U.S. patent application number 12/185480 was filed with the patent office on 2009-03-12 for catalytic antioxidants and methods of use.
This patent application is currently assigned to Florida Atlantic University. Invention is credited to Nathan Brot, Herbert Weissbach.
Application Number | 20090069428 12/185480 |
Document ID | / |
Family ID | 32393533 |
Filed Date | 2009-03-12 |
United States Patent
Application |
20090069428 |
Kind Code |
A1 |
Weissbach; Herbert ; et
al. |
March 12, 2009 |
CATALYTIC ANTIOXIDANTS AND METHODS OF USE
Abstract
The invention provides small molecules that act as catalytic
antioxidants and methods of use thereof. The compounds can
repeatedly bind and destroy reactive oxygen species by serving as
substrates for enzymes of the methionine sulfoxide reductase (Msr)
class. Some embodiments of the catalytic antioxidant compounds are
derived from drugs with anti-inflammatory activity due to
inhibition of cyclooxygenase enzymes.
Inventors: |
Weissbach; Herbert; (Boynton
Beach, FL) ; Brot; Nathan; (West Orange, NJ) |
Correspondence
Address: |
DARBY & DARBY P.C.
P.O. BOX 770, Church Street Station
New York
NY
10008-0770
US
|
Assignee: |
Florida Atlantic University
|
Family ID: |
32393533 |
Appl. No.: |
12/185480 |
Filed: |
August 4, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11512616 |
Aug 29, 2006 |
7414139 |
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12185480 |
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10723809 |
Nov 26, 2003 |
7129374 |
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11512616 |
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60429269 |
Nov 26, 2002 |
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Current U.S.
Class: |
514/562 ;
562/430 |
Current CPC
Class: |
A61K 38/00 20130101;
C07C 2602/08 20170501; C07C 317/50 20130101; C07C 323/60 20130101;
C07D 209/26 20130101; C07K 5/06078 20130101; C07C 317/44 20130101;
C07K 5/06026 20130101; A61P 25/00 20180101; C07D 307/58 20130101;
C07C 323/59 20130101 |
Class at
Publication: |
514/562 ;
562/430 |
International
Class: |
A61K 31/195 20060101
A61K031/195; C07C 317/06 20060101 C07C317/06; A61P 25/00 20060101
A61P025/00 |
Claims
1-2. (canceled)
3. A compound having formula 6, or a pharmaceutically acceptable
salt thereof; ##STR00008## wherein: the aromatic ring includes one
or more nitrogen atoms; the aromatic carboxyl group is oriented
ortho, meta, orpara to the methionine-based moiety; R.sub.1 is CH
of either t or S configuration; R.sub.2 is a normal or branched
alkyl or fluoroalkyl group having 1 to 6 carbons; R.sub.3 is methyl
or ethyl or a fluorinated derivative thereof; R.sub.4 is a hydrogen
or a normal or branched alkyl group having 1 to 6 carbons; R.sub.5
is a nitrogen with substituent R.sub.4 as defined herein, an
oxygen, or a sulfur; and X is S or Se in any oxidation state.
4. The compound of claim 3 having formula 6a, or a pharmaceutically
acceptable salt thereof: ##STR00009##
5. (canceled)
6. A compound having formula 7a, or a pharmaceutically acceptable
salt thereof: ##STR00010##
7. (canceled)
8. A compound having formula 8a, or a pharmaceutically acceptable
salt thereof: ##STR00011##
9-13. (canceled)
14. A method for reducing, preventing or reversing oxidative damage
in a cell, the method comprising the steps of: (a) providing a
non-naturally occurring compound comprising at least one methyl
sulfide or methyl sulfoxide moiety, the compound being a substrate
for at least one Msr enzyme; (b) providing a cell expressing at
least one Msr enzyme, said cell comprising or being exposed to
reactive oxygen species; and (c) contacting the cell with an amount
of the compound sufficient to reduce, prevent, or reverse oxidative
damage in the cell by said reactive oxygen species.
15. The method of claim 14, wherein the cell is within an animal
subject.
16. The method of claim 14, wherein the animal subject has a
condition or disorder associated with oxidative damage.
17. The method of claim 14, wherein the disorder involves
degeneration of a nerve cell.
18. The method of claim 14, wherein the condition is
age-related.
19. A method for extending the lifespan of an animal comprising
administering to the animal a therapeutically effective amount of a
non-naturally occurring compound according to any one of claims 3,
4, 6, or 8.
20. A method of treating a disease related to oxidative damage in a
cell consisting essentially of administering to a patient in need
thereof, a compound according to any one of claims 3, 4, 6, or 8,
wherein said disease is selected from: smokers, emphysema,
reperfusion damage, Alzheimer's disease, Parkinson's disease,
amyotrophic lateral sclerosis (ASL), heart attacks and stroke.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the priority of U.S.
provisional patent application No. 60/429,269 filed on Nov. 26,
2002.
FIELD OF THE INVENTION
[0002] The invention relates to the fields of biochemistry,
pharmacology, and medicine. More particularly, the invention
relates to methods and compositions for promoting health and
increasing longevity by reducing oxidative damage to cells and
tissues.
BACKGROUND
[0003] Oxygen is involved in a wide range of normal metabolic
reactions and is essential for the survival of all aerobic
organisms, including human beings. Reactive oxygen species (ROS),
such as superoxide, are produced in abundance as a byproduct of the
incomplete reduction of oxygen that has entered the respiratory
chain. Superoxide is the precursor of other damaging oxygen species
including hydrogen peroxide, the hypochlorite ion and the hydroxyl
radical. Oxidase enzymes in cells such as phagocytes and nitric
oxide synthases are other sources of ROS.
[0004] While low levels of ROS are present under normal
physiological conditions, in excess, ROS can cause oxidative damage
to cells and tissues by, for example, oxidizing cellular
macromolecules such as nucleic acids, lipids and proteins.
Cumulative damage to cells in this manner can result in pathology.
Not surprisingly then, oxidative damage has been implicated in a
wide variety of diseases and conditions including chronic
obstructive lung disorders such as smoker's emphysema, reperfusion
damage, neurodegenerative diseases such as Alzheimer's disease,
Parkinson's disease, and amyotrophic lateral sclerosis (ALS), heart
attacks, stroke, several autoimmune diseases, and aging.
[0005] Regarding the latter, oxidative damage to cellular
macromolecules has been postulated to accelerate the aging process
and shorten lifespan. For example, the level of oxidized methionine
in proteins in an animal has been observed to increase with the age
of the animal. Moreover, in Drosophila, greater resistance to ROS
via over-expression of superoxide dismutase and catalase has been
correlated with longer lifespan, whereas genetic disruption of
superoxide dismutase and catalase has been correlated with shorter
lifespan.
[0006] Although cells have evolved their own enzymatic antioxidant
systems (e.g., superoxide dismutase, catalase, and peroxidase) to
neutralize ROS, such systems may not function at ideal levels to
minimize the rate of aging and the development of disease.
Accordingly, there is a clear need for non-naturally occurring
compositions and methods that reduce oxidative damage to cells. One
approach to increase the antioxidant activity in cells is to
provide cells with compounds that directly scavenge ROS, e.g.,
vitamins C, E, and A, glutathione, ubiquinone, uric acid,
carotenoids, and the like. Such conventional antioxidant compounds,
however, lose activity after neutralizing only one or two ROS
molecules. They are thus limited by the relatively small quantities
of ROS that they can destroy.
SUMMARY
[0007] The invention relates to the development of methyl sulfoxide
or methyl sulfide containing catalytic antioxidants that can
repeatedly be oxidized by a ROS, reduced back to an unoxidized
form, and oxidized again by a ROS. Unlike a conventional
antioxidant molecule, a single catalytic antioxidant molecule of
the invention can neutralize a multitude of different ROS
molecules.
[0008] The regenerative capacity of the catalytic antioxidant
molecules of the invention is based on their ability to act as
substrates for the methionine sulfoxide reductase (Msr) class of
enzymes. Among the various amino acids found in proteins,
methionine (Met) is one of the most susceptible to oxidation.
Oxidation of methionine by ROS yields methionine sulfoxide
[Met(O)]. The Msr enzymes, including MsrA and MsrB prominent in
virtually all cells, including mammalian cells, act as repair
enzymes that catalyze the reversal of the oxidation reaction,
reducing Met(O) back to methionine. In addition to reducing
methionine, MsrA and several other forms of Msr enzymes known in
bacteria can reduce a variety of other substrates, but in all cases
the core functional group recognized by the enzymes is a methyl
sulfoxide moiety. By reducing methyl sulfoxide moieties back to
methyl sulfide, the Msr enzymes repair damaging oxidation reactions
to methionine in proteins. In addition the methionine residues in
proteins, via cyclic oxidation and reduction by the Msr system, can
act as scavengers of ROS. In these ways the Msr system is believed
to contribute to the longevity and health of cells by conferring
resistance to ROS (reviewed in Weissbach et al., Archiv. Biochem.
Biophys. 397:172 178, 2002).
[0009] The catalytic antioxidants of the invention are small
molecules that act as substrates for Msr enzymes. A scheme of
interaction of the compounds of the invention with the Msr pathway
is shown diagrammatically in FIG. 1. Methyl sulfide groups on the
antioxidant compounds can react with reactive oxygen species (ROS)
such as superoxide or hydrogen peroxide to form methyl sulfoxides,
for example methionine sulfoxide, which occurs in proteins and in
the free form in cells. Upon trapping and destruction of the ROS by
a catalytic antioxidant compound of the invention, the methyl
sulfoxide formed thereon can serve as a substrate for one or more
Msr enzymes. Nucleophilic attack of the methyl sulfoxide by a
cysteine residue in the Msr enzyme leads to transfer of the oxygen
from the compound to the enzyme, reducing the compound back to its
unoxidized state (FIG. 1). The compounds, thus regenerated, are
available for repeated reuse as antioxidants. Thus, the catalytic
antioxidant compounds of the invention function not only as typical
ROS scavengers, but also regenerate themselves by harnessing the
catalytic action of the Msr enzymes.
[0010] Accordingly, in one aspect, the invention features
non-naturally occurring (or purified, naturally occurring)
compounds including at least one methyl sulfide or methyl sulfoxide
moiety, the compounds being a substrate for at least one MsrA
enzyme and at least one MsrB enzyme, or a pharmaceutically
acceptable salt thereof, Certain embodiments of the compounds are
based on a backbone derived from the chemical structure of sulindac
(1(Z)-5-fluoro-2-methyl-1
[[4-(methylsulfinyl)phenyl)methylene]-1-H-indenyl-3-acetic
acid).
[0011] Other embodiments of the compounds are non-naturally
occurring (or purified, naturally occurring) compounds including at
least one methyl sulfide or methyl sulfoxide moiety, the compounds
being a substrate for at least one Msr enzyme and having a backbone
not based on sulindac. Various embodiments of these compounds have
a backbone based on several known cyclooxygenase (COX) inhibitors,
including acetyl salicylic acid, mefenamic acid, ibuprofen,
indomethacin, and rofecoxib (VIOXX.TM.). The invention also
includes compositions based on these compounds in a
pharmaceutically acceptable carrier.
[0012] In another aspect, the invention provides a method for
reducing, preventing or reversing oxidative damage in a cell. The
method includes the steps of: (a) providing a non-naturally
occurring (or purified, naturally occurring) compound including in
its chemical structure at least one methyl sulfide or methyl
sulfoxide moiety, the compound being a substrate for at least one
Msr enzyme; (b) providing a cell expressing at least one Msr
enzyme, the cell containing or being exposed to reactive oxygen
species; and (c) contacting the cell with an amount of the compound
sufficient to reduce, prevent, or reverse oxidative damage in the
cell by the reactive oxygen species.
[0013] The cell can be within an animal subject, such as a human
being. The animal subject can have a condition or disorder
associated with oxidative damage. The disorder can involve
degeneration of a nerve cell. The condition affecting the subject
can be age-related.
[0014] Yet another embodiment of the invention is a method for
extending the lifespan of an animal. The method involves
administering to the animal a therapeutically effective amount of a
non-naturally occurring (or purified, naturally occurring) compound
including at least one methyl sulfide or methyl sulfoxide moiety,
the compound being a substrate for at least one Msr enzyme.
[0015] As used herein, the terms "methionine moiety" and
"methionine analog" include all structures encompassed by general
methionine formula 1 described herein, including selenomethionine
derivatives.
[0016] As used herein, the term "catalytic antioxidant" refers to a
non-naturally occurring (or purified, naturally occurring)
antioxidant compound that can be enzymatically regenerated after it
is oxidized by an oxidizing agent (for example a ROS) such that
each equivalent of antioxidant compound can destroy more than one
equivalent of the oxidizing agent.
[0017] Unless otherwise defined, all technical terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. Although methods
and materials similar or equivalent to those described herein can
be used in the practice or testing of the present invention,
suitable methods and materials are described below. The particular
embodiments discussed below are illustrative only and not intended
to be limiting. All publications, patent applications, patents, and
other references mentioned herein are incorporated by reference in
their entirety. In the case of conflict, the present specification,
including definitions will control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a schematic diagram showing the mechanism of
action of a catalytic anti-oxidant, according to an embodiment of
the invention.
[0019] FIG. 2 is a schematic diagram showing the cycle of catalytic
antioxidant activity of sulindac, catalyzed by an MsrA enzyme,
according to an embodiment of the invention.
[0020] FIGS. 3A and 3B is two graphs showing kinetics of sulindac
sulfide production by MsrA, according to an embodiment of the
invention.
[0021] FIGS. 4A and 4B is a schematic diagram showing chemical
synthetic pathways for making methionine derivatives of sulindac
(compounds 2a and 3a), according to an embodiment of the
invention.
[0022] FIGS. 5A and 5B is a schematic diagram showing chemical
synthetic pathways for making methionine derivatives of sulindac
(compounds 4a and 5a), according to an embodiment of the
invention.
[0023] FIGS. 6A and 6B is a schematic diagram showing chemical
synthetic pathways for making catalytic antioxidants based on
salicylic acid and mefenamic acid (compounds 6a and 7a,
respectively), according to an embodiment of the invention.
[0024] FIGS. 7A C is a schematic diagram showing chemical synthetic
pathways for making catalytic antioxidants based on ibuprofen,
indomethacin and VIOXX.TM. (compounds 8a, 9a, and 10a,
respectively), according to an embodiment of the invention.
[0025] FIG. 8 shows a NMR spectrum of compound 2a of the
invention.
[0026] FIG. 9 is a micrograph of a TLC plate showing the presence
of reduction products of sulindac (S) and sulindac methionine
sulfoxide (SMO) following incubation with MsrA and MsrB enzymes.
Results demonstrate that S is a substrate for MsrA and that SMO is
a substrate for both MsrA and MsrB.
[0027] FIG. 10 is a graph showing enhanced survival of
sulindac-treated flies exposed to oxidative stress induced by
paraquat.
[0028] FIG. 11 is a graph showing enhanced survival of G93A
trausgenic mice over expressing a mutant superoxide dismutase with
neurodegenerative disease treated with sulindac.
[0029] FIG. 12 is a graph showing enhanced motor performance of
sulindac-treated transgenic G93A mice.
[0030] FIG. 13 is a graph showing neuronal cell counts in sections
of spinal cords of G93A mice. Neuronal cell survival is
significantly higher in animals receiving sulindac.
DETAILED DESCRIPTION
[0031] The invention encompasses compositions and methods relating
to catalytic antioxidants useful in reducing or preventing
oxidative damage in cells. The antioxidant compounds contain active
sites that capture ROS. The antioxidant ability of the compounds is
regenerated following capture of ROS by interaction with enzymes of
the Msr class that reduce methyl sulfoxide moieties back to the
methyl sulfide.
[0032] The below described preferred embodiments illustrate various
compositions and methods within the invention. Nonetheless, from
the description of these embodiments, other aspects of the
invention can be made and/or practiced based on the description
provided below.
Biological Methods
[0033] Methods involving conventional chemistry, cell biology and
molecular biology techniques are described herein. Such techniques
are generally known in the art and are described in detail in
methodology treatises such as Classics in Total Synthesis. Targets,
Strategies, Methods, K. C. Nicolaou and E. J. Sorensen, VCH, New
York, 1996; and The Logic of Chemical Synthesis, E. J. Coney and
Xue-Min Cheng, Wiley & Sons, New York, 1989. Molecular
biological and cell biological methods are described in treatises
such as Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1 3,
ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 2001; and Current Protocols in Molecular
Biology, ed. Ausubel et al., Greene Publishing and
Wiley-Interscience, New York, 1992 (with periodic updates).
Catalytic Antioxidants Having Methyl Sulfide or Methyl Sulfoxide
Groups
[0034] The invention provides small molecules containing at least
one (e.g., 1, 2, 3 or more) methyl sulfoxide or methyl sulfide
group that can enter cells and prevent oxidative damage by a
catalytic antioxidant mechanism. The methyl sulfide group on the
compounds reacts with ROS, forming methyl sulfoxide. The methyl
sulfoxide-bearing compounds, in turn, act as substrates for Msr
enzymes which reduce the compounds and thereby regenerate their
antioxidant properties. These compounds can be administered to
cells or animals to reduce cellular damage caused by ROS.
[0035] Referring to FIG. 1, these compounds serve 1) as ROS
scavengers (antioxidants) by virtue of the active groups within
their structures that destroy or react with ROS, and 2) as
catalytic antioxidants by acting as substrates for Msr enzymes that
reduce the oxidized compounds back to the unoxidized form capable
of further reaction with ROS. The catalytic nature of the
antioxidant compounds of the invention is due to their ability to
serve as substrates for Msr enzymes. The core functional group
recognized by these enzymes is methyl sulfoxide. In the case of
N-methionine-containing peptide and protein substrates, this
functional group is contained within the amino acid methionine.
[0036] Any compound having a methyl sulfide or methyl sulfoxide
functional group that is a substrate for a Msr enzyme can be used.
Sulindac, a non-steroidal anti-inflammatory drug and COX inhibitor,
is one example of a methyl sulfoxide-containing compound that
serves as a substrate for Msr enzymes. Sulindac is a pro drug, and
is only active as a COX inhibitor when the methyl sulfoxide moiety
on the molecule is reduced to the sulfide. Heretofore, sulindac was
not known to act as a substrate for a Msr. FIG. 2 shows the
reduction of sulindac to sulindac sulfide, catalyzed by Msr. As
described below, sulindac was tested as a substrate against six
known members of the Msr family identified in bacteria (E. coli)
and against Msr enzymes present in mammalian (bovine) tissues. MsrA
and a membrane-associated Msr of bacteria were shown to be able to
reduce sulindac to the active sulfide. In mammalian tissues,
reduction of sulindac was primarily attributable to the activity of
MsrA.
[0037] As further described below, sulindac administration (1)
protected Drosophila against the damage from paraquat-induced ROS
production, (2) prolonged the survival of spinal cord motor neurons
in mice with a neurodegenerative disease caused by oxidative
damage, and (3) extended the lifespan of the foregoing mice.
Methionine-Based Catalytic Antioxidants
[0038] In one aspect, the invention provides catalytic antioxidant
compounds having methionine moieties or analogs of methionine. Such
compounds are substrates for Msr enzymes that recognize the methyl
sulfoxide functional group in methionine (for example, MsrA and
MsrB). The methionine moiety or analog found in the
methionine-containing embodiments of the compounds has the
following general structure:
##STR00001##
[0039] Groups R.sub.1, R.sub.2, R.sub.3, and X in general structure
1 are defined as follows:
[0040] R.sub.1 may be CH (of either R or S configuration).
[0041] R.sub.2 may be a normal or branched alkyl or fluoroalkyl
group having 1 to 6 carbons.
[0042] R.sub.3 may be ethyl or preferably methyl, or a fluorinated
derivative thereof.
[0043] X may be either S or Se in any oxidation state.
[0044] As used herein, the terms "methionine moiety" and
"methionine analog" include all structures encompassed by general
formula 1, including selenomethionine analogs of methionine.
General structure 1 also includes esters and salts of the
carboxylic acid. Oligopeptides containing methionine for attachment
to small molecules are also encompassed by the invention.
Methionine-Based Catalytic Antioxidants Derived from COX
Inhibitors
[0045] Inflammation and oxidative damage are known to coexist in
many disease states and degenerative conditions. Accordingly,
particularly preferred embodiments of the methionine-containing
compounds of the invention are derivatives of anti-inflammatory
agents such as COX inhibitors. Specific examples of such compounds,
employing scaffolds based on several COX inhibitors, and methods
for their synthesis are provided in the examples below. Exemplary
compounds include those derived from the following scaffolds:
sulindac; acetyl salicylic acid (ortho-acetoxybenzoic acid),
mefenamic acid (2-[(2,3-Dimethylphenyl)amino]benzoic acid);
ibuprofen (.alpha.-methyl-4-(2-methylpropyl)-benzeneacetic acid);
indomethacin
(1-(p-chlorobenzoyl)-5-methoxy-2-methyl-indole-3-acetic acid); and
rofecoxib (4-[4-(methylsulfonyl)phenyl)-3-phenyl-2(5H)-furanone,
for example, VIOXX.TM., sold by Merck) and celecoxib
(4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-benzenesulfon-
-amide, for example, CELEBREX.TM. sold by Pfizer.
[0046] Embodiments of the invention that are sulindac derivatives
can have the following general formulas 2 5: Embodiments of the
invention that are sulindac derivatives have the following general
formulas 2-5:
##STR00002##
[0047] Groups R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
and X in general formulas 2, 3, 4 and 5 are defined as follows:
[0048] R.sub.1 may be CH (of either R or S configuration).
[0049] R.sub.2 may be a normal or branched alkyl or fluoroalkyl
group having 1 to 6 carbons.
[0050] R.sub.3 may be ethyl or preferably methyl, or a fluorinated
derivative thereof.
[0051] R.sub.4 may be a hydrogen or a normal or branched alkyl
group having 1 to 6 carbons.
[0052] R.sub.5 may be a CH (of either R or S configuration).
[0053] R.sub.6 is a may be a hydrogen or a normal or branched alkyl
or fluoroalkyl group having 1 to 6 carbons.
[0054] R.sub.7 may be a nitrogen (with substituent R.sub.4 as
defined above), a CH (of either R or S configuration), or a normal
or branched alkyl or fluoroalkyl group having 1 to 6 carbons.
[0055] X may be either S or Se in any oxidation state.
[0056] General structures 2, 3, 4 and 5 also include esters and
salts of the carboxylic acid group. The invention also encompasses
sulindac derivatives containing oligomeric methionine moieties and
analogs.
[0057] Embodiments of the invention that are acetyl salicylic acid
derivatives can have the following general formula:
##STR00003##
[0058] The aromatic ring of general structure 6 may contain one or
more nitrogen atoms (for example pyridine or pyrazine). The
aromatic carboxyl group in general structure 6 may be oriented
ortho, meta, orpara to the methionine-based moiety. Groups R.sub.1,
R.sub.2, R.sub.3, R.sub.4, R.sub.5, and X in the general structure
are defined as follows:
[0059] R.sub.1 may be CH (of either R or S configuration).
[0060] R.sub.2 may be a normal or branched alkyl or fluoroalkyl
group having 1 to 6 carbons.
[0061] R.sub.3 may be ethyl or preferably methyl or a fluorinated
derivative thereof.
[0062] R.sub.4 may be a hydrogen or a normal or branched alkyl
group having 1 to 6 carbons.
[0063] R.sub.5 may be a nitrogen (with substituent R.sub.4 as
defined above), an oxygen, or a sulfur.
[0064] X may be either S or Se in any oxidation state.
[0065] General structure 6 also includes esters and salts of the
carboxylic acid group, The invention also encompasses acetyl
salicylic acid derivatives containing oligomeric methionine
moieties and analogs.
[0066] Embodiments of the invention that are mefenamic acid
derivatives can have the following general formula:
##STR00004##
[0067] Both aromatic rings of general structure 7 may contain one
or more nitrogen atoms (for example pyridine or pyrazine). The
aromatic carboxyl group in general structure 7 may be oriented
ortho, meta, orpara to the aniline nitrogen. Groups R.sub.1,
R.sub.2, R.sub.3, R.sub.4, and X in the general structure are
defined as follows:
[0068] R.sub.1 may be CH (of either R or S configuration).
[0069] R.sub.2 may be a normal or branched alkyl or fluoroalkyl
group having 1 to 6 carbons.
[0070] R.sub.3 may be ethyl or preferably methyl or a fluorinated
derivative thereof.
[0071] R.sub.4 may be a hydrogen or a normal or branched alkyl
group having 1 to 6 carbons.
[0072] X may be either S or Se in any oxidation state.
[0073] General structure 7 also includes esters and salts of the
carboxylic acid group. Mefenamic acid derivatives containing
oligomeric methionine are also envisioned.
[0074] Embodiments of the invention that are ibuprofen derivatives
have the following general formula:
##STR00005##
[0075] The aromatic ring of general structure 8 may contain one or
more nitrogen atoms (for example pyridine or pyrazine). The
sec-butyl group in general structure 8 may be oriented ortho, meta,
or para to the methionine-based moiety. Groups R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, and X in the general structure are
defined as follows:
[0076] R.sub.1 may be CH (of either R or S configuration).
[0077] R.sub.2 may be a normal or branched alkyl or fluoroalkyl
group having 1 to 6 carbons.
[0078] R.sub.3 may be ethyl or preferably methyl or a fluorinated
derivative thereof.
[0079] R.sub.4 may be a hydrogen or a normal or branched alkyl
group having 1 to 6 carbons.
[0080] R.sub.5 may be a CH (of either R or S configuration).
[0081] X may be either S or Se in any oxidation state.
[0082] General structure 8 also includes esters and salts of the
carboxylic acid group. The invention also encompasses ibuprofen
derivatives containing oligomeric methionine moieties and
analogs.
[0083] Embodiments of the invention that are indomethacin
derivatives can have the following general formula:
##STR00006##
[0084] Groups R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
R.sub.7 and X in general structure 9 are defined as follows:
[0085] R.sub.1 may be CH (of either R or S configuration).
[0086] R.sub.2 may be a normal or branched alkyl or fluoroalkyl
group having 1 to 6 carbons.
[0087] R.sub.3 may be ethyl or preferably methyl or a fluorinated
derivative thereof.
[0088] R.sub.4 may be a hydrogen or a normal or branched alkyl
group having 1 to 6 carbons.
[0089] R.sub.5 may be a CH (of either R or S configuration).
[0090] R.sub.6 is a may be a hydrogen or a normal or branched alkyl
or fluoroalkyl group consisting of 1 to 6 carbons.
[0091] R.sub.7 may be any halogen oriented ortho, meta, or para to
the carbonyl group.
[0092] X may be either S or Se in any oxidation state.
[0093] General structure 9 also includes esters and salts of the
carboxylic acid group. The invention also encompasses indomethacin
derivatives containing oligomeric methionine moieties and
analogs.
[0094] Embodiments of the invention that are Vioxx.RTM. derivatives
can have the following general formula:
##STR00007##
[0095] The lactone ring in general structure 10 may be oriented
ortho, meta, or para to sulfonyl group. Groups R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and X in general structure 10 are defined as
follows:
[0096] R.sub.1 may be CH (of either R or S configuration).
[0097] R.sub.2 may be a normal or branched alkyl or fluoroalkyl
group having 1 to 6 carbons.
[0098] R.sub.3 may be ethyl or preferably methyl or a fluorinated
derivative thereof.
[0099] R.sub.4 may be a hydrogen or a normal or branched alkyl
group having 1 to 6 carbons.
[0100] X may be either S or Se in any oxidation state.
[0101] Ar may be phenyl, alkyl and halogen substituted phenyl, and
heteroaromatic compounds.
[0102] General structure 10 also includes esters and salts of the
carboxylic acid group. The invention also encompasses Vioxx.RTM.
derivatives containing oligomeric methionine moieties and
analogs.
Testing of Catalytic Antioxidant Compounds
[0103] The ability of any given molecule having a chemical
structure including at least one methyl sulfoxide- and/or methyl
sulfide-containing moiety, or at least one methionine and/or
methionine sulfoxide moiety to act as a catalytic antioxidant can
be determined empirically.
[0104] For example, a molecule containing a methyl sulfoxide group
to be tested (i.e., a test molecule) can be subjected to an
enzymatic assay that indicates if the test molecule can serve as a
substrate for MsrA, MsrB or other members of the Msr family (see,
for instance, the NADPH assay described in Example 1, and the
extraction assay described in Example 2, below). A test molecule
can also be subjected to an assay that indicates the molecule's
ability to increase resistance to oxidative stress in cells in
vitro (for example PC-12 cells subjected to insult with MTP+) or in
an animal subject (for example, Drosophila or a mammalian model of
oxidative damage) (see, for instance, the assays described in
Examples 7, 8 and 9 below).
Preventing/Reversing Oxidative Damage in a Cell
[0105] The catalytic antioxidant molecules of the invention can be
used to reduce, prevent or reverse oxidative damage in a cell (for
example, a cell in an animal). In this method, a non-naturally
occurring catalytic antioxidant compound is brought into contact
with the cell. After entering the interior of the cell, the
compound, if in the reduced (sulfide) form, will be oxidized to the
sulfoxide by ROS (i.e., act as a ROS scavenger). Subsequent
reduction catalyzed by an Msr enzyme will regenerate the original
sulfide. If the test molecule contains a methyl sulfoxide moiety,
it will be reduced to the sulfide by the Msr system within the cell
and subsequently act as an antioxidant. With either the sulfide or
the sulfoxide as the test molecule, the oxidation/reduction cycle
will permit the compound to destroy ROS catalytically, as shown in
FIG. 1.
[0106] The effectiveness of particular compounds can be assessed
using conventional in vitro and in vivo assays, for example,
determining a cell's, or an animal's response to a challenge with
an agent that produces ROS. For instance, to assess a test molecule
for the ability to prevent oxidative damage caused by ROS in a
cell, cells can be cultured by conventional means and challenged
with an agent that produces ROS within the cells. An exemplary
cellular system for testing the effect of ROS damage in nerve
cells, for example, is an assay employing PC-12 cells subjected to
insult with MPP+, an agent that generates superoxide and other
oxygen radicals. To assess the efficacy of a test compound in an
animal, Drosolphila melanogaster (fruit fly) is an excellent animal
model. The flies can be treated with an agent that produces ROS
(for example, paraquat) and then fed with a diet containing the
test molecule and monitored for their survival, compared to control
flies receiving Paraquat alone. Mammalian models of oxidative
damage are also well known and include inter alia a transgenic
mouse model of amyotrophic lateral sclerosis (ALS) based on a
mutation in the superoxide dismutase (SOD1) gene.
Animal Subjects
[0107] Because oxidative damage to cells is a ubiquitous
phenomenon, the invention is believed to be compatible with any
animal subject. A non-exhaustive list of examples of such animals
includes mammals such as mice, rats, rabbits, goats, sheep, pigs,
horses, cattle, dogs, cats, and primates such as monkeys, apes, and
human beings. Those animal subjects that have a disease or
condition that relates to oxidative damage are preferred for use in
the invention as these animals may have the symptoms of their
disease reduced or even reversed. In particular, human patients
suffering from inflammation, chronic obstructive lung diseases such
as emphysema, reperfusion damage after heart attack or stroke,
neurodegenerative diseases (for example, Parkinson's disease,
Alzheimer's disease, and ALS), autoimmune diseases such as
rheumatoid arthritis, lupus, and Crohn's disease, conditions
related to premature birth, conditions caused by exposure to
ultraviolet light, and age-related conditions (as but one example,
age-related degenerative conditions of the eye including
age-related macular degeneration and cataract formation) are
suitable animal subjects for use in the invention. In the
experiments described herein, animals used for demonstration of
beneficial effects of protection against ROS damage by the
compounds of the invention are the fruit fly and the mouse.
Nonetheless, by adapting the methods taught herein to other methods
known in medicine or veterinary science (for example, adjusting
doses of administered substances according to the weight of the
subject animal), the compounds and compositions of the invention
can be readily optimized for use in other animals.
Administration of Compositions
[0108] The catalytic antioxidant compositions of the invention may
be administered to animals including humans in any suitable
formulation. For example, the compositions may be formulated in
pharmaceutically acceptable carriers or diluents such as
physiological saline or a buffered salt solution. Suitable carriers
and diluents can be selected on the basis ofi mode and route of
administration and standard pharmaceutical practice. A description
of other exemplary pharmaceutically acceptable carriers and
diluents, as well as pharmaceutical formulations, can be found in
Remington's Pharmaceutical Sciences, a standard text in this field,
and in USP/NF. Other substances may be added to the compositions to
stabilize and/or preserve the compositions, or enhance the activity
of the Msr system. One such enhancing substance could be
nicotinamide which is part of the molecule, NADPH, that supplies
the reducing power to the reaction catalyzed by the members of the
Msr family.
[0109] The compositions of the invention may be administered to
animals by any conventional technique. Such administration may be
oral or parenteral (for example, by intravenous, subcutaneous,
intramuscular, or intraperitoneal introduction). The compositions
may also be administered directly to the target site by, for
example, surgical delivery to an internal or external target site,
or by catheter to a site accessible by a blood vessel. Other
methods of delivery, for example, liposomal delivery or diffusion
from a device impregnated with the composition, are known in the
art. The compositions may be administered in a single bolus,
multiple injections, or by continuous infusion (for example,
intravenously or by peritoneal dialysis). For parenteral
administration, the compositions are preferably formulated in a
sterilized pyrogen-free form.
[0110] Compositions of the invention can also be administered in
vitro to a cell (for example, to prevent oxidative damage during ex
vivo cell manipulation, for example of organs used for organ
transplantation or in in vitro assays) by simply adding the
composition to the fluid in which the cell is contained.
Effective Doses
[0111] An effective amount is an amount which is capable of
producing a desirable result in a treated animal or cell (for
example, reduced oxidative damage to cells in the animal or cell).
As is well known in the medical and veterinary arts, dosage for any
one animal depends on many factors, including the particular
animal's size, body surface area, age, the particular composition
to be administered, time and route of administration, general
health, and other drugs being administered concurrently. It is
expected that an appropriate dosage for parenteral or oral
administration of compositions of the invention would be in the
range of about 1 .mu.g to 100 mg/kg of body weight in humans. An
effective amount for use with a cell in culture will also vary, but
can he readily determined empirically (for example, by adding
varying concentrations to the cell and selecting the concentration
that best produces the desired result). It is expected that an
appropriate concentration would be in the range of about 0.0001 100
mM. More specific dosages can be determined by the method described
below.
[0112] Toxicity and efficacy of the compositions of the invention
can be determined by standard pharmaceutical procedures, using
cells in culture and/or experimental animals to determine the
LD.sub.50 (the dose lethal to 50% of the population) and the
ED.sub.50 (the dose that effects the desired result in 50% of the
population). Compositions that exhibit a large LD.sub.50/ED.sub.50
ratio are preferred. Although less toxic compositions are generally
preferred, more toxic compositions may sometimes be used in in vivo
applications if appropriate steps are taken to minimize the toxic
side effects.
[0113] Data obtained from cell culture and animal studies can be
used in estimating an appropriate dose range for use in humans. A
preferred dosage range is one that results in circulating
concentrations of the composition that cause little or no toxicity.
The dosage may vary within this range depending on the form of the
composition employed and the method of administration.
EXAMPLES
[0114] The present invention is further illustrated by the
following specific examples, which should not be construed as
limiting the scope or content of the invention in any way.
Example 1
Sulindac is a Substrate for MsrA Enzyme
[0115] The enzyme methionine sulfoxide reductase (MsrA) is known to
exhibit broad specificity for compounds that contain a methyl
sulfoxide group. This example provides evidence that sulindac, a
known antioxidant containing a methyl sulfoxide moiety, can act as
a substrate for MsrA.
[0116] Materials and Methods.
[0117] Reductase assay. Reaction mixtures were prepared containing
50 mM Tris-Cl pH 7.4, 15 .mu.g of E. coli thioredoxin, 1 .mu.g E.
coli thioredoxin reductase, 100 nmoles of NADPH, 1 .mu.mole of
sulindac and 100-400 ng of MsrA in a final volume of 500 .mu.l.
Incubations were performed at 37.degree. C. for various times.
[0118] The amount of product (sulindac sulfide) synthesized was
determined by measuring the oxidation of NADPH
spectrophotometrically at 340 nm. Because sulindac absorbs very
strongly at this wavelength, the loss of absorbance at 340 nm could
not be measured directly. To accomplish this, the sulindac and
sulindac sulfide were removed from the incubations by extraction
with ethyl acetate as follows. At the end of incubation, 500 .mu.l
of 0.5 M Bis-Tris-Cl pH 5.5 and 3 ml of ethyl acetate were added.
The tubes were mixed (vortexed) for 5 seconds (3 times). After
separation, the organic phase was removed and another 3 ml of ethyl
acetate were added. After mixing the organic phase was again
removed. The two extractions essentially removed all of the
sulindac and sulindac sulfide, leaving the NADPH in the aqueous
phase, which was measured at 340 nm. The loss of absorption at 340
nm, dependent on sulindac, is a measure of sulindac reduction. (
0.062 at 340 nm=10 nmoles of sulindac sulfide formed).
[0119] Results.
[0120] With a purified enzyme, sulindac reduction can be measured
by a modified NADPH oxidation assay. The results of a reductase
assay using MsrA from E. coli are summarized below in Table 1.
TABLE-US-00001 TABLE 1 Reduction of Sulindac to Sulindac Sulfide by
MsrA Sulindac MsrA Sulindac Thioredoxin MetS(o) Time .DELTA.
sulfide Tube # (100 ng/.mu.l) (0.2 M) (5 .mu.g/.mu.l) (0.2 M) (min)
OD.sub.340 OD.sub.340 (nmol) 1 8 .mu.l -- 3 .mu.l -- 0.657 0 2 -- 5
.mu.l 3 .mu.l -- 0.660 0 3 1 .mu.l 5 .mu.l 3 .mu.l -- 20 0.622
0.038 5.8 4 2 .mu.l 5 .mu.l 3 .mu.l -- 20 0.586 0.074 11.2 5 4
.mu.l 5 .mu.l 3 .mu.l -- 20 0.522 0.138 21.0 6 2 .mu.l 5 .mu.l --
-- 20 0.684 7 2 .mu.l 5 .mu.l 3 .mu.l -- 10 0.626 0.034 5.2 8 2
.mu.l 5 .mu.l 3 .mu.l -- 30 0.531 0.129 19.4
[0121] The results show that sulindac was reduced in a time- and
concentration-dependent manner by MsrA enzyme.
Example 2
Sulindac is a Substrate for Msr Enzymes in Bacteria and Mammals
[0122] This example demonstrates that sulindac is a substrate for
MsrA and membrane-bound Msr in E. coli and for MsrA and possibly
other Msr in mammalian tissues.
[0123] Material and Methods.
[0124] Chemicals, enzymes and substrates. Sulindac (S), sulindac
sulfide (SS) and all other chemicals and E. coli thioredoxin
reductase were obtained from Sigma Chemicals (St. Louis, Mo.),
unless noted otherwise. Thioredoxin (from E. coli) was purchased
from Promega (Madison, Wis.). N-acetyl-.sup.3H-met-R, S--(O),
met-R--(O), met-S--(O) DABS-met-R--(O) and DABS-met-S--(O) were
prepared as previously described (Brot N. et al., Anal. Biochem.
122 (1982) 291-294; Lavine, F. T. J. Biol. Chem. 169 (1947)
477-491; Minetti C. et al., Ital. J. Biochem. 43 (1994)
273-283).
[0125] Bacterial enzymes. Recombinant MsrA and MsrB from
Escherichia coli were obtained as described previously (Grimaud, R.
et al., J. Biol. Chem. 276 (2001) 48915-48920; Rahman, M. A. et
al., Cellular & Molecular Biology 38 (1992) 529-542). Partially
purified DEAE fractions of free-S-Msr (fSMsr), free-R Msr (fRMsr)
and MsrA1, and a membrane vesicle associated Msr (mern-R,S-Msr)
were prepared from an E. coli MsrA/B double mutant as described
(Etienne, F. et al., Biochem. & Biophys. Res. Comm. 300 (2003)
378-382; Spector, D. et al., Biochem. & Biophys. Res. Comm. 302
(2003) 284-289). The enzyme preparations had specific activities
similar those reported earlier.
[0126] Mammalian enzymes. Calf liver, kidney and brain extracts
were prepared at 4.degree. C. Thirty grams each of calf tissue
(liver, kidney, brain) were minced using a hand-held homogenizer in
5 volumes of buffer A containing 250 mM sucrose, 10 mM Tris-Cl pH
7.4 and 1 mM EDTA. The homogenates were dounced (6 strokes) and
spun at 1,500.times.g for 10 minutes and the pellet was discarded.
The supernatants (S-10) were spun at 10,000.times.g for 10 minutes.
The S-10 supernatants were centrifuged at 100,000.times.g for 12
hours and the resulting pellets and supernatants (S-100) were
saved. The S100 pellets were suspended in cold buffer A and
centrifuged at 100,000.times.g for 4 hours. The washed microsomal
pellets (containing all of the ribosomes) were suspended in 2 ml of
buffer A (S-100 pellet)
[0127] To prepare mitochondria, the S10 pellets were suspended in
20 ml buffer A. The suspension was layered on top of a
discontinuous Ficoll gradient made up of an equal volume of 12%
Ficoll in buffer A (lower layer) and 7.5% Ficoll in buffer A (upper
layer). The tubes were centrifuged at 24,000.times.g for 24 min.
The pellets were resuspended in buffer A and centrifuged at
20,000.times.g for 15 min. The pellets (containing mitochondria)
were suspended in 2 ml of buffer A. All fractions were stored at
-80.degree. C.
[0128] Reductase assay and quantitation of sulindac sulfide formed.
With crude cellular fractions when there is a large amount of NADPH
oxidation, the NADPH assay described in Example 1 above cannot be
used. For use with crude cellular fractions, an extraction assay
was developed based on the ability of sulindac sulfide to be
extracted into benzene. The reaction mixture for the reduction of
sulindac to sulindac sulfide contained in a total volume of 30
.mu.l: 100 mM Tris-Cl, pH 7.4; 0.6 .mu.moles glucose-6-phosphate;
50 ng glucose-6-phosphate dehydrogenase; 30 nmoles NADPH; 2.5 .mu.g
thioredoxin, 1 .mu.g thioredoxin reductase, 50 nmoles Sul and
varying amount of Msr enzymes. Unless stated otherwise, incubations
were for 1 hour at 37.degree. C. At the end of the incubation 370
.mu.l of 25 mM Tris-Cl pH 8.0, 100 .mu.l acetonitrile and 1 ml of
benzene were added to each tube. After vortexing for 30 seconds and
spinning for 1 min at room temperature, the benzene phase was
removed and the optical density was read at 350 nm Fifty nmoles of
SS or S, when carried through the extraction procedure, gave
optical density readings of 0.910 and 0.030, respectively. Under
these conditions, virtually all of the SS was extracted into the
benzene, while about 2.5% of S was extracted. In some experiments
using calf tissue extracts, the standard 30 .mu.l reaction mixture
volume was tripled (90 .mu.l) to obtain statistically significant
values. The extraction assay was not altered except for reduction
of the Tris buffer volume to 310 .mu.l.
[0129] To remove the S epimer of sulindac, the sulindac (R, S
mixture) was incubated with excess MsrA (4 .mu.g) and DTT for 60
minutes, or until the reaction reached completion. Upon completion,
any further reduction seen upon addition of an enzyme fraction in a
second incubation would be due to reduction of the R epimer of
sulindac.
[0130] In some experiments the product was also identified by thin
layer chromatography (TLC). After incubation, both the unreacted S
and SS product were extracted into 1 ml ethyl acetate. The ethyl
acetate phase was removed, dried in a speed vacuum at room
temperature and the residue was suspended in 5 .mu.l of ethyl
acetate which was then loaded onto a TLC plate. The plate was
developed with butanol:acetic acid:water (60:15:25) as the solvent.
The compounds were visualized by their yellow color. The Rf values
of S and SS were 0.80 and 0.95, respectively.
[0131] Results.
[0132] Using the extraction assay described above in Methods, it
was found that recombinant MsrA from E. coli could reduce S to SS.
FIG. 3A shows a time course for the reaction and FIG. 3B shows the
effect of MsrA concentration on reduction of S. The reaction was
dependent on the thioredoxin reducing system. The product, SS, was
independently identified by TLC.
[0133] S is a substrate for mem-R, S-Msr. E. coli is known to have
at least 6 members of the Msr family. Referring to Table 2, these
proteins differ in their stereo-specificity, substrate specificity,
i.e., free vs. protein-bound met(O), and location within the cell,
i.e., soluble or membrane-associated. Whereas the msrA and msrB
genes have been cloned and the recombinant proteins purified, the
other soluble E. coli Msr enzymes (i.e., fSMsr, fRMsr and MsrA1)
have been only partially purified, but have been separated by
conventional fractionation procedures using DEAF cellulose
chromatography (Etienne, F. et al., Biochem. & Biophys. Res.
Comm. 300 (2003) 378-382; Spector, D. et al., Biochem. &
Biophys. Res. Comm. 302 (2003) 284-289). The membrane associated
Msr, which has activity toward both the R and S forms of free and
peptide bound met(O), was present as a membrane vesicle
preparation.
TABLE-US-00002 TABLE 2 Substrate Specificity of Methionine
Sulfoxide Reductases in E. coli ENZYME SUBSTRATE TYPE Free-R-(o)
Free-S-(o) Peptide-R-(o) Peptide-S-(o) MsrA + + MsrB (+) + fRMsr +
fSMsr + MsrA1 + Membrane + + + + Msr Brackets ( ) indicate a very
weak activity.
[0134] Referring to Table 3, S was compared as a substrate for
highly purified MsrA and MsrB from E. coli and the partially
purified enzyme preparations. The results showed that MsrA and the
mem-R,S-Msr are able to reduce S to SS. Very weak activity was
observed with MsrA1. S was not a substrate for MsrB, which
recognizes peptide-bound met-S--(O).
TABLE-US-00003 TABLE 3 Activity of E. coli Msr Enzymes Using
Sulindac as a Substrate. ENZYME TYPE UNITS OF ACTIVITY MsrA 11.3
MsrB 0 fRMsr 0 fSMsr 0 MsrA1 <0.9 Membrane 5.1 Unit of activity
is defined as nmoles of SS formed per hour. Enzyme concentrations
used: 250 ng MsrA; 10 .mu.g MsrB; 290 .mu.g fRMsr; 200 .mu.g fSMsr;
40 .mu.g MsrA1; 50 .mu.g membrane fraction.
[0135] Referring now to Table 4, it is seen that the membrane bound
Msr of E. coli, which likely contains more than one Msr activity,
reduces primarily the R form of sulindac. In these experiments
either S, which is a mixture of the R and S epimers, or the R
epimer of sulindac (see Methods) were used as substrates. Both
exhibited similar activities. Although these results support the R
form being reduced, definitive proof may require the chemical
synthesis and assay of each epimer of S.
TABLE-US-00004 TABLE 4 Membrane Msr of E. coli Reduces Primarily
the R Epimer of Sulindac. SUBSTRATE NMOLES FORMED Sulindac (R, S)
2.75 Sulindac (R) 2.41 R epimer of sulindac was obtained by
incubating sulindac (R, S) with excess MsrA as described in Methods
to remove the S epimer. 35 .mu.g of membrane fraction was used.
[0136] Reduction of sulindac in mammalian (bovine) tissues. Results
shown in Table 5 reveal that crude homogenates (S-10 fractions, see
Methods) of calf liver, kidney and brain are able to reduce S. Of
the tissues tested, kidney has the highest specific activity and
brain the lowest.
TABLE-US-00005 TABLE 5 Sulindac Reductase Activity in Calf Tissues.
TISSUE SPECIFIC ACTIVITY Liver 4.39 Kidney 6.53 Brain 2.31 The
preparation of the various S-10 fractions are described in methods.
Specific activity is given as nmoles of product formed per hour per
mg of protein.
[0137] The liver extracts were fractionated and mitochondria, S-100
and S-100 pellet (microsomes) were prepared as described in
Methods. As shown in Table 6, all three cellular fractions were
able to reduce S to SS. The identity of the enzyme(s) responsible
for the activity was not been determined, but preliminary evidence
indicated that MsrA was largely responsible, based on the
observation that the addition of excess amounts of met-S--(O)
inhibited the activity in all three fractions, whereas the addition
of met R--(O) had only a slight effect. Thus the responsible enzyme
had met-S--(O) activity. Because free Met(O) Msr enzymes (i.e., FS
Msr and FRMsr) cannot reduce sulindac (Table 2), MsrA is most
likely the enzyme responsible for this activity.
TABLE-US-00006 TABLE 6 Subcellular Distribution Sulindac Reductase
Activity in Bovine Liver. LIVER FRACTIONS SPECIFIC ACTIVITY S-10
4.39 S-100 6.20 Mitochondria 2.44 Microsomes 1.31 The indicated
liver fractions were prepared as described in Methods. Specific
activity is given as nmoles of product synthesized per hour per mg
of protein.
Example 3
Synthesis of Sulindac Methionine Catalytic Antioxidants
[0138] As shown above, sulindac is a substrate for MsrA but not for
MsrB. Sulindac contains a methyl sulfoxide moiety which is
recognized by MsrA enzymes, but does not contain a N-methionine
sulfoxide moiety (see FIG. 2), the substrate recognized by both
MsrA and MsrB enzymes (Table 2). This example describes schemes for
the chemical synthesis of derivatives of sulindac that are improved
as substrates for multiple Msr enzymes including MsrB, by
modification to contain an N-substituted methionine, in which the
methionine amino group is in peptide or amide linkage.
[0139] Compound 2a contains a methionine group linked through the
amino group to the acetyl moiety of sulindac. Referring to FIG. 4A,
compound 2a
(1(Z)-5-fluoro-2-methyl-1-[4-(methylsulfinyl)phenyl]methylene]-1H-indene--
3-[1-methylthiomethylenyl-2-aminoacetyl]propanoic acid) can be
prepared starting from sulindac and methionine sulfoxide methyl
esters as follows. Compound 2a was synthesized as follows. To a 50
ml round bottom flask under an argon atmosphere fitted with a
teflon stir bar and rubber stopper, 1.4 mmol of sulindac was
dissolved in 20 ml DMF followed by the addition of 1.5 mol of
methionine sulfoxide methyl ester. Dicyclohexylcarbodiimide (1.2
mmol), triethylamine (2.0 mmol) and 4-dimethylaminopyridine (0.05
mmol) were placed in the reaction flask. After 12 hours, TLC
analysis (75% ethyl acetate in hexanes) showed the formation of the
product at R.sub.f=0.29. The reaction mixture was then placed onto
a 2.5 cm diameter flash column filled with approximately 6 inches
of silica gel and topped off with a quartz sand plug. The following
elution sequence was used 5% EtOAc/Hex (250 mL), 30% EtOAc/Hex (500
mL), 50% EtOAc/Hex (250 mL) and a final elution of 85% EtOAc/Hex
(250 mL). HPLC analysis of the compound (gradient elution from 5%
to 95% MeCN/H.sub.2O over 45 min) gave a peak at 22.5 min with 98%
purity. Proton NMR analysis of compound 2a is shown in FIG. 8.
[0140] Another methionine derivative of sulindac, i.e., compound 3a
is given in FIG. 4B. A suitable scheme for the synthesis of
compound 3a with control of the .alpha.-carbon stereochemistry is
also shown in FIG. 4B. In this particular synthetic method, the
synthesis begins with commercially available sulindac (racemic
form). The sulindac is converted to its methyl ester by treatment
with diazomethane (CH.sub.2N.sub.2). The methyl ester is then
treated with a strong base to form the enolate, followed by
quenching with N-bromosuccinimide (NBS) leading to the
.alpha.-bromoester (Kita et al., J. Am. Chem. Soc. 123.3214, 2001).
The ester group of this intermediate is then selectively reduced to
the primary alcohol using diisobutylaluminum hydride (DIBAL-H),
according to the method of Fukuyama et al., J. Am. Chem. Soc.
116:3125, 1994, to give intermediate compound 1-3a. Compound 1-3a
epoxidizes to give intermediate compound 2-3a. Treatment of
compound 2-3a with methyl sulfide is expected to lead to the
.beta.-hydroxysulfide compound 3-3a (Conte et al., Tetrahedron
Lett. 30:4859, 1989). Using para-toluene sulfonylchloride (TsCl),
the hydroxyl group in compound 3-3a is converted to the
corresponding tosylate (compound 4-3a). By an extension of the
method of O'Donnell (O'Donnell et al., J. Am. Chem. Soc. 111:2353,
1989), the tosylate on compound 4-3a reacts with a protected
diphenylimino-glycine derivative under the influence of a cinchona
alkaloid asymmetric phase-transfer catalyst. This reaction gives
the corresponding .alpha.-imino ester (compound 5-3a), with control
over the stereochemistry of the .alpha.-carbon. Subsequent aqueous
hydrolysis of the imino and tert-butyl ester groups gives the
desired compound 3a.
[0141] Referring now to FIG. 5A, sulindac contains a methylene
group adjacent to a carboxyl that is easily converted into enolate
1-4a. Lithium diisopropylamide (LDA) is a base typically used to
form these types of enolates. Intermediate 1-4a should react with
bromoacetyl methionine sulfoxide (A) to form the new carbon-carbon
bond found in 2-4a. Hydrolysis of this intermediate with lithium
hydroxide gives the corresponding carboxylic acid derivative
(compound 4a).
[0142] FIG. 5B illustrates yet another embodiment of an
N-methionine derivative of sulindac indicated as compound 5a. In
compound 5a, the sulindac structure and the N-acetyl methionine
group are tethered by a diamine chain that can be of varying
length. The use of such a linker molecule provides the ability to
generate a large variety of methionine derivatives through
combinatorial synthesis methods. Compound 5a may be obtained as
follows (FIG. 5B). Under the action of DCC, sulinidac is coupled to
tert-butoxycalbonyl (BOC) mono-protected diamine, followed by
removal of the BOC protecting group under acidic conditions using
trifluoroacetic acid (TFA). This intermediate is coupled to
N-acetyl methionine in the presence of DCC to give compound 5a.
Compound 5a can easily be obtained as the single enantiomer (or
epimers of the sulfoxide position). The addition of N-acetyl
methionine moieties is preferred, as these moieties are expected to
act as a substrate for enzymes that recognize N-blocked methionine
sulfoxide, (such as MsrA and MsrB). D amino acids may be preferred
to minimize metabolism. A racemic mixture of the sulfoxides (i.e.,
both R and S forms) is preferred if it is desired to have the
compound function as a substrate for most, if not all, known Msr
family enzymes that recognize free or protein-bound forms of
methionine sulfoxide (whether R or S epimers).
Example 4
Synthesis of Methionine Catalytic Antioxidants Derived From
Salicylic Acid and Mefenamic Acid
[0143] This example describes chemical synthetic schemes suitable
for preparing bifunctional compounds that can serve both as
catalytic antioxidants and anti-inflammatory agents (COX
inhibitors).
[0144] As described above, sulindac is one example of a COX
inhibitor. This example describes methionine derivatives of other
COX inhibitors, i.e., acetyl salicylic acid and mefenamic acid.
These bifunctional antioxidant compounds contain the amino group of
methionine in the form of an amide and preferably retain the
carboxyl group found in the parent compounds that may be critical
to their inhibitory action.
[0145] Referring to FIG. 6A, starting from the methyl ester of
salicylic acid, the phenol hydroxy group is expected to react with
the carbon bearing the bromine in bromoacetylmethionine sulfoxide
(BAMS) to form the oxygen-carbon bond of intermediate 1-6a. In the
case of mefenamic acid, the reaction with BAMS is likely to occur
at the amine nitrogen to give intermediate 1-7a (FIG. 6B). The
salicyclic and mefenamic methionine sulfoxide derivatives can be
converted to the respective carboxylic acid products 6a and 7a
using a mild hydrolysis reaction with lithium hydroxide (LiOH).
Example 5
Synthesis of Methionine Catalytic Antioxidants Derived from
Ibuprofen, Indomethacin and Rofecoxib/Vioxx.RTM.
[0146] Referring now to FIG. 7, ibuprofen (FIG. 7A), indomethacin
(FIG. 7B), and rofecoxib/Vioxx.RTM. (FIG. 7C) each contain a
methylene group adjacent to a carboxyl or a sulfonyl group that is
easily converted into enolate, shown for intermediates 1-8a and
1-10a. Lithium diisopropylamide (LDA) is a typical base used to
form enolates. Intermediates 1-8a, 1-9a, and 1-10a are shown to
react with bromoacetyl methionine sulfoxide to form the new
carbon-carbon bonds in intermediates 2-8a, 2-9a, and 2-10a,
Hydrolysis of these intermediates with lithium hydroxide gives the
corresponding carboxylic acid derivatives (compounds 8a, 9a, and
10a).
Example 6
Sulindac Methionine Sulfoxide is a Substrate for MsrA and MsrB
[0147] As shown above, sulindac is a substrate for MsrA but not for
MsrB. Referring to FIG. 4A, unmodified sulindac contains a methyl
sulfoxide moiety, but does not include within its structure a
methionine sulfoxide moiety, the required substrate for Msr B
enzymes. Sulindac methionine sulfoxide (SMO), an N-acetyl
methionine sulfoxide derivative of sulindac described in Example 4
above includes both a methyl sulfoxide and a methionine sulfoxide
(see, for instance, compound 2a in FIG. 4A). This example
demonstrates that SMO can serve as a substrate for both MsrA and
MsrB enzymes.
[0148] Materials and Methods.
[0149] Synthesis of SMO. Sulindac methionine sulfoxide (SMO) was
synthesized according to the synthetic pathway described in Example
3 supra. Compound 2a was used for these experiments.
[0150] Reductase assay and thin layer chromatography (TLC).
Reaction mixtures were prepared in duplicate for assay of the
reduction of sulindac (S) and sulindac methionine sulfoxide (SMO).
Mixtures contained in a total volume of 30 .mu.l: 2M Tris-Cl pH
7.4, 200 mM DTT, 100 nmoles of S or SMO, 3 .mu.g of MsrA enzyme, or
21 .mu.g of MsrB enzyme. Incubation was carried out for 2 hours at
37.degree. C., at the end of which the duplicate samples were
combined and dried in a speed-vacuum unit at room temperature. The
residue was suspended in 50 .mu.l of ethanol, which was then loaded
onto a silica gel TLC plate. The plate was developed with butanol:
acetic acid:water (60:15:25) as the solvent. The compounds were
visualized by their yellow color.
[0151] Results:
[0152] As discussed above, it is known that MsrA can reduce methyl
sulfoxide moieties that occur as functional groups within free and
peptide-bound methionine (i.e., Met(O)), but also within other
molecules. By contrast, MsrB can only reduce Met(O), and works best
with Met(O) in peptide linkage (see Table 2). Accordingly, based on
the known substrate specificity of MsrA and MsrB, several different
products would be predicted upon reaction of sulindac and SMO with
MsrA and MsrB. For example, because the structure of sulindac (S)
contains only a methyl sulfoxide (as seen in FIG. 2), reduction of
S by MsrA results in SS. Reduction of S by MsrB would not be
expected to generate a product, due to the absence of methionine
sulfoxide in S. In contrast to unmodified S, SMO includes both the
methyl sulfoxide group of S as well as the methyl sulfoxide
included in the methionine group (see, for example, compound 2a in
FIG. 4A). Accordingly, reaction of SMO with MsrA could generate
several possible products having one or the other, or both methyl
sulfoxide groups reduced, i.e.: sulindac sulfide methionine
sulfoxide (SSMO), sulindac methionine (SM), or sulindac sulfide
methionine (SSM). With MsrB, however, only the methionine sulfoxide
should be reduced and the expected product is SM.
[0153] FIG. 9 shows TLC results from the various incubations, i.e.,
MsrA+S (lane 1); MsrA+SMO (lane 2); MsrB+S (lane 3) and MsrB+SMO
(lane 4). In FIG. 9, the indicated substrates and reaction products
are as follows: S--sulindac; SS--sulindac sulfide; SM--sulindac
methionine; SSM--sulindac sulfide methionine; SMO--sulindac
methionine sulfoxide; SSMO--sulindac sulfide methionine sulfoxide.
The positions where the substrates, products and standards migrate
on the TLC plate are indicated by arrows.
[0154] The results of the enzyme assays demonstrate the following.
Lane 1 shows the presence of SS, indicating that sulindac is a
substrate for MsrA. Lane 2 reveals formation of SSM, SM, and SSMO,
demonstrating that SMO is a substrate for MsrA and that both methly
sulfoxide groups can be reduced. Lane 3 shows only S, demonstrating
that unmodified sulindac is not a substrate for MsrB. By contrast,
lane 4 reveals that SMO is a substrate for MsrB, shown by the
formation of SM (FIG. 9) Thus it is shown that a methionine
derivative of sulindac, i.e., SMO, can act as a substrate for both
MsrA and MsrB enzymes.
Example 7
Sulindac Increases Resistance to Oxidative Stress in Drosophila
[0155] This example demonstrates that sulindac, an antioxidant
containing a methyl sulfoxide moiety, can extend the lifespan of
flies subjected to an agent known to kill flies via production of
ROS.
[0156] Materials and Methods.
[0157] Paraquat is a cytotoxic compound known to form superoxide
radicals intracellularly. Three different concentrations of
paraquat (i.e., 10 mM, 5 mM and 2.5 mM) were tested. Flies
(Drosophila) were raised for 3 days on apple juice medium (33%
apple juice, 1.7% sucrose and 2.7 mg/ml methylparaben, a mold
inhibitor, in 3.5% agar) containing various concentrations of
sulindac or no supplement (Controls). After 3 days at 25.degree.
C., flies were transferred to test vials for counting.
[0158] Results.
[0159] In the group treated with 2.5 mM paraquat, approximately 80%
and 25% of the flies in the untreated control group, respectively,
were alive after 3 and 6 days of paraquat exposure. By contrast,
approximately 95% and 60%, respectively, of the flies treated with
2 ml-M sulindac remained alive at the 3 day and 6 day time points
(FIG. 10). Similar results were observed in the groups exposed to
higher concentrations of paraquat. For example, in groups exposed
to 10 mM paraquat, the respective survival rates after 2 and 3 days
were approximately 50% and 17% in the controls and 85% and 57% in
the sulindac treated groups. These results demonstrate that
administration of a methyl sulfoxide-containing compound that is a
substrate for MsrA can lengthen the lifespan of paraquat-exposed
flies. Earlier studies showed that over expressing MsrA enzyme in
transgenic flies extended their lifespan. The present data provide
evidence that increasing the intracellular level of a substrate for
the Msr system can also provide a protective effect against
damaging ROS species, leading to increased longevity under
conditions of oxidative stress.
Example 8
Sulindac Promotes Cell Survival in Neuronal Cells Subjected to
Oxidative Damage by MPP+
[0160] This example demonstrates a protective effect of sulindac on
PC-12 cells following insult with MPP+, a toxic compound that
selectively destroys dopaminergic neurons in vitro, and in an in
vivo animal model of Parkinson's disease.
[0161] Materials and Methods.
[0162] MPP+ neurotoxin. The neurotoxin 1-methyl-4
phenyl-1,2,3,6-tetrahydropyridine (MPTP) when given to both humans
and primates results in a clinical syndrome closely similar to
Parkinson's disease. The compound is metabolized to
1-methyl-4-phenylpyridinium (MPP+) by monamine oxidase B and is
subsequently selectively taken up by dopaminergic terminals and
concentrated in the neuronal mitochondria in the substantia nigra.
MPP+ inhibits complex 1 of the electron transport chain and is
thought to cause irreversible inactivation of the complex by
generating free radicals (Hartley A., Stone J. M., Heron C, Cooper
J. M., and Schapira A. H. V. J. Neurosci. 63:1987-1990, 1994) MPP+
increases superoxide synthesis in vivo and in vitro. MPP+ damage is
decreased in transgenic mice overexpressing superoxide dismutase,
suggesting that free radicals are involved in its
neurotoxicity.
[0163] Cell culture. PC-12 cells were initially grown overnight in
Dulbecco's modified Eagle's medium containing high glucose (Gibco #
11195-065) 5% fetal calf serum and 10% horse serum in 9 cm dishes.
They were then transferred to 6 cm dishes and grown in the same
medium without glucose but using sodium pyruvate as the sole energy
source (Gibco # 11966-025) These cells were pretreated with
sulindac(Sigma) at concentrations of 0.1, 0.2, or 0.5 mM for 48
hours, the medium containing the sulindac was removed and replaced
with fresh medium The cells were then incubated for 24 hours in
medium containing MPP+ at a final concentrations of 0.2 mM. Control
cells were incubated in MPP+-free medium. At the end of the 24 hr
period, cell viability was assayed by trypan blue exclusion.
[0164] Results.
[0165] Referring to Table 7, the results showed that 0.2 mM MPP+
was highly toxic to PC-12 cells, causing approximately 85% of the
cells to die (15% cell survival) following a 24 hour treatment with
this compound. Pretreatment with sulindac prior to MPP+ insult was
protective against cell death, exhibiting a dose-response with
approximately 35% cell death (65% cell survival) following
pretreatment with the maximum concentration tested, i.e., 0.5 mM.
In the absence of MPP, sulindac had no effect on the viability of
the cells.
TABLE-US-00007 TABLE 7 Effect of Sulindac on the Viability of PC-12
Cells Treated with MPP+. Sulindac (mM) Dead cells (%) Exp 1 Dead
cells (%) Exp 2 0 85 87 0.1 67 74 0.2 55 39 0.5 34 35
Example 9
Sulindac Extends the Lifespan of a Transgenic Mouse Model of
Familial Amyotrophic Lateral Sclerosis (ALS)
[0166] This example provides evidence that sulindac, a methyl
sulfoxide containing compound that acts as a substrate for MsrA
enzymes can significantly extend lifespan, increase motor neuron
cell count and improve motor performance in a mouse model of ALS
based on a mutation in superoxide dismutase (SOD1).
[0167] Materials and Methods:
[0168] ALS is an adult onset neurodegenerative disease with an
unknown etiology. ALS is most commonly sporadic with about 10% of
cases being inherited as an autosomal dominant familial form. It is
now known that about 20% of the familial cases are associated with
a mutant form of Cu/Zn SOD (Rosen, D. R., et. al., (1993) Nature
362:59-62) Although the protein harbors a mutation (over 100
different SOD mutations having been documented in ALS patients), it
is still enzymatically active. Oxidative damage is one of the main
hypotheses for the toxicity of the mutant protein. The animals used
in this study express a mutant form of SOD similar to patients with
the disease.
[0169] Transgenic mice expressing a mutant form of SOD similar to
that found in human ALS patients were used for this study.
Transgenic male mice with a G93A human SOD1 (G1H/+) mutation
(B6SJL-TgN(SOD1-G93A)1 Gur; Jackson Laboratories, ME) were used to
breed with female B6SJL mice (Jackson Laboratories, ME). The F1
generations were genotyped for the G93A mutation with polymerase
chain reaction (PCR) using tail DNA, and two specific primers from
the SOD1 gene.
[0170] Sulindac administration. C93A mice were treated with
sulindac at two different doses, i.e., 300 PPM and 450 PPM, which
was mixed into their food beginning on postnatal day 30. Three
groups were examined (i.e., 300 PPM, 450 PPM sulindac and
controls). Motor performance was assessed by Rotarod testing for
each group and survival time was recorded.
[0171] Motor function testing. Mice were trained for 2-3 days to
become acquainted with the Rotarod apparatus (Columbus instruments,
Columbus, Ohio). Rotarod performances were assessed in G93A mice
starting at 60 days of age. The testing began with placing the mice
on a rod that rotates at 12 rpms. The time period that the mice
stayed on the rod before falling off was recorded as a measurement
of the competence of their motor function. Three trials were
performed, and the best result of the three trials was recorded
representing the status of the motor performance. Mice were tested
twice a week until they could no longer perform the task.
[0172] Survival times. The initial sign of disease in G93A
transgenic mice is a resting tremor that progresses to gait
impairment, asymmetrical or symmetrical paralysis of the hind
limbs, and ultimately complete paralysis at the end stage. Mice
were sacrificed when they were unable to roll over within 20
seconds after being pushed on their side. This time point was
determined to be the time of survival, at which time mice were
sacrificed.
[0173] Light microscopic immunocytochemistry. Mice were perfused
transcardially with cold 0.1M phosphate-buffered saline (PBS) for 1
minute followed by cold 4% paraformaldehyde in PBS for 10 minutes.
The spinal cords were removed rapidly, blocked coronally, and
post-fixed in 4% paraformaldehyde in PBS for 6 hours. Blocks were
cryo-protected in 30% sucrose for 24 hours and were sectioned on a
cryostat at a thickness of 35 micrometers. All protocols were
conducted within NIH guidelines for animal research and were
approved by the Institutional Animal Care and Use Committee
(IACUC).
[0174] Serial transverse sections (50 .mu.m thick) were cut on a
cryostat and collected for Nissl staining and very fourth section
was analyzed for neuronal volume and number using the optical
fractionator and nucleator probes of the Stereo Investigator System
(Microbrightfield, Colchester, Vt.). Six tissue sections of the
lumbar spinal cord from each mouse were analyzed. All cells were
counted from within the ventral horn below a horizontal line across
the gray matter through the ventral border of the central canal.
Photomicrographs were taken on a Zeiss Axiophot II microscope.
[0175] Statistical analysis. Statistical analysis of survival was
performed using Kaplan-Meier test for survival measured in
postnatal days, Fisher's Test for mean age of death analysis, and
Scheffe test for motor performance.
[0176] Results:
[0177] Referring to FIG. 11, G93A mice treated with 450 PPM
sulindac survived an average of 131.17.+-.10.9 days. This was a 7%
increase over control mice, which survived an average of
123.16.+-.11 days (P=0.083). G93A mice treated with 300 PPM
sulindac also exhibited extended survival (a 10% increase) relative
to the controls, with mean survival time of 135.17.+-.11.4 days
(P=0.02).
[0178] The results of several statistical tests of the data shown
in FIG. 11 are presented in Table 8.
TABLE-US-00008 TABLE 8 Rank Test Chi-Square DF P-Value LogranK
(Mantel-Cox) 6.744 2 .0343 Breslow-Gehan-Wilcoxon 7.796 2 .0203
Tarone-Ware 7.374 2 .0250 Peto-Peto-Wilcoxon 7.661 2 .0217
Harrington-Fleming (rho = .5) 7.374 2 .0250
[0179] The sulindac-treated groups showed a significant improvement
of motor performance, as evaluated by Rotarod performance times
(FIG. 2). Microscopic analysis of spinal cord sections revealed
that the sulindac-treated mice had significantly higher counts of
motor neurons as compared with G93A controls (FIG. 13). Differences
between the 300 PPM and 450 PPM sulindac groups were not
significant (FIG. 13 and Table 9).
TABLE-US-00009 TABLE 9 Scheffe for Motor Performance Mean Diff.
Crit. Diff. P-Value Controls, Sulindac 300 PPM -59.443 54.695 .0308
S Controls, Sulindac 450 PPM -73.119 53.271 .0053 S Sulindac 300
PPM, Sulindac -13.676 56.815 .8267 450 PPM
OTHER EMBODIMENTS
[0180] This description has been by way of example of how the
compositions and methods of the invention can be made and carried
out. Various details may be modified in arriving at the other
detailed embodiments, and many of these embodiments will come
within the scope of the invention. Therefore, to apprise the public
of the scope of the invention and the embodiments covered by the
invention, the following claims are made.
* * * * *