxyflavone; Acacetin diacetate;
Anisindione; Apigenin; Apigenin triacetate; Biochanin a; Biochanin
a diacetate; Chlorpropham; Chrysophanol; Daidzein;
Dehydrovariabilin; Derrubone; Derrusnin; Derrustone;
Dibenzoylmethane; Fenbendazole; Formononetn; Genistein; Ginkgetin;
Indoprofen; Ipraflavone; Liquiritigenin dimethyl ether;
Methoxyvone; Methyl robustone; Phenazopyridine hydrochloride;
Phenindione; Pinosylvin; Pinosylvin methyl ether, Piperine;
Pramoxine hydrochloride; Resveratrol 4'-methyl ether, Retusin
7-methyl ether; Robustone; Spironolactone; Tilorone; Tranilast; or
Xanthone.
[0051] In one embodiment, the method comprises administering to the
human an effective amount of any one or any combination of the
following compounds: Pinosylvin; Derrustone; Methoxyvone;
Dehydrovariabilin; or Chrysophanol.
[0052] In another embodiment, the method comprises administering to
the human an effective amount of any one or any combination of the
following compounds: Resveratrol 4'-methyl ether; Derrubone;
Ginkgetin; or Methyl robustone.
[0053] In yet another embodiment, the method comprises
administering to the human an effective amount of any one or any
combination of the following compounds: Tilorone; Phenindione;
Pramoxine hydrochloride; Indoprofen; Phenazopyridine hydrochloride;
Piperine; 6,3'-dimethoxyflavone; Anisindione;
5,4'-dimethoxyflavone; Pinosylvin; Derrustone;
4,7-dimethoxyflavone; Daidzein; 4'-methoxychalcone; Tranilast;
Biochanin a diacetate; Resveratrol 4'-methyl ether, Derrubone;
Chlorpropham; Genistein; Dehydrovariabilin; Retusin 7-methyl ether,
Xanthone ; Pinosylvin methyl ether, Chrysophanol; Apigenin;
2-methoxyxanthone; Apigenin triacetate; Fenbendazole;
Dibenzoylmethane; Methoxyvone; Ginkgetin, k salt; Methyl robustone;
Liquiritigenin dimethyl ether, Derrusnin; Biochanin a;
5,7-dimethoxyisoflavone; Formononetin; 4'-methoxyflavone; or
Acacetin diacetate.
[0054] In a further embodiment, the method comprises administering
to the human an effective amount of any one or any combination of
the following compounds: Pinosylvin;
[0055] Derrustone; Daidzein; 4'-methoxychalcone; Tranilast;
Biochanin a diacetate; Resveratrol 4'-methyl ether,
Dehydrovariabilin; Chrysophanol; or Methoxyvone.
[0056] In yet a further embodiment, the method comprises
administering to the human an effective amount of any one or any
combination of the following compounds: Daidzein or
Methoxyvone.
[0057] In another embodiment, the method comprises administering to
the human an effective amount of a compound comprising a
9H-xanthen-9-one selected from a group consisting of:
2-hydroxyxanthone, 2-methoxyxanthone, and Xanthone.
[0058] In yet another embodiment, the method comprises
administering to the human an effective amount of a compound
comprising a 4H-chromen-4one selected from a group consisting of:
4,7-dimethoxyflavone; 4'-methoxyflavone; 5,4'-dimethoxyflavone;
5,7,4'-trimethoxyflavone; 5,7-dimethoxyisoflavone;
6,3'-dimethoxyflavone; Acacetin diacetate; Apigenin; Apigenin
triacetate; Biochanin a; Biochanin a diacetate; Daidzein;
Derrubone; Derrustone; Formononetn; Genistein; Ginkgetin;
Ipraflavone; Liquiritigenin dimethyl ether; Methoxyvone; and
Retusin 7-methyl ether.
[0059] In a further embodiment, the method comprises administering
to the human an effective amount of a compound comprising a
(4-methoxyphenyl)4H-chromene-4-one selected from a group consisting
of: 4,7-dimethoxyflavone; 4'-methoxyflavone; 5,4'-dimethoxyflavone;
5,7,4'-trimethoxyflavone; Acacetin diacetate; Biochanin a;
Biochanin a diacetate; Formononetn; and Retusin 7-methyl ether.
[0060] In still another embodiment, the method comprises
administering to the human an effective amount of a compound
comprising a 1,3-benzodioxol selected from a group consisting of:
Derrubone; Derrusnin; Derrustone; Methyl robustone; Piperine; and
Robustone.
[0061] In yet a further embodiment, the method comprises
administering to the human an effective amount of a compound
selected from any one or a combination of compounds listed in FIG.
2 and/or FIG. 3.
[0062] In another aspect of the invention, the method comprises
administering to the human an effective amount of Lansoprazole.
[0063] Disorders and diseases in which enhancing arginase activity
is desired for treatment include ischemia, hypoxia,
neurodegenerative disease or condition, or stroke. Additional
disorders and diseases in which enhancing arginase activity is
desired for treatment traumatic disorders (including but not
limited to spinal cord injuries, spinal cord lesions, or other CNS
pathway lesions), surgical nerve lesions, damage secondary to
infarction, infection, exposure to toxic agents, malignancy,
paraneoplastic syndromes, or patients with various types of
neurodegenerative disorders of the central nervous system.
Method for Treating Ischemia
[0064] In one embodiment, the invention provides a method for
treating ischemia in a human in need thereof. The method comprises
administering to the human a compound that enhances arginase
activity as described above, including those compounds grouped in
the various genera and sub-genera.
[0065] Any mammal suffering from ischemia can be treated in
accordance with the method of the present invention. Ischemia
generally refers to a condition of decreased blood flow to an
organ, tissue and/or cell. The decrease in blood flow can be caused
by, for example, constriction (e.g., hypoxemic vasoconstriction) or
obstruction (e.g., clot, atherosclerotic plaque) of a blood
vessel.
[0066] Ischemia can occur in any cell, organ, and/or tissue.
Examples of cells, organs, and/or tissues which can be subjected to
ischemia include neuronal cells (e.g., neurons, ganglia, Schwann
cells, astrocytes, oligodendrocytes and microglia), brain, spinal
cord, intestinal cells, kidney cells, heart and cardiac muscle
cells such as myocytes, etc.
Method for Treating Hypoxia
[0067] In yet another embodiment, the invention provides a method
for treating hypoxia in a human in need thereof The method includes
administering to the human a compound that enhances arginase
activity as described above, including those compounds grouped in
the genera and various sub-genera.
[0068] Any mammal suffering from hypoxia can be treated in
accordance with the method of the present invention. Hypoxia
generally refers to a lack of oxygen to cells, organs, and/or
tissues. Hypoxia can be caused by, for example, ischemia, anemia
and chemical modification of blood, such as carboxyhemoglobin,
etc.
[0069] Hypoxia can occur in any cell, organ, and/or tissue.
Examples of cells, organs, and/or tissues which can be subjected to
hypoxia include neuronal cells (e.g., neurons, ganglia, Schwann
cells, astrocytes, oligodendrocytes and microglia), brain, spinal
cord, kidney cells, intestinal cells, heart and cardiac muscle
cells such as myocytes, skin cells, etc.
Method for Treating Neurodegenerative Disease or Condition
[0070] In still another embodiment, the invention provides a method
for treating a neurodegenerative disease or condition in a human in
need thereof The method includes administering to the human a
compound that enhances arginase activity as described above,
including those compounds grouped in the genera and various
sub-genera.
[0071] A neurodegenerative disease or condition typically refers to
a disorder generally characterized by gradual and progressive loss
of cells, tissue and/or organ of the central or peripheral nervous
system. Examples of such cells, tissues and organs include, the
brain, spinal cord, neurons, ganglia, Schwann cells, astrocytes,
oligodendrocytes and microglia.
[0072] Any mammal suffering from any neurodegenerative disease or
condition can be treated in accordance with the method of the
present invention. For example, the neurodegenerative disease or
condition can be an acute condition. Acute conditions generally
occur as a result of trauma to a cell, tissue and/or organ of the
nervous system. The trauma can, for example, partially or
completely block blood flow to the cell, tissue and/or organ.
Examples of acute neurodegenerative conditions include head injury
and brain injury.
[0073] Alternatively, the neurodegenerative disease or condition
can be a chronic neurodegenerative condition. Examples of chronic
neurodegenerative diseases and conditions include Parkinson's
disease, Alzheimer's disease, Huntington's disease and Amyotrophic
Lateral Sclerosis (also known as Lou Gherig's disease).
[0074] Additional examples of neurodegenerative disorders and
diseases that can be treated by the invention include but are not
limited to Alexander disease, Alper's disease, Alzheimer's disease,
Amyotrophic lateral sclerosis, Ataxia telangiectasia, Batten
disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease),
Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne
syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease,
Huntington disease, HIV-associated dementia, Kennedy's disease,
Krabbe disease, Lewy body dementia, Machado-Joseph disease
(Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple
System Atrophy, Neuroborreliosis, Parkinson disease,
Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral
sclerosis, Prion diseases, Refsum's disease, Sandhoff disease,
Schilder's disease, Schizophrenia, Spielmeyer-Vogt-Sjogren-Batten
disease (also known as Batten disease), Spinocerebellar ataxia
(multiple types with varying characteristics), Spinal muscular
atrophy, Steele-Richardson-Olszewski disease, Tabes dorsalis, and
other dementias.
Method for Treating Stroke
[0075] In a further aspect, the invention provides a method for
treating stroke in a human in need thereof. The method includes
administering to the human a compound that enhances arginase
activity as described above, including those compounds grouped in
the genera and various sub-genera.
[0076] Any mammal suffering from stroke can be treated in
accordance with the method of the present invention. Stroke is a
type of cardiovascular disease that generally involves the
interruption of blood flow to and/or within the brain. The
interruption of blood flow can be due to, for example, a blockage
or rupture of an artery or vessel. The blockage typically occurs
from a blood clot. As a result of the interruption of blood flow,
the brain does not receive sufficient amounts of blood.
Method for Treating Trauma of the CNS or PNS
[0077] In still a further embodiment, the invention provides a
method for treating trauma of the central nervous system (CNS) or
peripheral nervous system (PNS) in a human in need thereof. The
method includes administering to the human a compound that enhances
arginase activity as described above, including those compounds
grouped in the genera and various sub-genera.
[0078] Any type of trauma to the nervous system may be treated by
the methods of the claimed invention. As described above, trauma of
the CNS or PNS include, but are not limited to, spinal cord
injuries, spinal cord lesions, other CNS pathway lesions, as well
as injuries to the PNS, such as injuries to a nerve or neuron of
the PNS and axon damage resulting in demyelination of the PNS. Such
trauma can arise from either physical injury or disease. Any mammal
suffering from a trauma of the CNS or PNS can be treated in
accordance with the methods of the present invention.
[0079] For example, spinal cord injury refers to any damage to the
spinal cord. The damage typically results in loss of function, such
as mobility or feeling. Damage to the spinal cord can occur, for
example, as a result or trauma (car accident, gunshot, falls, etc.)
or disease (polio, spina bifida, Friedreich's Ataxia, etc).
[0080] Any injury to the spinal cord can be treated in. accordance
with the method of the present invention. For example, the injury
can be a complete injury to the spinal cord. Complete injury
typically refers to the lack of function (e.g., no sensation and no
voluntary movement) below the site of injury. Both sides of the
body are usually affected.
[0081] Alternatively, the injury may be an incomplete injury to the
spinal cord. An incomplete injury generally refers to some function
below the site of injury. For instance, a person with an incomplete
injury may be able to move one limb more than another, may be able
to feel parts of the body that cannot be moved, or may have more
functioning on one side of the body than the other, etc.
Method for Promoting Regeneration of a Neural Cell in a Human in
Need Thereof
[0082] In another aspect, the invention provides a method for
promoting regeneration of a neural cell in a human in need thereof
As described in Lange, et aL, J Nutr. 2004 October ;134(10
Suppl):2812S-2817S; discussion 2818S-2819S, arginase has a role in
the axonal regeneration pathway. Arginase also has neuroprotective
properties. Arginase is sufficient in protecting neurons against
several apoptosis-inducing stimuli. Moreover, arginase acts as a
nitric oxide-independent inhibitor of neuronal apoptosis.
[0083] Several investigators have found that molecules or drugs
that prevent injury in the PNS or CNS have no effect on or worse,
negatively impact the ability of the nervous system to regenerate
or repair. There is thus an urgent need to identify targets whose
activation would provide an environment that is simultaneously
instructive for neuronal protection and repair. It is proposed that
such an intervention would provide greater latitude in the timing
of initiation of treatment.
[0084] Arginine can be metabolized by nitric oxide synthase to
produce nitric oxide. It can also be degraded by arginase to
produce urea and ornithine, which in turn is a precursor for the
synthesis of polyamines. These two pathways compete for arginine.
Arginase thus produces polyamines at the expense of nitric
oxide.
[0085] Polyamines have been implicated in neuronal growth and
development, axonal regeneration after injury, and in would healing
outside of the CNS. Arginase I is upregulated and polyamine
synthesis increases in neurons in response to either dbCAMP or
BDNF.
[0086] By producing polyamines, arginase can overcome the effects
of myelin-associated glycoprotein (MAG) and myelin on neurite
outgrowth. Arginase is an enzyme that mediates repair by reducing
its substrates (L-arginine) and increasing its ultimate products
(polyamines), respectively.
[0087] As toxic levels of nitric oxide (NO) are implicated in acute
ischemic cortical injury and motor neuron loss due to absence of
trophic factors, arginase can mediate neuroprotection. By producing
polyamines, arginase can overcome the effects of myelin-associated
glycoprotein (MAG) and myelin on neurite outgrowth Arginase is thus
a bi-functional enzyme that mediates neuroprotection or repair by
reducing its substrates (L-arginine) and increasing its ultimate
products (polyamines), respectively.
[0088] As used herein, the phrase "neural cell" includes nerve
cells (i.e., neurons, e.g., uni-, bi-, or mulipolar neurons) and
their precursors and glial cells (e.g., macroglia such as
astrocytes, oligodendrocytes, ependymal cells, radial glia, Schwann
cells, Satellite cells, and microglia) and their precursors.
Microglia are specialized macrophages capable of phagocytosis that
protect neurons of the central nervous system. The term "precursor"
refers to cells which are capable of developing into a specific
cell type. For example, a neural cell precursor is a cell which is
capable of developing into a mature neural cell (i.e., a cell
having the characteristic morphology and function of a neural
cell).
[0089] Accordingly, the claimed invention provides methods for
promoting regeneration of a neural cell in a human in need
thereof.
[0090] The method includes administering to the human an effective
amount of a compound that enhances arginase activity, wherein the
compound is any one of the following: 2-hydroxyxanthone;
2-methoxyxanthone;. 3-methylcholanthrene; 4,7-dimethoxyflavone;
4'-methoxychalcone; 4'-methoxyflavone; 5,4'-dimethoxyflavone;
5,7,4'-trimethoxyflavone; 5,7-dimethoxyisoflavone;
6,3'-dimethoxyflavone; Acacetin diacetate; Anisindione; Apigenin;
Apigenin triacetate; Biochanin a; Biochanin a diacetate;
Chlorpropham; Chrysophanol; Daidzein; Dehydrovariabilin; Derrubone;
Derrusnin; Derrustone; Dibenzoylmethane; Fenbendazole; Formononetn;
Genistein; Ginkgetin; Indoprofen; Ipraflavone; Liquiritigenin
dimethyl ether, Methoxyvone; Methyl robustone; Phenazopyridine
hydrochloride; Phenindione; Pinosylvin; Pinosylvin methyl ether;
Piperine; Pramoxine hydrochloride; Resveratrol 4'-methyl ether;
Retusin 7-methyl ether, Robustone; Spironolactone; Tilorone;
Tranilast; or Xanthone.
[0091] In one embodiment, the method comprises administering to the
human an effective amount of any one or any combination of the
following compounds: Pinosylvin; Derrustone; Methoxyvone;
Dehydrovariabilin; or Chrysophanol.
[0092] In another embodiment, the method comprises administering to
the human an effective amount of any one or any combination of the
following compounds: Resveratrol 4'-methyl ether; Derrubone;
Ginkgetin; or Methyl robustone.
[0093] In yet another embodiment, the method comprises
administering to the human an effective amount of any one or any
combination of the following compounds: Tilorone; Phenindione;
Pramoxine hydrochloride; Indoprofen; Phenazopyridine hydrochloride;
Piperine; 6,3'-dimethoxyflavone; Anisindione;
5,4'-dimethoxyflavone; Pinosylvin; Derrustone;
4,7-dimethoxyflavone; Daidzein; 4'-methoxychalcone; Tranilast;
Biochanin a diacetate; Resveratrol 4'-methyl ether, Derrubone;
Chlorpropham; Genistein; Dehydrovariabilin; Retusin 7-methyl ether,
Xanthone ; Pinosylvin methyl ether; Chrysophanol; Apigenin;
2-methoxyxanthone; Apigenin triacetate; Fenbendazole;
Dibenzoylmethane; Methoxyvone; Ginkgetin, k salt; Methyl robustone;
Liquiritigenin dimethyl ether; Derrusnin; Biochanin a;
5,7-dimethoxyisoflavone; Formononetin; 4'-methoxyflavone; or
Acacetin diacetate.
[0094] In a further embodiment, the method comprises administering
to the human an effective amount of any one or any combination of
the following compounds: Pinosylvin; Derrustone; Daidzein;
4'-methoxychalcone; Tranilast; Biochanin a diacetate; Resveratrol
4'-methyl ether; Dehydrovariabilin; Chrysophanol; or
Methoxyvone.
[0095] In yet a further embodiment, the method comprises
administering to the human a an effective amount of n effective
amount of any one or any combination of the following compounds:
Daidzein or Methoxyvone.
[0096] In another embodiment, the method comprises administering to
the human an effective amount of a compound comprising a
9H-xanthen-9-one selected from a group consisting of:
2-hydroxyxanthone, 2-methoxyxanthone, and Xanthone.
[0097] In yet another embodiment, the method comprises
administering to the human an effective amount of a compound
comprising a 4H-chromen4-one selected from a group consisting of:
4,7-dimethoxyflavone; 4'-methoxyflavone; 5,4'-dimethoxyflavone;
5,7,4'-trimethoxyflavone; 5,7-dimethoxyisoflavone;
6,3'-dimethoxyflavone; Acacetin diacetate; Apigenin; Apigenin
triacetate; Biochanin a; Biochanin a diacetate; Daidzein;
Derrubone; Derrustone; Formononetn; Genistein; Ginkgetin;
Ipraflavone; Liquiritigenin dimethyl ether; Methoxyvone; and
Retusin 7-methyl ether.
[0098] In a further embodiment, the method comprises administering
to the human an effective amount of a compound comprising a
(4-methoxyphenyl)4H-chromene-4-one selected from a group consisting
of: 4,7-dimethoxyflavone; 4'-methoxyflavone; 5,4'-dimethoxyflavone;
5,7,4'-trimethoxyflavone; Acacetin diacetate; Biochanin a;
Biochanin a diacetate; Formononetn; and Retusin 7-methyl ether.
[0099] In still another embodiment, the method comprises
administering to the human an effective amount of a compound
comprising a 1,3-benzodioxol selected from a group consisting of:
Derrubone; Derrusnin; Derrustone; Methyl robustone; Piperine; and
Robustone.
[0100] In yet a further embodiment, the method comprises
administering to the human an effective amount of a compound
selected from any one or a combination of compounds listed in FIG.
2 and/or FIG. 3.
[0101] In another aspect of the invention, the method comprises
administering to the human an effective amount of Lansoprazole.
Methods for Protecting a Neural Cell in a Human in Need Thereof
[0102] In yet another aspect, the invention provides a method for
protecting a neural cell in a human in need thereof. The method
includes administering to the human a compound that enhances
arginase activity, wherein the compound comprises any one of the
following compounds: 2-hydroxyxanthone; 2-methoxyxanthone;
3-methylcholanthrene; 4,7-dimethoxyflavone; 4'-methoxychalcone;
4'-methoxyflavone; 5,4'-methoxyflavone; 5,7,4'-trimethoxyflavone;
5,7-dimethoxyisoflavone; 6,3'-dimethoxyflavone; Acacetin diacetate;
Anisindione; Apigenin; Apigenin triacetate; Biochanin a; Biochanin
a diacetate; Chlorpropham; Chrysophanol; Daidzein;
Dehydrovariabilin; Derrubone; Derrusnin; Derrustone;
Dibenzoylmethane; Fenbendazole; Formononetn; Genistein; Ginkgetin;
Indoprofen; Ipraflavone; Liquiritigenin dimethyl ether;
Methoxyvone; Methyl robustone; Phenazopyridine hydrochloride;
Phenindione; Pinosylvin; Pinosylvin methyl ether; Piperine;
Pramoxine hydrochloride; Resveratrol 4'-methyl ether, Retusin
7-methyl ether; Robustone; Spironolactone; Tilorone; Tranilast; or
Xanthone.
[0103] In another aspect of the invention, the method comprises
administering to the human an effective amount of Lansoprazole. The
method includes administering to the human any one or a combination
of compounds that enhances arginase activity included in the genera
and sub-genera of compounds described above.
Compounds
[0104] Compounds useful in the methods of the present invention
include 2-hydroxyxanthone; 2-methoxyxanthone; 3-methylcholanthrene;
4,7-dimethoxyflavone; 4'-methoxychalcone; 4'-methoxyflavone;
5,4'-dimethoxyflavone; 5,7,4'-trimethoxyflavone;
5,7-dimethoxyisoflavone; 6,3'-dimethoxyflavone; Acacetin diacetate;
Anisindione; Apigenin; Apigenin triacetate; Biochanin a; Biochanin
a diacetate; Chlorpropham; Chrysophanol; Daidzein;
Dehydrovariabilin; Derrubone; Derrusnin; Derrustone;
Dibenzoylmethane; Fenbendazole; Formononetn; Genistein; Ginkgetin;
Indoprofen; Ipraflavone; Liquiritigenin dimethyl ether,
Methoxyvone; Methyl robustone; Phenazopyridine hydrochloride;
Phenindione; Pinosylvin; Pinosylvin methyl ether; Piperine;
Pramoxine hydrochloride; Resveratrol 4'-methyl ether; Retusin
7-methyl ether; Robustone; Spironolactone; Tilorone; Tranilast; or
Xanthone.
[0105] Another compound useful in the methods of the present
invention includes Lansoprazole.
[0106] Further examples compounds useful in the methods of the
present invention include any one or a combination of compounds
listed in FIG. 2 and/or FIG. 3.
[0107] These compounds are known in the art. The chemical formula,
structures, and references for a genus of compounds described above
are shown in FIG. 16.
[0108] The chemical formula, structures, and references for
Pinosylvin, Derrustone, Methoxyvone, Dehydrovariabilin, and
Chrysophanol are shown in FIG. 1.
[0109] The compounds can be in the form of a pharmaceutically
acceptable salt The term "pharmaceutically acceptable salt" refers
to a well-tolerated, nontoxic salt prepared from any basic or
acidic compound mentioned above, and an acid or base, respectively
The acids may be inorganic or organic acids of any one of the
compounds mentioned above. Examples of inorganic acids include
hydrochloric, hydrobromic, nitric hydroiodic, sulfuric, and
phosphoric acids. Examples of organic acids include carboxylic and
sulfonic acids. The radical of the organic acids may be aliphatic
or aromatic. Some examples of organic acids include formic, acetic,
phenylacetic, propionic, succinic, glycolic, glucuronic, maleic,
furoic, glutamic, benzoic, anthranilic, salicylic, phenylacetic,
mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic,
panthenoic, benzenesulfonic, stearic, sulfanilic, alginic,
tartaric, citric, gluconic, gulonic, arylsulfonic, and galacturonic
acids. Appropriate organic bases may be selected, for example, from
N,N-dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and
procaine.
[0110] Throughout this specification, parameters are defined by
maximum and minimum amounts. Each minimum amount can be combined
with each maximum amount to define a range.
Administration
[0111] The compounds are administered to a human. The compound is
administered to the human in an amount effective in achieving its
purpose. The effective amount of the compound to be administered
can be readily determined by those skilled in the art during
pre-clinical trials and clinical trials by methods familiar to
physicians and clinicians. Typical daily doses include
approximately 1 mg to 1000 mg.
[0112] Any method known to those in the art for contacting a cell,
organ or tissue with a compound may be employed. Suitable methods
include in vitro, ex vivo, or in vivo methods. In vitro methods
typically include cultured samples. For example, a cell can be
placed in a reservoir (e.g., tissue culture plate), and incubated
with a compound under appropriate conditions suitable for enhancing
arginase activity. Suitable incubation conditions can be readily
determined by those skilled in the art.
[0113] Ex vivo methods typically include cells, organs or tissues
removed from a mammal, such as a human. The cells, organs or
tissues can, for example, be incubated with the compound under
appropriate conditions. The contacted cells, organs or tissues are
normally returned to the donor, placed in a recipient, or stored
for future use. Thus, the compound is generally in a
pharmaceutically acceptable carrier.
[0114] In vivo methods are typically limited to the administration
of a compound, such as those described above, to a mammal,
preferably a human. The compounds useful in the methods of the
present invention are administered to a mammal in an amount
effective in enhancing arginase activity or treating the mammal.
The effective amount is determined during pre-clinical trials and
clinical trials by methods familiar to physicians and
clinicians.
[0115] An effective amount of a compound useful in the methods of
the present invention, preferably in a pharmaceutical composition,
may be administered to a mammal in need thereof by any of a number
of well-known methods for administering pharmaceutical compounds.
The compound may be administered systemically or locally.
[0116] For example, the compound may be administered orally,
intravenously, intranasally, intramuscularly, subcutaneously, or
transdermally. Other routes of administration include
intracerebroventricularly or intrathecally.
Intracerebroventiculatly refers to administration into the
ventricular system of the brain. Intrathecally refers to
administration into the space under the arachnoid membrane of the
spinal cord. Thus intracerebroventricular or intrathecal
administration may be preferred for those diseases and conditions
which affect the organs or tissues of the central nervous
system.
[0117] The compounds useful in the methods of the invention may
also be administered to mammals by sustained release, as is known
in the art. Sustained release administration is a method of drug
delivery to achieve a certain level of the drug over a particular
period of time. The level typically is measured by serum or plasma
concentration.
[0118] A description of methods for delivering a compound by
controlled release can be found in international PCT Application
No. WO 02/083106. The PCT application is incorporated herein by
reference in its entirety. Other controlled release agents are
described, for example, in U.S. Pat. Nos. 5,567,439; 6,838,094;
6,863,902; and 6,905,708. The controlled release agents and methods
for making them in these patents are incorporated herein by
reference.
[0119] Any formulation known in the art of pharmacy is suitable for
administration of the compounds useful in the methods of the
present invention. For oral administration, liquid or solid
formulations may be used. Some examples of formulations include
tablets, gelatin capsules, pills, troches, elixirs, suspensions,
syrups, wafers, chewing gum and the like. The compounds can be
mixed with a suitable pharmaceutical carrier (vehicle) or excipient
as understood by practitioners in the art. Examples of carriers and
excipients include starch, milk, sugar, certain types of clay,
gelatin, lactic acid, stearic acid or salts thereof, including
magnesium or calcium stearate, talc, vegetable fats or oils, gums
and glycols.
[0120] For systemic, intracerebroventricular, intrathecal, topical,
intranasal, subcutaneous, or transdermal administration,
formulations of the compounds useful in the methods of the present
inventions may utilize conventional diluents, carriers, or
excipients etc., such as those known in the art to deliver the
compounds. For example, the formulations may comprise one or more
of the following: a stabilizer, a surfactant, preferably a nonionic
surfactant, and optionally a salt and/or a buffering agent. The
compound may be delivered in the form of an aqueous solution, or in
a lyophilized form.
[0121] The stabilizer may, for example, be an amino acid, such as
for instance, glycine; or an oligosaccharide, such as for example,
sucrose, tetralose, lactose or a dextran. Alternatively, the
stabilizer may be a sugar alcohol, such as for instance, mannitol;
or a combination thereof Preferably the stabilizer or combination
of stabilizers constitutes from about 0.1% to about 10% weight for
weight of the compound. The surfactant is preferably a nonionic
surfactant, such as a polysorbate. Some examples of suitable
surfactants include Tween20, Tween80; a polyethylene glycol or a
polyoxyethylene polyoxypropylene glycol, such as Pluronic F-68 at
from about 0.001% (w/v) to about 10% (w/v).
[0122] The salt or buffering agent may be any salt or buffering
agent, such as for example, sodium chloride, or sodium/potassium
phosphate, respectively. Preferably, the buffering agent maintains
the pH of the pharmaceutical composition in the range of about 5.5
to about 7.5. The salt and/or buffering agent is also useful to
maintain the osmolality at a level suitable for administration to a
human or an animal. Preferably the salt or buffering agent is
present at a roughly isotonic concentration of about 150 mM to
about 300 mM.
[0123] The formulations of the compounds useful in the methods of
the present invention may additionally contain one or more
conventional additive. Some examples of such additives include a
solubilizer such as, for example, glycerol; an antioxidant such as
for example, benzalkonium chloride (a mixture of quaternary
ammonium compounds, known as "quats"), benzyl alcohol, chloretone
or chlorobutanol; anaesthetic agent such as for example a morphine
derivative; or an isotonic agent etc., such as described above. As
a further precaution against oxidation or other spoilage, the
pharmaceutical compositions may be stored under nitrogen gas in
vials sealed with impermeable stoppers.
EXAMPLES
Example 1
Screening for Arginase I Upregulators
[0124] A 2000 compound library was tested to identify arginase-1
upregulators. The library tested was The Spectrum Collection.TM.
from MicroSource Discovery System, Inc. (Groton, Conn.). The 2000
compounds in the library are primarily Food and Drug Administration
(FDA)-approved compounds or natural products. An alphabetical list
of the compounds is available at the MicroSource Discovery website
at www.msdiscovery.com/spect.html. The compounds are supplied as 10
mM solutions in dimethyl sulfoxide (DMSO).
[0125] The library was screened using murine hippocampal HT22 cells
transfected with a luciferase-arginase I construct on 96 well
plates. Total protein was also measured to use in normalization.
The luciferase assay result for each compound is normalized to
protein content and is expressed as luciferase/.mu.g protein. The
fold increase was measured relative to untreated control,
represented as sample #1 in each HT22 Arg plate.
[0126] Ratios of the results from the "luciferase assay" and
"protein assay" were tabulated. A comparison of these normalized
responses indicates amount of enhanced arginase activity. Compounds
and their respective Chem ID numbers that upregulated arginase 1
above or near 2 fold were identified. See FIG. 2. The protocol that
was used to screen for the Arginase I upregulators is described in
detail below.
Materials
[0127] T75 flask, Corning 430641 vented [0128] 96 well plate (for
tissue culture), Mictotest Primaria, flat bottom, #35-3872 [0129]
96 well plate (for chemical compound), Mictotest.TM. 96, flat
bottom, #35-3072 [0130] Dulbecco's Phosphate Buffered Saline
(1.times.PBS), from Gibco #14190-144 [0131] 0.25% Trypsin-EDTA
solution, from Sigma #T4049 [0132] DMEM (Dulbecco's Modified
Eagle's Medium), from Gibco #11995-040 & #11965-092 [0133]
Fetal Bovine Serum (FCS), from Gibco #10082-147 [0134] Penicillin
10,000 IU/ml & Streptomycin 10,000 ug/ml (P/S) from Cellgro
#30-002-C1 [0135] Puromycin 1 mg/ml stock [0136] 10 mM stock of
chemical compounds in DMSO, The Spectrum Collection.TM. from
MicroSource Discovery System. Inc; stored at -80.degree. C. [0137]
Lysis Reagent (5.times.), from Promega #E153A [0138] Luciferase
Assay Substrate, from Promega #E151A [0139] Luciferase Assay
Buffer, from Promega #E 152A [0140] LmaxII.sup.384 from Molecular
Device [0141] 70% Ethanol [0142] 96-well NUNC 236107 white plate
(Luciferase Assay) [0143] UV Plate for plate reader (Protein
Assay)
Reagents
TABLE-US-00001 [0144] Ingredient Stock Final conc. Volume DMEM for
HT22 - 4.8Kb Arg - Puro DMEM (#11965-092) 445 ml FCS 10% 50 ml P/S
3 ml Puromycin 1 mg/ml 4 .mu.g/ml 2 ml Total 500 ml Lysis Reagent
(10 ml per plate) Lysis Reagent 5x 1x 8 ml ddH.sub.2O 32 ml Total
40 ml
Day 1. Harvest cell and seeding
Materials:
TABLE-US-00002 [0145] 12x 96 well plates (#35-3872) Two types of
DMEM medium 8x T75 Flasks Trypsin solution 2x Glass sterile pipette
Timer Sterile pipette (25 ml, 10 ml, 5 ml) 6x 50 ml centrifuge
tubes Repeat pipette & tips
Steps
[0146] 1. Remove old medium from T.sub.75 flask by suction
[0147] 2. Rinse once with 5 ml 1.times.PBS; discard 1.times.PBS
[0148] 3. Add 3 ml Trypsin to treat cells and incubate at
37.degree. C. incubator for 3 min
[0149] 4. Observe under microscope to see if all cells detached
[0150] 5. Stop trypsin reaction by adding 10 ml DMEM and 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 to each
flask
[0153] 8. Prepare two 50 ml centrifuge tubes by adding 30 ml fresh
medium to each tube
[0154] 9. Discard supernatant by suction and add 8 ml 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 50
ml centrifuge tube.
[0156] 11. Use repeat pipette and sterile tips to dispense 100
.mu.l/well to six 96-well plates (for duplicate three chemical
plate) from two 50 ml centrifuge tubes
[0157] 12. Repeat step 1-9 for another cell line (total is twelve
96-well plates)
[0158] 13. Label all plates and T.sub.75 flask and incubate at
37.degree. C. incubator for 24 hours or until at least 50%
confluence
[0159] 14. Leave three chemical plates in 4.degree. C. refrigerator
to thaw overnight
Day 2. Chemical Treatment
Materials:
TABLE-US-00003 [0160] 3x 96 well plates (#35-3072) 3x Chemical
plates Repeat pipette & tips cAMP stock (2.5 mM) (positive
control) 10 .mu.l tips (12x regular and Medium (for HT22 Luc) 3x
long "reach")
Steps
[0161] 1. Check if T75 flasks reach 50-70% confluence under
microscope.
[0162] 2. Thaw chemical plates at room temperature for 10
minutes
[0163] 3. On a sterile 96 well plate, add 100 .mu.l/well DMEM
medium (for HT22 Luc)
[0164] 4. Prepare 500 .mu.M secondary chemical stocks by adding 5
.mu.l original 10 mM chemical stocks to 100 .mu.l DMEM medium
(1:20)
[0165] 5. Add 2 .mu.l/well chemical to duplicate plates (column
#2-11) and two cell lines (HT22 Luc and HT22Arg); the final
concentration is 10 .mu.M (1:50)
[0166] 6. Treat and prepare the other two chemical plates the same
way
[0167] 7. Use repeat pipette to add 2 .mu.l/well 2.5 mM cAMP stock
(positive control--f.c. 50 .mu.M DFO) to column#12 for all plates;
column#1 is negative control (Blank).
[0168] 8. Label and incubate at 37.degree. C. incubator for 24
hours
[0169] 9. Wrap and store two secondary stock plates at 4.degree. C.
refrigerator
[0170] 10. Leave original chemical plates at basement -80.degree.
C. freezer
Day 3. Lysis and Read
Materials:
TABLE-US-00004 [0171] 2x Glass sterile pipette 1x Lysis Reagent (10
ml for each plate) 12x 96-well NUNC white plate Luciferase Assay
Substrate & Buffer 12x 96-well UV plate (1 set per plate) 10
.mu.l tips (24x regular) Repeat pipette & tips Timer
Steps
[0172] 1. After 24 hrs incubation, remove all medium by suction and
add 100 .mu.l/well 1.times.Lysis Reagent <not sterile>; set
timer to record incubation time
[0173] 2. Work on one plate at a time, leave it on shaker and then
work on the second plate. Move the first plate to freezer when the
second plate is done. Repeat for all plates.
[0174] 3. The plates have to keep in freezer at least 15 minutes or
until it's ready for next step.
[0175] 4. Thaw plate at room temperature
[0176] 5. Prepare Substrate solution by mixing Luciferase Assay
Substrate (keep in freezer) with 10 ml Luciferase Assay Buffer
(thaw at room temperature)
[0177] 6. Use multiple channel pipette to transfer10 .mu.l/well
cell lysate /NUNC white plate for reading with Luciferase Assay
supernatant to a 96-well
[0178] 7. Use multiple channel pipette to transfer 2.5 .mu.l/well
cell lysate /supernatant to a 96-well UV plate for reading with
Protein Assay
[0179] 8. Stored the plate in 4.degree. C. refrigerator or basement
freezer
[0180] 9. Set up and run LmaxII.sup.384 ATP Assay: [0181]
<control><injector-M> [0182] a. Wash with 70% Ethanol
[0183] b. Wash with ddH.sub.2O [0184] c. Reverse to release waste
[0185] d. Prime LmaxII.sup.384 with Luciferase Assay Substrate
Solution [0186] e. leave plate in the equipment [0187] <Wash
steps need to be done once per day; Prime the machine if leave
without running for a long time> [0188] f. Read--ATP Assay
(.about.30 min /plate) [0189] Integrate: 5 sec/Shake: 5 sec [0190]
M-injection: volume: 100 .mu.l [0191] delay: 5sec
[0192] 10. When the plate is half done, start step 6-7 for
continuous reading.
[0193] 11. Save and calculate data
##STR00001##
Example 2
Quantitative RT-PCR and Immunoblot Analysis
[0194] In another analysis, 40 compounds were assayed in HT22 cells
on a custom plate ordered from MicroSource Discovery System, Inc.
(Groton, Conn.).
[0195] FIG. 3 lists the forty compounds that were tested, along
with their Microsource Discovery System, Inc. ChemID numbers, their
respective plate locations, and their "compound number" for the
purpose of these experiments. An "X" mark in FIG. 3 indicates
whether the compound enhanced arginase activity in the respective
assay.
[0196] Western blots were performed to measure the level of
enhanced arginase activity, i.e., arginase I expression, and MRNA
levels of arginase I. In addition, .beta.-actin messages were
measured. Western blot results were repeated, and the results of
the two experiments are shown in FIGS. 4 and 5. The sample numbers
listed on the top of each blot of FIGS. 4 and 5 correspond to the
sample number and compound listed in FIG. 3.
[0197] In FIGS. 6 through 9, the levels of Arginase I messenger RNA
(mRNA) upregulated by each of the forty compounds, normalized to
.beta.-actin, are presented. A comparison of the levels of Arginase
I MRNA upregulated by each of the forty compounds normalized to
actin indicates amount of enhanced arginase activity. Phosphate
buffered saline (PBS) was used as a negative control, and cAMP was
used as a positive control. The sample numbers (e.g., c1, c2)
listed on the bottom of each graph of FIGS. 6 through 9 correspond
to the sample number and compound listed in FIG. 3.
[0198] The protocols used for the quantitative RT-PCR and
Immunoblot analyses are described in detail below.
[0199] Quantitative RT-PCR'Total RNA was prepared from primary
mixed cortical neurons using TriZOL (Invitrogen) and cDNA
generating using a SuperScript III First-Strand Synthesis System
for RT-PCR kit (Invitrogen), according to the manufacturer's
protocol. Real time PCRs were performed as a duplex reaction using
arginase gene expression assay which uses a FAM-labeled probe, and
P-actin gene expression assay which uses a VIC-labeled probe
(Applied Biosystems, Foster City, Calif.) so that arginase
amplification could be normalized to .beta.-actin. Real time PCRs
were performed using a 7500 Real Time PCR System (Applied
Biosystems) using standard PCR protocol and amplification
conditions. See FIGS. 6 through 9.
[0200] Immunoblot Analysis--Cell lysates were obtained by rinsing
neurons with cold PBS followed by lysis in NP40 lysis buffer
(Boston Bioproducts, Worcester, Mass.). Protein concentrations in
lysates were quantified by Bradford assay (Bio-Rad, Hercules,
Calif.). Nuclear and cytoplasmic protein extractions were obtained
using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce
Biotechnology, Rockford, Ill.) according to the manufacturer's
protocol. Samples were boiled in Laemmli buffer and electrophoresed
under reducing conditions on 12% (or 7.5% for pRb immunoblots)
polyacrylamide gels. Proteins were transferred to a nitrocellulose
membrane (Bio-Rad) by electroblotting. Nonspecific binding was
inhibited by incubation in Tris-buffered saline with Tween 20
(TBST: 50 mM Tris-HCl, pH 8.0, 0.9% NaCl, and 0.1% Tween 20)
containing 5% nonfat milk for at least 1.5 h. Primary antibodies
against arginase, a-tubulin (Sigma), were diluted in TBST
containing 5% milk overnight at 4.degree. C. followed by incubation
with respective horseradish peroxidase-conjugated secondary
antibodies (Bio-Rad) for 2 hours at room temperature.
Immunoreactive proteins were detected according to the enhanced
chemiluminescent protocol (Amersham Biosciences, Sunnyvale,
Calif.). See FIGS. 4 and 5.
Example 3
MAG Inhibition Analysis; Comparison with Rho Kinase Inhibitor
[0201] MAG is a well-characterized protein of the central nervous
system (CNS) and the peripheral nervous system (PNS). MAO has been
identified as an inhibitor of axonal regeneration and neurite
outgrowth. A consequence of elevated cAMP is the synthesis of
polyamines, resulting from an up-regulation of Arginase I, a key
enzyme in their synthesis. Inhibiting polyamine synthesis blocks
the cAMP effect on regeneration. Either over-expression of Arginase
I or exogenous polyamines can overcome inhibition by MAG and by
myelin in general, as described by Cai, et al., Neuron. 2002 Aug.
15;35(4):711-9.
[0202] Compounds were tested for their ability to overcome
myelin-associated glycoprotein (MAG) inhibition in P7 rat
cerebellar neurons.
[0203] In experiments performed in the Roman Giger laboratory at
the University of Rochester School of Medicine and Dentistry,
twelve compounds were tested, using the materials and methods
described by Venkatesh, et al., J Neurosci 2005 Jan.
26;25(4):808-22. The compounds and their respective post-treatment
results are listed in FIG. 10.
[0204] The twelve compounds tested were Acetaminphen; Pinosylvin;
Resveratol 4-methyl ether, Chrysophanol (low dose); Daidzein;
Anisomycin; Methoxyvone; Dehydrovariabilin; Phenethyl caffeate
(cape); Fenbendazole; Derrustone; Epicatechin pentaacetate;
Lansoprazole.
[0205] The twelve compounds were tested in two different dilutions
(10 nM and 40 nM final concentration). FIG. 11 shows the assay
results for the compounds at 10 nM and FIG. 12 shows the assay
results for the compounds at 40 nM. The compound sample numbers
listed on the bottom of FIGS. 11 and 12 correspond to the compound
sample numbers listed in FIG. 10.
[0206] MAG-overexpressing CHO cells and control CHO feeder layers
were used in the assay. All experiments were done with Percoll
purified P7 rat cerebellar neurons. The twelve compounds were
tested in a "post-treatment" manner. Post-treatment refers to
administering the compounds after the neurons were plated.
[0207] After 24 h in culture, cells were fixed and stained with
TuJ1 (cells with neurites longer than 1 cell body diameter were
quantified). Untreated cells (first column of FIGS. 11 and 12) and
DMSO only (second column of FIGS. 11 and 12) were used as a
negative controls. RHO-kinase inhibitor Y27632 (15 microM) (third
column of FIGS. 11 and 12) was used as a positive control. All
experiments have been repeated 3-5 times independently.
[0208] In FIGS. 11 and 12, there are two bars for each compound
tested. One bar represents the results on the CHO-MAG cells
(labeled "MAG"), and the other bar (labeled "r2") is the result on
control CHO cells.
[0209] The following compounds were found to have no effect on
releasing MAG inhibition: #1 (Acetaminphen), #3 (Resveratol
4-methyl ether), #5 (Daidzein), #7 (Methoxyvone), #9 CAPE, #1
(Derrustone), and #12 (epicatechin pentaacetate).
[0210] The experiment was repeated three times with the most
promising compounds, which were compound #6 (Anisomycin), #8
(Dehydrovariabilin), #10 (Fenbendazole), #2 (Pinosylvin) and #4
(Chrysophanol at low doses).
Example 4
MAG Inhibition Analysis
[0211] MAG has been identified as an inhibitor of axonal
regeneration and neurite outgrowth. A consequence of elevated cAMP
is the synthesis of polyamines, resulting from an up-regulation of
Arginase I, a key enzyme in their synthesis. Inhibiting polyamine
synthesis blocks the cAMP effect on regeneration. Either
over-expression of Arginase I or exogenous polyamines can overcome
inhibition by MAG and by myelin in general, as described by Cai, et
al., Neuron. 2002 Aug. 15;35(4):71 1-9.
[0212] Compounds were tested for their ability to overcome
myelin-associated glycoprotein (MAG) inhibition in primary rat
neurons.
[0213] Inhibiting polyamine synthesis blocks the cAMP effect on
regeneration. Either over-expression of Arginase I or exogenous
polyamines can overcome inhibition by MAG and by myelin in
general.
[0214] In experiments performed in the Marie Filbin laboratory at
Hunter College on p5 rat cerebellar neurons, twelve compounds were
tested, using the materials and methods described by Mukhopadhyay,
et a, Neuron. 1994 Sep;13(3):757-67. The compounds and their
respective post-treatment results are listed in FIG. 13.
[0215] The twelve compounds were Acetaminphen; Pinosylvin;
Resveratol 4-methyl ether; Chrysophanol (low dose); Daidzein;
Anisomycin; Methoxyvone; Dehydrovariabilin; Phenethyl caffeate
(cape); Fenbendazole; Derrustone; Epicatechin pentaacetate;
Lansoprazole. Two experiments were done.
[0216] In a first experiment, the twelve compounds were tested for
their ability to overcome inhibition by MAG in culture. The
compounds were tested for their ability to inhibit neurite
outgrowth of MAG-expressing CHO cells. Cerebellar neurons were
cultured on MAG-expressing CHO cells and neurite length is compared
to neurons growing on control CHO cells, not expressing MAG.
[0217] The cells were assessed for their ability to overcome MAG
inhibition when added directly to the cultures at a range of
concentrations from 2 - 20 uM. Neurons were plated at a density of
about 10,000 neurons per well of an 8-well culture dish containing
a monolayer of either MAG-expressing or control CHO cells. The
co-cultures were, incubated for 16-18 h and then immunostained for
.beta.III tubulin. See FIG. 14 A-E. Neurite outgrowth was
quantified as previously described (Mukhopadhyay et al., 1994).
When added directly to the co-cultures, none of the compounds had
any effect on inhibition by MAG. MAG inhibited neurite outgrowth as
potently as when the compound was absent
[0218] Next, the compounds were tested for their ability to
overcome inhibition by MAG when the neurons were exposed to the
compounds prior to being exposed to MAG--a procedure termed,
"priming." Neurons were primed with the individual compounds at
various concentrations ranging from 2-20 uM overnight and were then
plated onto either the MAG-expressing or control CHO cell
monolayers. Of the 12 compounds tested, three were able to overcome
inhibition by MAG completely: Daidzein (20 .mu.M), Lanzoprazole (20
.mu.M) and Methoxyvone (5 .mu.M). See FIGS. 13 and 14.
[0219] In FIG. 14, P5 cerebellar neurons were plated on CONT and
MAG expressing CHO cells. See FIG. 14A-E, which are pictures
representative of .beta.III tubulin positive cells. Neurons are
plated either with DMSO (0.1%) on control CHO monolayers (FIG.
14A), or on substrate inhibitor (MAG) (FIGS. 14B-E) and treated
with DMSO (0.1%) (FIG. 14B), methoxyvone (5 .mu.M) (FIG. 14C),
daidzein (20 .mu.M) and Lanzoprazole (20 .mu.M). Graph in FIG. 14
depicts the average length of the longest neurite (percentage of
the longest neurite of the control). At least 400 neurons were
measured in each assay and the experiment was carried out at least
twice.
[0220] The other compounds did not show as strong of an effect in
overcoming MAG inhibition as the compounds Daidzein (20 .mu.M),
Lanzoprazole (20 .mu.M) and Methoxyvone (5 .mu.M). See FIG. 13. The
overcoming of MAG inhibition occurred when the cells were
"preconditioned." Preconditioning treatment means that neurons were
first treated overnight with the compound and then trypsinized,
plated, and allowed to grow for neurite extension for 16-18 h on
the top of the CHO monolayers. Neurons that were directly treated
with the different compounds on the top of the monolayers did not
show any overcoming of MAG inhibition.
[0221] In a second experiment, the twelve compounds were tested for
their ability to upregulate arginase I protein. Neurons were
treated with, diazein (20 uM), methoxyvone (5 uM) or lanzoprazole
(20 uM) for 18 hours and were then lysed and subjected to gel
electrophoresis, followed by western blotting and immunostaining
for Arginase I protein. See FIG. 15. As a positive control, neurons
were treated with 1 mM db cAMP (FIG. 15). Db-cAMP was previously
shown to increased Arginase I protein 3-fold as described by Cai,
et a, Neuron. 2002 Aug. 15;35(4):71 1-9.
[0222] As was shown previously (Cai et al., 2002), Arginase I is
up-regulated in response to treatment with dbcAMP. As shown
presently, Arginase I protein level is also up-regulated after the
treatment with diadzein (20 .mu.M), methoxyvone (5 .mu.M) or
Lanzoprazole (20 .mu.M).
* * * * *
References