U.S. patent application number 11/056011 was filed with the patent office on 2005-08-04 for methods for identifying agents that inhibit serum aging factors and uses and compositions thereof.
This patent application is currently assigned to Purdue Research Foundation. Invention is credited to Morre, D. James, Morre, Dorothy M..
Application Number | 20050169903 11/056011 |
Document ID | / |
Family ID | 34425532 |
Filed Date | 2005-08-04 |
United States Patent
Application |
20050169903 |
Kind Code |
A1 |
Morre, Dorothy M. ; et
al. |
August 4, 2005 |
Methods for identifying agents that inhibit serum aging factors and
uses and compositions thereof
Abstract
The invention described herein encompasses methods of preventing
or treating disorders caused by oxidative damage by an
aging-specific isoform of NADH oxidase (AR--NOX). The invention
encompasses methods of assaying, screening, and identifying agents
that inhibit AR--NOX, as well as methods using ubiquinone to
inhibit the ability of AR--NOX to generate reactive oxygen species.
These agents may be formulated into pharmaceutical compositions in
the prevention and treatment of disorders caused by oxidative
damage.
Inventors: |
Morre, Dorothy M.; (West
Lafayette, IN) ; Morre, D. James; (West Lafayette,
IN) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST ST
NEW YORK
NY
10017
US
|
Assignee: |
Purdue Research Foundation
|
Family ID: |
34425532 |
Appl. No.: |
11/056011 |
Filed: |
February 11, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11056011 |
Feb 11, 2005 |
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09536551 |
Mar 28, 2000 |
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6878514 |
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60126894 |
Mar 30, 1999 |
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Current U.S.
Class: |
424/94.1 ;
514/690 |
Current CPC
Class: |
A61K 31/122 20130101;
Y02A 50/411 20180101; Y02A 50/30 20180101 |
Class at
Publication: |
424/094.1 ;
514/690 |
International
Class: |
A61K 038/43; A61K
031/12 |
Claims
1. A method of preventing a complication of a primary disorder in
patients wherein said complication results from oxidative damage
resulting from the generation of reactive oxygen species by
AR--NOX, which comprises: administering to a patient having said
primary disorder, in an amount effective to prevent said
complication, one or more ubiquinones in a pharmaceutically
acceptable carrier.
2. A method for preventing secondary disorders in patients having a
primary disorder that causes oxidative damage resulting from the
generation of reactive oxygen species by AR--NOX which comprises:
administering to a patient having a primary disorder, in an amount
effective to prevent said secondary disorder, one or more
ubiquinones in a pharmaceutically acceptable carrier.
3. The method of claim 1 or 2 wherein the total daily amount of
ubiquinones administered is from about 1 to about 500 mg of a
composition comprising ubiquinones.
4. The method of claim 3 wherein the total daily amount of
ubiquinones administered is from 1 to 100 mg of the compositions
comprising ubiquinones.
5. The method of claim 1 or 2 wherein the ubuqionone is coenzyme
Q.sub.10.
6. The method of claim 1 or 2 wherein said ubiquinone is not
coenzyme Q.sub.10.
7. The method of claim 1 or 2 wherein coenzyme Q.sub.10 is
administered with a ubiquinone selected from a group consisting of
coenzyme Q.sub.6, coenzyme Q.sub.7, coenzyme Q.sub.8, or coenzyme
Q.sub.9.
8. The method of claim 1 or 2 wherein the primary disorder is old
age, rheumatoid arthritis, arthritis associated with age, or
fatigue associated with age.
9. The method of claim 1 or 2 wherein the primary disorder is a
result of aged cells.
10. The method of claim 1 or 2 wherein the primary disorder is
cancer.
11. The method of claim 1 or 2 wherein the primary disorder is
selected from a group comprising myocardial infarction, alcoholism,
favism, malaria, sickle cell anemia, Fanconi's anemia,
protoporphyrin photo-oxidation, nutritional deficiencies,
Kwashiorkor, thalassemia, dietary iron overload, idiopathic
hemochromatosis, metal ion-mediated nephrotoxicity, aminoglycoside
nephrotoxicity, autoimmune nephrotic syndromes, oral iron
poisoning, endotoxin liver injury, free fatty acid-induced
pancreatitis, nonsteroidal antiinflammatory drug induced
gastrointestinal tract lesions, glomerulonephritis, autoimmune
diseases, vasculitis, hepatitis B virus, Parkinson's disease,
neurotoxins, allergic encaphalomyelitis, potentiation of traumatic
injury, hypertensive cerebrovascular injury, vitamin E deficiency,
adriamycin cardiotoxicity, Keshan disease, selenium deficiency,
alcohol cardiomyopathy, photic retinopathy, occular hemorrhage,
cataractogenesis, degenerative retinal damage, amyotrophic lateral
sclerosis, aged-related macular degeneration, diabetes,
atherogenesis, and atherosclerosis.
12-54. (canceled)
Description
[0001] This application claims benefit of U.S. provisional
application serial No. 60/126,894 filed Mar. 30, 1999.
1. INTRODUCTION
[0002] The present invention relates to methods for the prevention
or treatment of disorders and complications of disorders resulting
from cell damage caused by an aging-related isoform of NADH oxidase
(AR--NOX). The invention comprises assays for screening for agents
that bind AR--NOX and inhibit the ability of AR--NOX to generate
reactive oxygen species as well as methods of using ubiquinone to
inhibit the ability of AR--NOX to generate reactive oxygen species.
The invention also encompasses using the therapeutic compounds
detected in the screening assays of the invention in a
pharmaceutically acceptable carrier.
2. BACKGROUND OF THE INVENTION
2.1. Mitochondrial Theory of Aging
[0003] The mitochondrial theory of aging proposes that accumulation
of spontaneous somatic mutations of mitochondrial DNA (mtDNA) leads
to errors of mtDNA-encoded polypeptide chains (Harman, 1956, J.
Gerontol. 11:298-300; Harman, 1972, J. Am. Geriatr. Soc.
20:145-147; Miquel et al., 1980, Exp. Gerontol. 15:575-591; Linnane
et al., 1989, Lancet, 1:642-645; Arnheim and Cortopassi, 1992,
Mutat. Res. 275:157-167; Ozawa, 1995, Biochim. Biophys. Acta
1271:177-189; de Grey, 1997, BioEssays 19:161-166; de Grey, 1998,
J. Anti-Aging Med. 1:53-66; Lenaz et al., 1997, Mol. Cell. Biochem.
174:329-333; and Lenaz et al., 1998, BioFactors 8:195-204). These
errors occurring in mtDNA-encoded polypeptide chains are stochastic
and randomly transmitted during mitochondrial division and cell
division. The consequence of these alterations, affecting
exclusively the four mitochondrial complexes involved in protein
translocation, is defective oxidative phosphorylation. Respiratory
chain defects may become associated with increased oxidative
stress, thus establishing a vicious cycle with amplification of the
original damage (Ozawa, 1995, Biochim. Biophys. Acta 1271:177-189
and Lenaz, 1998, Biochim. Biophys. Acta 1366:53-67). In this view,
therefore, mutated mitochondrial DNA, despite being present only in
very small quantities in the body, may be the main generator of
oxidative stress. Mutations in mtDNA are mainly represented by
deletions and are unevenly distributed throughout the body. Each
type of deletion is usually found at a very small percentage of
total mtDNA. However, considering that the total number of
deletions may be several hundreds in the different copies of mtDNA,
they may account for such an amount to overcome the threshold
required for decreasing the respiratory chain activity (Ozawa,
1995, Biochim. Biophys. Acta 1271:177-189; Yoneda et al., 1995,
Biochem. Biophys. Res. Comm. 209:723-729; and Ozawa, 1997, Physiol.
Rev. 77:425464; Lenaz, 1998, Biochim. Biophys. Acta 1366:53-67 and
Schon et al., 1996, Cellular Aging and Cell Death, J. Wiley &
Sons, Inc., New York, pp. 19-34).
[0004] Aging was proposed to result from an ever-increasing level
of destructive chemical reactions involving free radicals, with
mitochondria as the principal mediators of the process (Harman,
1956, J. Gerontol. 11:298-300 and Harman, 1972, J. Am. Geriatr.
Soc. 20:145-147). The main line of reasoning to support this idea
is that, of all subcellular components, mitochondria is both a
major source of free radicals and a major direct victim of free
radical damage. As a result, loss of mitochondrial function may be
the driving intracellular change underlying aging, and the cause of
other pro-oxidant changes such as slower protein turnover. There is
considerable indirect as well as direct experimental support for
the theory. For example, a decline in ATP synthesis capacity and of
energy-depending processes during aging has been reported (Syrovy
and Gutmann, 1997, Exp. Gerontol. 12:31-35; Sugiyama et al., 1993,
Biochem. Mol. Biol. In t. 30:937-944; Boffoli et al., 1996,
Biochim. Biophys. Acta 1226:73-82; and Lenaz et al., 1998,
BioFactors 8:195-204).
[0005] The mitochondrial theory of aging is currently among the
most popular theories of aging as it takes into account one of the
most common sources of genetic lesions associated with senescence,
that of mtDNA. Mitochondrial DNA is located at the inner
mitochondrial membrane near the sites of formation of highly
reactive oxygen species and their products. The mitochondrial
genome encodes several subunits of the electron transport chain as
well as components of the ATP synthase and mitochondrial tRNAs and
rRNAs. Up to 2-4% of the oxygen metabolized by mitochondria is
estimated to be converted to oxygen radicals because the flow
process of electrons of the mitochondrial electron transport chain
is not fully efficient (Boveris et al., 1972, Biochem. J.
128:617-630 and Richter et al., 1988, Proc. Natl. Acad. Sci. U.S.A.
85:6465-6467). In contrast to the nuclear genome, mtDNA may be
unable to counteract the damage inflicted by those products of
respiration because mitochondria lack excision and recombination
repair (Miquel, 1992, Mutat. Res. 275:209-216). After an oxidative
stress to cultured cells, the damage to mtDNA is higher and
persists longer than that to nuclear DNA (Yakes and Van Houten,
1997, Proc. Natl. Acad. Sci. U.S.A. 94:514-519). The steady state
level of mtDNA oxidative change is about ten to sixteen times
greater than that of nuclear DNA as indicated by the amount of
8-oxo-2'-deoxyguanosine (a biomarker of oxidative DNA damage)
formed by the reaction of hydroxyl free radicals with guanine in
mtDNA compared to nuclear DNA (Richter et al., 1988, Proc. Natl.
Acad. Sci. U.S.A. 85:6465-6467 and Shigenaga et al., 1994, Proc.
Natl. Acad. Sci. U.S.A. 91:10771-10778). Also, lipid peroxidation
of mitochondrial membranes seems to damage mtDNA as indicated by
altered electrophoretic mobility (Balcavage, 1982, Mech. Aging Dev.
19:159-170).
[0006] However, alterations of mtDNA of themselves have been
difficult to link to other forms of cellular and tissue changes
related to aging. Chief among these is low density lipoprotein
(LDL) oxidation and atherogenesis (Steinberg, 1997, J. Biol. Chem.
272:20963-20966).
2.2. Plasma Membrane Redox System
[0007] A consistent correlate of aging cells is the accumulation of
somatic mutations of mitochondrial DNA (mtDNA) leading to defective
oxidative phosphorylation through alterations that affect
exclusively the four mitochondrial complexes involved in proton
translocation (Harman, 1956, J. Gerontol. 11:298-300; Harman, 1972,
J. Am. Geratr. Soc. 20:145-147; Miquel et al., 1980, Exp. Gerontol.
15:575-591; Linnane et al., 1989, Lancet I:652-645; Arnheim and
Cortopassi, 1992, Mutat. Res. 275:157-167; Ozawa, 1995, Biochim.
Biophys. Acta 1271:177-189; de Grey, 1997, BioEssays 19:161-166; de
Grey, 1998, J. Anti-Aging Med. 1:53-66; Lenaz et al., 1997, Mol.
Cell. Biochem. 174:329-333; and Lenaz et al., 1998, BioFactors
8:195-204). A major piece of the puzzle missing from our
information is how mitochondrial lesions are propagated to adjacent
cells and blood components in the aging cascade. A plasma membrane
oxido-reductase (PMOR) system has been suggested to augment
survival of mitochondrially deficient cells through regeneration of
oxidized pyridine nucleotide. The pyridine nucleotide is required
to sustain glycolytic ATP production in the presence of diminished
respiratory chain activity (de Grey, 1997, BioEssays 19:161-166; de
Grey, 1998, J. Anti-Aging Med. 1:53-66); Yoneda et al, 1995,
Biochem. Biophys. Res. Comm. 209:723-729; Schon et al., 1996,
Cellular Aging and Cell Death, J. Wiley and Sons, New York, pp.
19-34; Ozawa, 1997, Physiol. Rev. 77:425464; and Lenaz, 1998,
BioFactors 8:195-204).
[0008] A cell surface protein with hydroquinone (NADH) oxidase
activity (designated NOX) that functions as a terminal oxidase of
PMOR has been discovered by the Inventors. Thus, a complete
electron transport chain involving a cytosolic hydroquinone
reductase, plasma membrane located quinones and the NOX protein was
elucidated by the Inventors (Kishi et al., 1999, Biochem. Biophys.
Acta 1412:66-77 and Morr, 1998, Plasma Membrane Redox Systems and
their Role in Biological Stress and Disease, Klewer Academic
Publishers, Dordrecht, The Netherlands, pp. 121-156). This system
provides a rational basis for operation of the mitochondrial theory
of aging and for propagation of aging related mitochondrial
lesions, including a decline in mitochondrial ATP synthetic
capacity and other energy-dependent processes during aging (Boffoli
et al., 1996, Biochem. Biophys. Acta 1226:73-82; Lenaz et al.,
1998, BioFactors 8:195-204; de Grey, 1997, BioEssays 19:161-166;
and de Grey, 1998, J. Anti-Aging Med. 1:53-66).
[0009] Alterations in mitochondria DNA are by far the most common
sources of genetic lesion associated with aging and senescence. It
has been widely noted that mitochondrial DNAs are located at the
inner mitochondrial membrane near sites where highly reactive
oxygen species and their products might be formed. Several subunits
of the electron transport chain as well as components of the ATP
synthase and mitochondrial tRNAs and rRNAs are encoded by the
mitochondrial genome. Since the flow of electrons of the
mitochondrial electron transport chain is not fully efficient, up
to 2-4% of the oxygen metabolized by mitochondria has been
estimated to be converted to oxygen radicals (Boveris et al., 1972,
Biochem. J. 128:617-630 and Richter et al., 1988, Proc. Natl. Acad.
Sci. U.S.A. 85:6465-6467). A major tenet of the mitochondrial
theory of aging is that mtDNA may be unable to counteract the
damage inflicted by oxygen radicals and their products due to a
lack of excision and recombination repair mechanisms (Miquel, 1992,
Mutat. Res. 275:209-216). This has been demonstrated in cultured
cells where damage to mtDNA resulting from oxidase stress is not
only higher but persists longer than does damage to nuclear DNA
(Yakes and Van Houten, 1997, Proc. Natl. Acad. Sci. U.S.A.
94:514-519). Using the amount of 8-oxo-2'-deoxyguanosine as a
bio-marker of oxidative DNA damage formed by the reaction of
hydroxyl free radicals with guanine in mtDNA, the steady state
level of mtDNA oxidative change is about 10 to 16 times greater
than that of nuclear DNA (Richter et al., 1988, Proc. Natl. Acad.
Sci. U.S.A. 85:6465-6467 and Shigenaga et al., 1994, Proc. Natl.
Acad. Sci. U.S.A. 91:10771-10778). Even lipid peroxidation of
mitochondrial membranes seems to lead to damage of mtDNA
(Balcavage, 1982, Mech Aging Dev. 19:159-170).
[0010] Nevertheless, alterations of mtDNA and other forms of
cellular and tissue changes, related to aging, have been difficult
to link. Chief among these is the oxidation of low density
lipoproteins (LDLs) and its implications as causal to atherogenesis
(Steinberg, 1997, J. Biol. Chem. 272:20963-20966).
[0011] A model to link accumulation of lesions in mtDNA to
extracellular responses, such as the oxidation of lipids in low
density lipoprotein (LDLs) and the attendant arterial changes, was
first proposed with rho.sup.o cells (Larm et al., 1994, J. Biol.
Chem. 269:30097-30100; Lawen et al., 1994, Mol. Aspects. Med.
15:s13-s27; de Grey, 1997, BioEssays 19:161-166; and de Grey, 1998,
J. Anti-Aging Med. 1:53-66). These cells lack mtDNA and are unable
to carry out oxidative phosphorylation. It has been demonstrated
that the PMOR system actually functions to regenerate NAD.sup.+
from NADH (Larm et al., 1994, J. Biol. Chem. 269:30097-30100 and
Lawen et al., 1994, Mol. Aspects. Med. 15:s13-s27). In the absence
of functional mitochondrial respiratory chain, NADH accumulates as
the result of glycolytic production of ATP. The rho.sup.o cells
lacking functional mitochondria apparently survive though enhanced
electron flow to molecular oxygen via PMOR. The PMOR is accordingly
over-expressed in these cells. Oxidative stress and LDL oxidation
are common complicating features in diabetics (Kennedy and Lyons,
1998, Metabolism 56:14-21).
[0012] The capacity of cells to generate ATP is determined either
by reoxidation of NADH by mitochondrial respiratory mechanisms
(reduction of pyruvate and uridine are provided, cells can grow
without a functional mitochondrial electron transfer from oxygen to
water) or by cytosolic glycolytic mechanisms (reduction of pyruvate
to lactate). Transformed human cells in culture provided with
excess pyruvate grow anaerobically on a glucose medium where
NAD.sup.+ is regenerated from the NADH that is produced during
glycolysis (Vaillant et al., 1996, J. Bioenerg. Biomemb.
28:531-540). This continual regeneration of NAD.sup.+ ensures that
the glycolytic pathway will provide sufficient ATP to sustain cell
growth and viability.
2.3. Plasma Membrane Oxido-Reductase (PMOR) Chain: Role in
Aging
[0013] Aging cells expressing mitochondrial lesions require a
functional PMOR, as observed in rho.sup.o cells. Mitochondrial DNA
encodes respiration and oxidative phosphorylation enzymes
exclusively so that cells with functionally-deficient mitochondria
become anaerobic if they are to survive. In such cells, the PMOR,
as demonstrated by the Inventors, regenerate sufficient export of
reducing equivalents to maintain the NAD.sup.+/NADH homeostasis,
ensuring survival of cells completely deficient in aerobic
respiration.
[0014] Work by the Inventors done in collaboration with Prof T.
Kishi, Kobe-Gakuin University, Japan, has described a cell surface
NADH oxidase protein, designated NOX, capable of oxidizing
hydroquinones (Kishi et al., 1999, Biochem. Biophys. Acta
1412:66-77). This protein, which is located at the exterior of the
cell, appears to be multifunctional but may have a major function
as a terminal oxidase of the PMOR (Morr, 1995, Biochem. Biophys.
Acta 1240:201-208 and DeHahn et al., 1997, Biochem. Biophys. Acta
1328:99-108). These findings define a complete electron transfer
chain of the plasma membrane capable of transfer of electrons from
NADH to an external electron acceptor via a reduced quinone
intermediate. The Inventors demonstrate that in cells where plasma
membrane oxidoreductase (PMOR) is over-expressed/activated
electrons are transferred from NADH to external acceptors via a
defined electron transport chain. The resultant transfer could
result subsequently in the generation of reactive oxygen species
(ROS) at the cell surface. Such cell surface-generated ROS then
would be capable of propagating an aging cascade originating in
mitochondria to both adjacent cells as well as to circulating blood
components such as low density lipoproteins.
[0015] Mammalian plasma membranes are enriched in coenzyme Q
(ubiquinone) and the plasma membrane at the cytosolic surface
contains a quinone reductase capable of oxidizing NADH and reducing
coenzyme Q. The electron acceptor is either molecular oxygen, or
under certain conditions, both molecular oxygen and protein
disulfides (Morr, 1994, J. Bioenerg. Biomemb. 26:421-433; Chueh et
al., 1997, J. Biol. Chem. 272:11221-11227; and Morr et al., 1998,
J. Bioenerg. Biomemb. 30:477-487). The enzyme can alternate between
the two acceptors (Morr, 1998, Plasma Membrane Redox Systems and
their Role in Biological Stress and Disease, Klewer Academic
Publishers, Dordrecht, The Netherlands, pp. 121-156). Hormones and
growth factors stimulate NADH oxidation and favor protein disulfide
reduction at the expense of oxygen consumption (Brightman et al.,
1992, Biochim. Biophys. Acta 1105:109-117; Morr, 1994, J. Bioenerg.
Biomemb. 26:421-433; and Chueh et al., 1997, J. Biol. Chem.
272:11221-11227). Stoichiometric relationships have been
demonstrated among protein disulfide reduction, NADH oxidation and
protein-thiol formation using isolated plasma membranes from a
plant source stimulated by an auxin plant growth factor, 2,4-D
(Chueh et al., 1997, J. Biol. Chem. 272:11221-11227). A similar
stoichiometry has been shown for NADH oxidation in HeLa cells (Morr
et al., 1998, J. Bioenerg. Biomemb. 30:477-487).
[0016] As a terminal oxidase of the PMOR electron transport chain,
the NOX protein is responsible not only for maintaining
NAD.sup.+/NADH homeostasis in anaerobic cells but appears to play a
role in the enhanced generation of ROS in aged cells expressing
mitochondrial mutations. Oxygen appears to be the principal natural
electron acceptor for cytosolic NADH oxidation in the resting
state. However, a number of parameters, including metals (iron or
copper), could interrupt the orderly two-electron flow to molecular
oxygen that ordinarily forms water and initiate a one-electron
process producing superoxide (O.sub.2.sup.-) radicals (Table 3 in
Section 6.2). Superoxide then initiates a reaction that generates
H.sub.2O.sub.2 and other aggressive oxidants such as the hydroxy
radical (OH.sup.-) (Papa and Skulachev, 1997, Mol. Cell. Biochem
174:305-319). These ROS appear to be released into the environment
to react with neighboring cells and circulating molecules such as
LDL (Steinberg, 1997, J. Biol. Chem. 272:20963-20966).
[0017] Clearly, there is a need to find agents which reduce the
ability of AR--NOX to generate reactive oxygen species (ROS) for
the purposes of reducing or treating the resultant physiological
conditions, such as oxidation of lipids in low density lipoprotein
(LDLs) and attendant arterial changes.
3. SUMMARY OF THE INVENTION
[0018] The present invention provides pharmaceutical compositions,
methods of use, and pharmaceutial kits for the treatment of
disorders resulting from oxidative changes in cells that result in
aging by targeting an aging-related isoform of NADH oxidase
(AR--NOX), shed into the sera by aging cells.
[0019] The invention is based in part, on the Inventors' discovery
that ubiquinones inhibit the activity of an aging-related isoform
of NADH oxidase (AR--NOX) shed into the sera by aging cells. The
inhibition of AR--NOX by ubiquinones results in a decrease in the
generation of reactive oxygen species by AR--NOX. A decrease in
reactive oxygen species should result in a decrease of oxidative
damage resulting from said reactive oxygen species.
[0020] In another embodiment, the invention comprises methods and
compositions for screening assays to identify agents that sequester
AR--NOX. In one embodiment, the invention encompasses methods for
detecting cell-membrane associated AR--NOX and soluble AR--NOX in
sera.
[0021] The pharmaceutical compositions further comprise varying
modes of administration of compounds that sequester AR--NOX. The
modes of administration of compounds includes but is not limited to
capsules, tablets, soft gels, solutions, suppositories, injections,
aerosols, or a kit. In yet another embodiment, the invention
comprises the isolation and characterization of AR--NOX.
3.1. DEFINITIONS
[0022] As used herein, the term "disorder" refers to an ailment,
disease, illness, clinical condition, or pathological
condition.
[0023] As used herein, the term "reactive oxygen species" refers to
oxygen derivatives from oxygen metabolism or the transfer of free
electrons, resulting in the formation of free radicals (e.g.,
superoxides or hydroxyl radicals).
[0024] As used herein, the term "antioxidant" refers to compounds
that neutralize the activity of reactive oxygen species or inhibit
the cellular damage done by said reactive species.
[0025] As used herein, the term "pharmaceutically acceptable
carrier" refers to a carrier medium that does not interfere with
the effectiveness of the biological activity of the active
ingredient, is chemically inert, and is not toxic to the patient to
whom it is adminstered
[0026] As used herein, the term "pharmaceutically acceptable
derivative" refers to any homolog, analog, or fragment
corresponding to the ubiquinone formulations described in Section
5.1. infra which exhibits antioxidant activity and is relatively
non-toxic to the subject.
[0027] The term "therapeutic agent" refers to any molecule,
compound, or treatment, preferably an antioxidant, that assists in
the prevention or treatment of the disorders, or complications of
disorders caused by reactive oxygen species.
[0028] The term "agent that sequesters AR--NOX" refers to any
molecule, compound, or treatment that interacts with AR--NOX, thus
decreasing the reaction of AR--NOX with other substrates and
inhibits the ability of AR--NOX to generate reactive oxygen
species.
[0029] The antioxidants, cellular components, and target proteins
defined herein are abbreviated as follows:
1 mitochondrial DNA mtDNA nicotinamide adenine dinucleotide NADH
cell surface hydroquinone (NADH) oxidase with NOX protein
disulfide-thiol isomerase activity NOX specific to non-cancer cells
CNOX NOX specific to aged cells AR-NOX NOX specific to cancer cells
tNOX low density lipoproteins LDLs plasma membrane oxido-reductase
chain PMOR ubiquinone or coenzyme Q CoQ coenzyme Q.sub.10 Q.sub.10
reactive oxygen species ROS
4. BRIEF DESCRIPTION OF TIE DRAWINGS
[0030] FIG. 1. Oxidation of reduced ubiquinol at 37.degree. C. by
an enzymatic preparation with NADH oxidase activity solubilized
from HeLa cells by low pH treatment, followed by heat and
proteinase K The disappearance of reduced ubiquinol was determined
from the increase in absorbance at 410 nm as a function of time in
the presence of 1 mg of the HeLa protein. The concentration of
quinol was 50 mM, pH 7.
[0031] FIG. 2. The activity of the oxidase is periodic as shown
here for the oxidation of NADH by samples of sera from a young (A)
and an aged (B) patient The maxima in the time course of NADH
oxidation measured as a decrease in absorbance at 340 nm over 1
minute at 1.5 minute intervals marked by single arrows have an
average period lent of 24 minutes and are present in all sera thus
for tested In the aged subject, which is representative of both
male and female aged subjects 75 to 98, the maxima indicated by the
double arrows reflect an average period length of about 26 min and
are characteristic of a NOX isoform associated with aging.
[0032] FIG. 3. Time course of cytochrome c reduction by sera
determined from the A.sub.550-A.sub.540 determined at 10 sec
intervals over 450 sec. After 200 sec either 15 mg superoxide
dismutase (SOD) or 45 mg ubiquinone (Q.sub.10) were added and the
reaction was continued. A. 40 year old female.+-.SOD. B. 98 year
old female.+-.SOD. C. 83year old female.+-.Q.sub.10. D. 94 year old
female.+-.Q.sub.10. Results from multiple patients, both male and
female, are summarized in Table 4 in Section 6.2.3. Line slopes are
in nmoles/min/ml sera.
5. DETAILED DESCRIPTION OF THE INVENTION
[0033] The present invention provides pharmaceutical compositions,
methods of use, and pharmaceutial kits for the treatment of
disorders resulting from oxidative changes in cells that result in
aging by targeting an aging-related isoform of NADH oxidase
(AR--NOX), shed into the sera by aging cells.
[0034] The invention is based in part, on the Inventors' discovery
that ubiquinones inhibit the activity of an aging-related isoform
of NADH oxidase (AR--NOX) shed into the sera by aging cells. The
inhibition of this hyperactive form of NOX, AR--NOX, by ubiquinones
results in a decrease in the generation of reactive oxygen species
by AR--NOX. A decrease in reactive oxygen species should result in
a decrease of oxidative damage resulting from said reactive oxygen
species.
5.1. Plasma Membrane Hydroquinone (NADH) Oxidase (NOX)
[0035] The plasma membrane NADH oxidase (NOX) is a unique cell
surface protein with hydroquinone (NADH) oxidase and protein
disulfide-thiol interchange activities that normally responds to
hormone- and growth factors (Brightman et al., 1992, Biochim.
Biophys. Acta 1105:109-117; Morr, 1994, J. Bioenerg. Biomemb.
26:421-433, and Morr, 1998, Plasma Membrane Redox Systems and their
Role in Biological Stress and Disease, Klewer Academic Publishers,
Dordrecht, The Netherlands, pp. 121-156). A hormone-insensitive and
drug-responsive form of the activity designated tNOX also has been
described which is specific for cancer cells (Bruno et al., 1992,
Biochem. J. 284:625-628; Morr and Morr, 1995, Protoplasma
184:188-195; Morr et al., 1995, Proc. Natl. Acad. Sci. U.S.A.
92:1831-1835; Morr et al., 1995, Biochim. Biophys. Acta 1240:11-17;
Morr et al., 1996, Eur. J. Cancer 32A:1995-2003; Morr et al., 1997,
J. Biomemb. Bioenerg. 29:269-280; and U.S. Pat. No. 5,605,810.
which is incorporated by reference in its entirety for all
purposes).
[0036] Because the NOX protein is located at the external plasma
membrane surface and is not transmembrane, a functional role as an
NADH oxidase is not considered likely (Morr, 1994, J. Bioenerg.
Biomemb. 26:421-433; DeHahn et al., 1997, Biochem. Biophys. Acta
1328:99-108; and Morr, 1998, Plasma Membrane Redox Systems and
their Role in Biological Stress and Disease, Klewer Academic
Publishers, Dordrecht, The Netherlands, pp. 121-156). While the
oxidation of NADH provides a basis for a convenient method to assay
the activity, the ultimate electron physiological donor appears to
be hydroquinones with specific activities for hydroquinone
oxidation greater than or equal to that of NADH oxidation and/or
protein thiol-disulfide interchange (Kishi et al., 1999, Biochem.
Biophys. Acta 1412:66-77).
[0037] The NOX protein partially purified from the surface of HeLa
cells also exhibits ubiquinol oxidase activity (Kishi et al., 1999,
Biochem. Biophys. Acta 1412:66-77). These preparations completely
lack NADH: ubiquinone reductase activity and oxidize
Q.sub.10H.sub.2 at a rate of 3 to 6 nanomoles/min/mg protein. The
K.sub.m for reduced Q.sub.10H.sub.2 is 30 mM. Activities are
inhibited competitively by the cancer cell specific NADH oxidase
inhibitors capsaicin (8-methyl-N-vanillyl-6-noneamide) and the
antitumor sulfonylurea
N-4-methylphenylsulfonyl)-N'-(4-chlorophenyl)u- rea (LY181984)
(Morr et al., 1995, Proc. Natl. Acad. Sci. U.S.A. 92:1831-1835;
Morr et al., 1996, Eur. J. Cancer 32A:1995-2003; and Morr et al.,
1995, Biochim. Biophys. Acta 1240:11-17). The oxidation of
Q.sub.10H.sub.2 proceeds with what appears to be a normal
two-electron transfer in keeping with the participation of the
plasma membrane NADH oxidase as a terminal oxidase of plasma
membrane electron transport from cytosolic NAD(P)H via coenzyme Q
to acceptors at the cell surface.
[0038] Evidence that NOX proteins under certain conditions are
capable of the production of ROS is presented in Table 3 in Section
6.2.2. Ultraviolet light as a source of oxidative stress in
cultured cells is used to initiate superoxide generation (Morr et
al., 1999, Biofactors 9:179-187). Such generation is due to the
NADH oxidase because in cell lines (HeLa, human cervical carcinoma
and BT-20 human mammary carcinoma), that contain a
capsaicin-responsive NADH oxidase, the response to UV is inhibited
by capsaicin. In the MCF10A cell line (human mammary epithelia), a
non-cancerous cell line lacking tNOX activity, the UV-induced
generation of superoxide is unaffected by capsaicin and the
resultant effects on the plasma membrane CNOX (Table 3 in Section
6.2.2). The switch whereby the oxidase may reduce oxygen by a
one-electron or four-electron mechanism is not understood at
present but may reside in a delicate redox balance of the carriers
involved. Such a balance may be broken by oxidative stress or cell
damage. Metal ions, such as iron and copper, released by tissue
damage also may play a role (Hersbko, 1992, Molec. Aspects Med.
13:113-165) in maintaining redox homeostasis.
5.2. Plasma Membrane Levels of Coenzyme Q
[0039] Plasma membrane ubiquinone or coenzyme Q (CoQ) plays a major
role in the PMOR system. Ubiquinone or coenzyme Q (CoQ) occurs
ubiquitously among tissues. In rat liver, the highest amount is
found in the Golgi apparatus but it is also concentrated in the
plasma membrane (Table 1) (Crane and Morr, 1977, Biomedical and
Clinical Aspects of Coenzyme Q, Elsevier Scientific,
Amsterdam-Oxford-New York, pp. 3-14 and Kaln et al., Lipids
25:93-99). The ubiquinone content of plasma membrane is two to five
times that of microsomes and only half that of mitochondria
2TABLE 1 Distribution of ubiquinone in subcellular fractions from
rat liver. The values are means .+-. seven experiments (Kaln et
al., 1987, Biochem. Biophys. Acta 926: 70-78). Fractions Ubiquinone
9 (mg/mg protein) Homogenate 0.79 .+-. 0.08 Golgi apparatus 2.62
.+-. 0.15 Lysosomes 1.86 .+-. 0.18 Mitochondria 1.40 .+-. 0.16
Inner mitochondrial membranes 1.86 .+-. 0.13 Microsomes 0.15 .+-.
0.02 Peroxisomes 0.29 .+-. 0.04 Plasma membranes 0.74 .+-. 0.07
Supernatant 0.02 .+-. 0.004
[0040] Ubiquinone has long been considered to have both pro- and
antioxidant roles over and above its more conventional role in
mediating electron transport between NADH and succinic
dehydrogenase and the cytochrome system of mitochondria (Ernster
and Dallner, 1995, Biochim. Biophys. Acta 127:195-204 and Crane and
Barr, 1985, Coenzyme Q, John Wiley & Sons, Chichester, pp.
1-37). Both pro- and antioxidant as well as electron transport
roles should be considered for ubiquinone in the plasma
membrane.
[0041] CoQ is normally a product of cellular biosynthesis and
provides a potentially important source of one-electron pro-oxidant
oxygen reduction (Andersson et al., 1994, Biochim. Biophys. Acta
1214:79-87 and Appelkvist et al., 1994, Molec. Aspects Med.
15S:37-46). In its reduced hydroquinone form (ubiquinol), it is a
powerful antioxidant acting directly upon either superoxide or
indirectly on lipid radicals alone or together with vitamin E
(.alpha.-tocopherol) (Crane and Barr, 1985, Coenzyme Q, John Wiley
& Sons, Chichester, pp. 1-37; Beyer and Ernster, 1990,
Highlights of Ubiquinone Research, Taylor & Francis, London,
pp. 191-213;and Beyer, 1994, J. Bioenerg. Biomemb. 26:349-358;
Kagan et al., 1990, Biochem. Biophys. Res. Comm. 169:851-857; and
Ernster et al., 1992, BioFactors 3:241-248).
[0042] The antioxidant action of ubiquinol normally yields the
ubisemiquinone radical. The latter is converted back to ubiquinol
by re-reduction through the electron transfer chain in mitochondria
or by various quinone reductases in various cellular compartments
including the plasma membrane (Takahashi et al., 1995, Biochem. J.
309:883-890; Takahashi et al., 1996, J. Biochem. (Tokyo)
119:256-263; Beyer et al., 1996, Proc. Natl. Acad Sci. U.S.A.
93:2528-2532; Beyer et al., 1997, Molec. Aspects Med. 18:s15-s23;
Navarro et al., 1995, Biochem. Biophys. Res. Comm. 212:138-143;
Villalba et al., 1995, Molec. Aspects Med. 18:s7-s13; and Arroyo et
al., 1998, Protoplasma 205:107-113). Thus, ubiquinone may transform
from a beneficial one-electron carrier to a superoxide generator if
the ubisemiquinone anion becomes protonated (Nohl et al., 1996,
Free Rad. Biol. Med. 20:207-213).
[0043] In perfused rat liver and in isolated rat hepatocytes, the
anti-cancer quinone glycoside, adriamycin, induces oxidative stress
by enhancing ROS production (Valls et al., 1994, Biochem. Mol.
Biol. Int. 33:633-642 and Beyer et al., 1996, Proc. Natl. Acad.
Sci. U.S.A. 93:2528-2532). Exogenous CoQ addition prevents this ROS
production and concomitantly protects the cells from oxidative
damage. Similar effects of exogenous CoQ on NOX-mediated ROS
production have been observed (Table 3 in Section 6.2.2). The
antioxidant effect at the plasma membrane may ameliorate LDL
oxidation by scavenging ROS by PMOR produced at the cell surface
(Thomas et al., 1997. Molec. Aspects Med 18:s85-s103).
[0044] Some studies have shown that overall CoQ levels decrease
with age (Beyer et al., 1985, Mech Aging Dev. 32:267-281; Kaln et
al., 1990, Lipids 25:93-99; and Genova et al., 1995, Biochem. J.
311:105-109). However this is not true for all tissues and
especially for the brain, where high CoQ levels are maintained
throughout aging (Soderberg et al., 1990, J. Neorochem. 54:415423
and Battino et al., 1995, Mech Aging Dev. 78:173-187). Thus, the
invention also encompasses particular therapeutic levels of
coenzyme Q for inhibiting or reducing the effects caused by
overactive or aberrant cell surface PMOR system and for
sequestering NOX isoforms.
5.3. Evidence for an Aging-Related CNOX Protein
[0045] The NOX protein is anchored in the outer leaflet of the
plasma membrane (Morr, 1995, Biochem. Biophys. Acta 1240:201-208
and DeHahn et al., 1997, Biochem. Biophys. Acta 1328:99-108).
Subsequently, the activity was shown to be shed in soluble form
from the cell surface (Morr et al., 1996, Biochim. Biophys. Acta
1280:197-206). The presence of the shed form in the circulation
provides an opportunity to use patient sera as a source of the NOX
protein for large scale isolation and characterization studies and
to examine the NOX activity in sera of subjects of advanced age in
a simple and non-invasive procedure that permits side-by-side
comparisons with sera of young adults. A serum form of the CNOX
activity which is specific to sera from elderly subjects (AR--NOX)
has been identified by the Inventors. Results are shown in Table 4
in Section 6.2.3 for elderly individuals 80-94 years of age. This
sera has a superoxide-generating and aging-related enzymatic
activity, which is substantially reduced by addition of 0.1 mM
coenzyme Q.
[0046] The source of the circulating age-related form of the
superoxide-generating activity is hypothesized to result from
shedding from cells, as observed in other NOX forms. Consistent
with this interpretation was the appearance of a coenzyme Q
inhibitable age-related reduction of ferric cytochrome c with a
buffy coat fraction (lymphocytes) comparing young and aged
patients.
[0047] Thus, in one embodiment, the invention is directed to
utilizing drugs which sequester, neutralize, bind, or otherwise
block or eliminate, the AR--NOX protein and inhibit its ability to
generate reactive oxygen species so that the cells undergo
apoptosis (Morr et al., 1995, Proc. Natl. Acad. Sci. U.S.A.
92:1831-1835; Vaillant et al., 1996, J. Bioenerg. Biomemb.
28:531-540; and Dai et al., 1997, Mol. Cell. Biochem. 166:101-109).
Additionally, based on the presence of an age related PMOR system
capable of generating ROS at the cell surface, an approach to
ablation of anaerobic cells in aged tissues is feasible. The
benefits of such an approach include the fact that: (1) while
normally only a small percentage of muscle fibers become anaerobic
even in severely affected tissues, the elimination of these cells
would not be expected to have deleterious side effects; (2)
apoptosis of anaerobic cells results in the of lowering serum
levels of oxidized lipoproteins and an overall reduction of the
oxidative stress to surrounding healthy cells. The cells displaying
all of the characteristics listed supra are hereby defined as aged
cells. Generally, the characteristics of aged cells includes those
that express and/or shed AR--NOX and include, but are not limited
to, those exhibiting one or more of the following characteristics:
an age-related PMOR system, the ability to generate reactive oxygen
species, and have functionally defective mitochondria.
[0048] In another embodiment, the invention is directed to
utilizing agents, e.g., drugs or supplements, which switch the NOX
protein from oxygen reduction to protein disulfide reduction. The
advantage of such an approach has already been observed with plant
cells in response to auxins (Chueh et al., 1997, J. Biol. Chem.
272:11221-11227).
5.4. Methods of Detecting AR--NOX
[0049] The invention further contemplates using AR--NOX as a
diagnostic tool when oxidative damage to cells and/or tissue is
suspected. As such, AR--NOX in tissue, cells, or circulation may be
detected. In one embodiment, detection may be achieved
immunologically, by employing antibodies specific to AR--NOX
(described supra in Section 5.4). Said antibodies may be conjugated
to a wide variety of labels, e.g., radioisotopes, enzymes,
fluorescers, chemiluminescers, and the like, wherein the label
provides a detectable signal.
[0050] Alternatively, in another embodiment, detection is based
upon assays that recognize that sera with AR--NOX exhibits a higher
rate of cytochrome c reduction than sera without AR--NOX. In this
embodiment, AR--NOX reacts with a substrate capable of generating
reactive oxygen species, e.g., superoxide dismutase, which results
in cytochrome c reduction. The detection of cytochrome c may be
detected spectrophotometrically by measuring the absorbance at
about 540 nm to 550 nm.
[0051] In yet another embodiment, AR--NOX is detected in an assay
which measures the disappearance of the ascorbate radical
spectrophotometrically by measuring the absorbance at about 265 nm
since AR--NOX reduces an electron acceptor, e.g. ascorbate radical.
In another embodiment, a similar spectrophomometric assay may be
carried out by measuring the reduction of NAD.sup.+ by AR--NOX
using methods known in the art (Morr et al., 1995, Proc. Natl.
Acad. Sci. U.S.A. 92:1831-1835).
[0052] Other embodiments of the invention include assays based on
the unique oscillation property of AR--NOX since NOX from healthy
cells and AR--NOX exhibit varying oscillations, which are given by
the oxidation of NADH, ubiquinol, or reduced vitamin K.sub.1. As
detailed in Section 6.2.3., varying oscillations have been observed
for several NOX activity forms such that the oscillations serve as
a diagnostic feature to identify NOX activity forms during their
purification (Morr, 1998, Plasma Membrane Redox Systems and their
Role in Biological Stress and Disease, Klewer Academic Publishers,
Dordrecht, The Netherlands, pp. 121-156, which is incorporated by
reference in its entirety).
[0053] In yet another embodiment of the invention, AR--NOX is
detected by resistance to retinoic acid, since NOX from healthy
cells is inhibited by retinoic acid and AR--NOX is not inhibited by
retinoic acid (Morr, 1998, Plasma Membrane Redox Systems and their
Role in Biological Stress and Disease, Klewer Academic Publishers,
Dordrecht, The Netherlands, pp. 121-156).
[0054] Still other embodiments include a method using AR--NOX to
identify cells where mitochondrial functions are depressed and
consequently, PMOR is overexpressed. Such cells may be identified
in the presence of overexpressed AR--NOX In cells where PMOR is
overexpressed as a result of decreased electron input firm the
respiratory chain, overcompensation by AR--NOX may represent a
diagnostic feature.
[0055] AR--NOX may also be assayed for disulfide-thiol interchange
activity, by using dithio-dipyridyl substrates. Examples of
derivatives of dithio-dipyridyl substrates include
2-pyrimidineyione, 2,2'-ditiopyridine, and 6,6'-ditionicotinic
acid, which may be used to assay the disulfide-thiol interchange
activity of AR--NOX (Morr et al., 1999, Mol. Cell. Biochem.
200:7-13).
5.5. Methods of Identifying Agents that Interact with AR--NOX
[0056] The present invention relates to in vitro and in vivo
methods for screening for agents which target AR--NOX. Within the
broad category of in vitro selection methods, several types of
methods are likely to be particularly convenient and/or useful for
screening test agents. These include, but are not limited to,
methods which measure a binding interaction between two or more
components, and methods which measure the activity of an enzyme
which is one of the interacting components, i.e., AR--NOX.
[0057] Binding interactions between two or more components can be
measured in a variety of ways known in the art. One approach is to
label one of the components with an easily detectable label place
it together with the other component(s) in conditions under which
they would normally interact (e.g., ubiquinone), perform a
separation step which separates bound labeled component from
unbound labeled component, and then measure the amount of bound
component. The effect of a test agent included in the binding
reaction can be determined by comparing the amount of labeled
component which binds in the presence of this agent to the amount
which binds in its absence.
[0058] The test agent may be labeled with a detectable marker,
using methods for labeling known in the art A "detectable marker"
refers to a moiety, such as a radioactive isotope or group
containing same, or nonisotopic labels, such as enzymes, biotin,
avidin, streptavidin, digoxygenin, luminescent agents, dyes,
haptens, and the like. Luminescent agents, depending upon the
source of exciting energy, can be classified as radioluminescent,
chemiluminescent, bioluminescent, and photoluminescent (including
fluorescent and phosphorescent).
[0059] The separation step in this type of approach can be
accomplished in various ways. In one approach, (one of) the binding
partner(s) for the labeled component can be immobilized on a solid
phase prior to the binding reaction, and unbound labeled component
can be removed after the binding reaction by washing the solid
phase. Attachment of the binding partner to the solid phase can be
accomplished in various ways known to those skilled in the art,
including but not limited to, chemical cross-linking, non-specific
adhesion to a plastic surface, interaction with an antibody
attached to the solid phase, interaction between a ligand attached
to the binding partner (e.g., biotin), and a ligand-binding protein
(e.g., avidin or streptavidin) attached to the solid phase.
[0060] Alternatively, the separation step can be accomplished after
the labeled component has been allowed to interact with its binding
partner(s) in solution. If the size differences between the labeled
component and its binding partner(s) permit such a separation, the
separation can be achieved by passing the products of the binding
reaction through an ultrafilter whose pores allow passage of
unbound labeled component but not of its binding partner(s) or of
labeled component bound to its partner(s). Separation can also be
achieved using any reagent capable of capturing a binding partner
of the labeled component from solution, such as an antibody against
the binding partner, a ligand-binding protein which can interact
with a ligand previously attached to the binding partner.
[0061] Another in vitro selection method which may be used is the
screening of combinatorial chemistry libraries, using ubiquinone or
ubiquinone derivatives as a base molecule. The methods for the
generation and screening of combinatorial libraries are described
in U.S. Pat. No. 5,565,324, which is incorporated by reference in
its entirety. Briefly, the synthesis of the ubiquinone derivatives,
using combinatorial chemistry, involves syntheses with a plurality
of stages, wherein each stage involves a plurality of choices,
where large numbers of products with varying compositions are
obtained. The substrates carrying the final product compounds may
be screen for AR--NOX binders.
[0062] Test methods which rely on measurements of AR--NOX enzymatic
activity have been described in the previous subsection, supra.
[0063] The invention also comprises in vivo screening methods to
identify test agents that interact with AR--NOX. In this approach,
coding sequences encoding part or all of a component(s) would be
introduced into a selected type of cell. Coding sequences for this
approach include cloned genes, cDNAs, fragments of either,
fragments amplified by the polymerase chain reaction, natural RNAs,
transcribed RNAs, or the like. For example, coding sequences for
two or more components which are known to interact with each other
(e.g., AR--NOX and ubiquinone) are introduced into a cell, and
agents are tested for their ability to moderate and/or displace the
interaction between these two components.
[0064] In another embodiment, proteins that interact with AR--NOX
may be identified by a yeast two-hybrid assay (Fields and Song,
1989, Nature 340:245-246). The yeast two-hybrid assay takes
advantage of the properties of the GAL4 protein of the yeast
Saccharomyces cerevisiae. GAL4 is a transcriptional activator
required for the expression of genes encoding enzymes of galactose
utilization, which consists of two separable and functionally
essential domains: an N-terminal domain which binds to specific DNA
sequences (UASG) and a C-terminal domain containing acidic regions,
which is necessary to activate transcription. The two-hybrid screen
comprises a system of two hybrid proteins containing parts of GAL4:
the GAL4 DNA-binding domain fused to a protein `X` and a GAL4
activating region fused to a protein `Y`. If X and Y can form a
protein-protein complex and reconstitute proximity of the GAL4
domains, transcription of a gene regulated by UASG (e.g.,
.beta.-galactosidase) occurs. In this embodiment, AR--NOX may be
used as the "bait" to screen for the "prey," i.e., putative
interacting agents, present as cDNAs from a cDNA library. In both
cases, the AR--NOX and putative interacting agents are fused in
frame with the two parts of GAL4, or other transcriptional
activator. Kits for two-hybrid assays are readily available, e.g.,
Hybrid Hunter.TM. Two-Hybrid system from Invitrogen.
[0065] Gene therapy approaches may also be used in accordance with
the present invention to inhibit AR--NOX. Among the compounds which
may interact with AR--NOX, and therefore disrupt its activity, are
antisense and ribozyme molecules. Such molecules are designed to
inhibit the expression of the target gene, AR--NOX. Techniques for
the production and use of antisense and/or ribozyme molecules are
well known to those of skill in the art and can be designed with
respect to the nucleotide sequence of AR--NOX.
[0066] Antisense RNA and DNA molecules act to directly block the
translation of mRNA by hybridizing to targeted mRNA and preventing
protein translation. With respect to antisense DNA,
oligodeoxynucleotides derived from the translation initiation site,
e.g., between the -10 and +10 regions of the target gene nucleotide
sequence of interest, are preferred.
[0067] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of mRNA (reviewed in Rossi, 1994, Current
Biology 4:469-471). The mechanism of ribozyme action involves
sequence specific hybridization of the ribozyme to complementary
target RNA, followed by an endonucleolytic cleavage. The
composition of ribozyme molecules must include one or more
sequences complementary to the target gene mRNA, and must include
the catalytic sequence responsible for mRNA cleavage (U.S. Pat. No.
5,093,246).
[0068] The invention further encompasses methods for monitoring
patient response to the agents identified by the methods described
supra. By monitoring circulating AR--NOX activity in patient sera,
it will be possible to determine therapeutic dosages and to monitor
therapeutic benefit. The response to the subject compositions may
be monitored by assaying the blood or urine of the patient for the
AR--NOX activity that is responsive to the agents that interact
with AR--NOX. By following the above monitoring procedures, an
effective dosage of the subject compositions may be administered in
accordance with the requirement of the individual patient.
5.6. Inhibition of AR--NOX by Ubiquinones
[0069] The invention comprises the administration of a
therapeutically effective amount of ubiquinones to a patient with a
disorder or a complication of a disorder caused by oxidative damage
resulting from the generation of reactive oxygen species by
AR--NOX. In a preferred embodiment, the total daily amount
administered is from about 1 to about 500 mg of ubiquinones. In a
more preferred embodiment, the total daily amount administered is
from about 1 to 100 mg of ubiquinones.
[0070] The invention is based on studies by the Inventors that have
identified a serum form of AR--NOX which is specific to sera from
elderly patients, and absent from sera of younger patients. Not
only is there a superoxide-generating and aging-related enzymatic
activity present in sera of the elderly patients, but also, the
aging-related enzymatic activity is reduced by the addition of
ubiquinone.
[0071] In one embodiment, the invention is used to identify
patients suffering from disorders associated with reactive oxygen
species who may be responsive to treatment with ubiquinones. Such
responsive patients may be identified by assay of serum or urine
for ubiquinone responsive superoxide generation. The generation of
superoxide may be followed by reduction of cytochrome c (described
in Section 6.1.3) or any other suitable biological or chemical
method.
[0072] The ubiquinones are benzoquinones with a base structure of
2,3-dimethoxy-s-methylbenzoquinone nucleus ("Q.sub.n") and differ
in the number of carbon atoms in the side chain of the 6-position,
wherein n is the number of carbon units in the side chain (n is 1
to 12). The differences in properties are due to the difference in
length of the side chain (Merck Index 10.sup.th Edition, 1983,
Merck & Co., Inc., Rahway, N.J., p. 1407). Naturally occurring
derivatives of ubiquinone are the coenzymes Q.sub.6 to Q.sub.10,
wherein Q.sub.10 is naturally occurring in humans and Q.sub.9 is
naturally occurring in rats.
[0073] The invention comprises a treating a patient with a
pharmacologically effective amount of ubiquinones to inhibit the
generation of reactive oxygen species. In a preferred embodiment,
the ubiquinones are of the human derivative Q.sub.10. In another
embodiment, the ubiquinones comprise the naturally occurring
derivatives Q.sub.6, Q.sub.7, Q.sub.8, and Q.sub.9. In another
embodiment, the ubiquinones comprise other derivatives Q.sub.1,
Q.sub.2, Q.sub.3, Q.sub.4, Q.sub.5, Q.sub.11, and Q.sub.12. In
another embodiment, the invention comprises mixtures of the
ubiquinone derivatives described supra. The invention further
comprises all pharmaceutically acceptable derivatives of the
compositions listed supra for methods of treating a patient with an
AR--NOX related disorder, with ubiquinone administration in the
range of 0.1 to 100 mg per kg body weight.
[0074] In addition, the invention provides a method for screening
for test compounds that interact with AR--NOX further comprising
comparing the interaction of AR--NOX of the test compound to the
interaction of AR--NOX with ubiquinone, wherein the interaction of
AR--NOX with ubiquinone serves as a positive control.
[0075] The invention also encompasses methods for monitoring
patient response to ubiquinones. By monitoring circulating AR--NOX
activity in patient sera, it will be possible to determine
therapeutic dosages and to monitor therapeutic benefit from
ubiquinones. The response to the subject compositions may be
monitored by assaying the blood or urine of the patient for the
AR--NOX activity that is responsive to the ubiquinone compositions.
By following the above monitoring procedures, an effective dosage
of the subject compositions may be administered in accordance with
the requirement of the individual patient.
5.7. Isolation and Characterization of AR--NOX
[0076] The AR--NOX protein has been purified by the Inventors from
lymphocytes and sera from aged individuals. The AR--NOX protein was
purified by standard protein preparative techniques well known to
those skilled in the art including ammonium sulfate precipitation,
anion exchange, gel filtration, preparative SDS-PAGE and
hydrophobic interaction chromatography, HPLC, and FPLC (Morr et
al., 1996, Biochem. Biophys. Acta 1280)197-206 and Current
Protocols in Molecular Biology, Green Publishing Associates and
Wiley Intersciences, N.Y.). The ability to generate superoxide as
evidenced by reduction of cytochrome c and inhibition by coenzyme Q
were used as selection criteria Sufficient AR--NOX amino acid
sequence was obtained by the Inventors to generate degenerate
oligonucleotide primers for the amplification of a portion of the
AR--NOX nucleic acid sequence.
[0077] The Inventors have also generated a NOX-specific polyclonal
antibody to the AR--NOX protein from lymphocytes. Once the amino
acid sequence of AR--NOX is deduced from the corresponding cDNA
sequence, the amino acid sequence may be used to strategically
generate peptide sera with therapeutic potential as probes specific
to AR--NOX to investigate and ameliorate NOX responses to aging.
Three criteria of the known protein sequence may be used in
selecting sequences for antibody production: 1. hydrophilicity as
calculated according to the algorithm of Hopp and Woods (Hopp and
Woods 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); 2. surface
probability as calculated according to the formula of Emini et al.
(Emini et al., 1985, J. Virol. 53:836-839); and 3. the antigenic
index measuring the probability that a region is antigenic as
calculated by summing several weighted measures of secondary
structure (Jamieson and Wolf, 1988, CABIOS 4:181-186). Peptide
antibodies can be affinity-purified using immobilized peptide. The
peptide antisera may be employed in co-incubation experiments with
isolated lymphocytes from aged individuals and isolated lipoprotein
particles to demonstrate that specific AR--NOX inhibition can
ablate propagation of oxidative stress in both in vitro and in vivo
systems.
[0078] The present invention also relates to methods for cloning of
AR--NOX. Using methods which are well known to those skilled in the
art, recombinant cDNA libraries may be constructed using RNA
prepared from cells known to express AR--NOX. The cDNA libraries
may be constructed using a variety of vector systems, including but
not limited to, bacteriophage vectors, plasmid vectors or mammalian
expression vectors. See, for example, the techniques described in
Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory, N.Y. and Current Protocols in Molecular
Biology, Green Publishing Associates and Wiley Intersciences, N.Y.
Alternatively, a human cDNA library library may be obtained from a
commercial source, e.g., Stratagene.
[0079] The recombinant cDNA libraries may be screened using a
number of different techniques which are well known to those
skilled in the art For example, a mixture of degenerate
oligonucleotide probes may be designed utilizing the partial amino
acid sequence of AR--NOX. The oligonucleotides may be labeled and
used directly to screen a cDNA library for clones containing
inserts with sequence homology to the oligonucleotide sequences.
Alternatively, the oligonucleotides may be used as primers in a
polymerase chain reaction. The template for the reaction is
cDNA-may be obtained by reverse transcription of mRNA prepared from
cells known to express AR--NOX, i.e., lymphocytes from aged
individuals. Alternatively, the template may be cDNA from a human
cDNA library library obtained from a commercial source, e.g.,
Stratagene. The amplified DNA fragment may be labeled and used to
screen a library for isolation of full length clones. In another
example, an expression library may be immunologically screened
using antibodies directed against AR--NOX. In yet another
embodiment of the invention, a cDNA library may be engineered into
a mammalian expression vector and screened by transfection into the
appropriate mammalian cell line followed by assaying for AR--NOX
activity in the tissue culture supernatant.
[0080] In yet another embodiment of the invention, a method for
cloning AR--NOX by means of polymerase chain reaction may be used
to clone a cDNA coding for AR--NOX. Such a method may be utilized
using RNA prepared from lymphocytes of aged individuals.
Alternatively, AR--NOX may be cloned by polymerase chain reaction
amplification of a human cDNA library obtained from a commercial
source (e.g., Stratagene).
[0081] In addition, gene expression assays using gene expression
arrays or microarrays are now practicable for identifying changes
in gene expression patterns between different cells or tissue types
(see, e.g., Schena et al., 1995, Science 270:467-470; Lockhart et
al., 1996, Nature Biotechnology 14:1674-1680; and Blanchard et al.,
1996, Nature Biotechnology 14:1649). Thus, in another, alternative
embodiment of the invention, such gene expression arrays or
microarrays may be used to compare mRNA expression patterns in
cells that exhibit AR--NOX activity (e.g., as determined by one of
the assays of the present invention) to mRNA expression patterns in
cells that do not exhibit AR--NOX activity and thus, do not express
AR--NOX.
5.8. Target Disorders
[0082] Disorders that can be treated by the methods of the present
invention include any clinical condition in which oxidative species
have been implicated.
[0083] Examples of clinical conditions in which oxidative species
have been implicated include, but are not limited to,
ischemia-reperfusion injury (e.g., stroke/myocardial infarction and
organ transplantation), cancer, aging, arthritis associated with
age, fatigue associated with age, alcoholism, red blood cell
defects (e.g., favism, malaria, sickle cell anemia, Fanconi's
anemia, and protoporphyrin photo-oxidation), iron overload (e.g.,
nutritional deficiencies, Kwashiorkor, thalassemia, dietary iron
overload, idiopathic hemochromatosis), kidney (e.g., metal
ion-mediated nephrotoxicity, aminoglycoside nephrotoxicity, and
autoimmune nephrotic syndromes), gastrointestinal tract (e.g., oral
iron poisoning, endotoxin liver injury, free fatty acid-induced
pancreatitis, nonsteroidal antiinflammatory drug induced
gastrointestinal tract lesions, and diabetogenic actions of
alloxan), inflammatory-immune injury (e.g., rheumatoid arthritis,
glomerulonephritis, autoimmune diseases, vasculitis, and hepatitis
B virus), brain (e.g., Parkinson's disease, neurotoxins, allergic
encephalomyelitis, potentiation of traumatic injury, hypertensive
cerebrovascular injury, and vitamin E deficiency), heart and
cardiovascular system (e.g., atherosclerosis, adriamycin
cardiotoxicity, Keshan disease (selenium deficiency) and alcohol
cardiomyopathy, eye (e.g., photic retinopathy, occular hemorrhage,
cataractogenesis, and degenerative renal damange), amyotrophic
lateral sclerosis, and age-related macular degeneration (Slater,
1989, Free Rad. Res. Comm. 7:119-390; Deng et al., 1993, Science
261:1047-1051; Seddon et al., 1994, JAMA 272:1413-1420; Brown,
1995, Cell 80:687-692; and Jenner, 1991, Acta Neurol. Scand.
84:6-15).
[0084] The invention is also directed to preventing or alleviating
complications of diabetes, atherogenesis, atherosclerosis, and
related diseases. Oxidative stress and LDL oxidation are common
complicating features in diabetics and circulating AR--NOX offers
opportunities for redox modulation of blood constituents important
to aging, atherogenesis, and atherosclerosis (Kennedy and Lyons,
1998, Metabolism 56;14-21).
[0085] In one embodiment, the invention is directed towards a
method of preventing a complication of a primary disorder in
patients wherein said complication results from oxidative damage
resulting from the generation of reactive oxygen species by
AR--NOX. The method comprises administering to a patient with a
primary disorder, in an amount effective to prevent said
complication, an agent or agents that sequesters AR--NOX in a
pharmaceutically acceptable carrier.
[0086] In another embodiment, the invention is directed towards a
method of preventing a secondary disorder in patients having a
primary disorder that causes oxidative damage resulting from the
generation of reactive oxygen species by AR--NOX. The method
comprises administering to a patient having a primary disorder, in
an amount effective to prevent said secondary disorder, an agent or
agents that sequesters AR--NOX, in a pharmaceutically acceptable
carrier.
5.9. Pharmaceutical Formulations
[0087] Agents that interact with AR--NOX identified by the methods
listed supra may be formulated into pharmaceutical preparations for
administration to mammals for prevention or treatment of disorders
in which oxidative species have been implicated. In a preferred
embodiment, the mammal is a human.
[0088] Compositions comprising a compound of the invention
formulated in a compatible pharmaceutical carrier may be prepared,
packaged, and labeled for treatment.
[0089] If the complex is water-soluble, then it may be formulated
in an appropriate buffer, for example, phosphate buffered saline or
other physiologically compatible solutions. Alternatively, if the
resulting complex has poor solubility in aqueous solvents, then it
may be formulated with a non-ionic surfactant such as Tween, or
polyethylene glycol. Thus, the compounds and their physiologically
acceptable solvates may be formulated for administration by
inhalation or insufflation (either through the mouth or the nose)
or oral buccal, parenteral rectal administration or, in the case of
tumors, directly injected into a solid tumor.
[0090] For oral administration, the pharmaceutically preparation
may be in liquid form, for example, solutions, syrups or
suspensions, or may be presented as a drug product for
reconstitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, or fractionated vegetable
oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates
or sorbic acid). The pharmaceutical compositions may take the form
of, for example, tablets or capsules prepared by conventional means
with pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinized maize starch, polyvinyl pyrrolidone or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well-known in the art.
[0091] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound.
[0092] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0093] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0094] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0095] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0096] The compounds may also be formulated as a topical
application, such as a cream or lotion.
[0097] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administer by implantation (for example,
subcutaneously or intramuscularly) or by intramuscular injection
Thus, for example, the compounds may be formulated with suitable
polymeric or hydrophobic materials (for example, as an emulsion in
an acceptable oil) or ion exchange resins, or as sparingly soluble
derivatives, for example, as a sparingly soluble salt. Liposomes
and emulsions are well known examples of delivery vehicles or
carriers for hydrophilic drugs.
[0098] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
[0099] The invention also provides kits for carrying out the
therapeutic regimens of the invention. Such kits comprise in one or
more containers therapeutically or prophylactically effective
amounts of the compositions in pharmaceutically acceptable form.
The composition in a vial of a kit of the invention may be in the
form of a pharmaceutically acceptable solution, e.g., in
combination with sterile saline, dextrose solution, or buffered
solution, or other pharmaceutically acceptable sterile fluid.
Alternatively, the complex may be lyophilized or desiccated; in
this instance, the kit optionally further comprises in a container
a pharmaceutically acceptable solution (e.g., saline, dextrose
solution, etc.), preferably sterile, to reconstitute the complex to
form a solution for injection purposes.
[0100] In another embodiment, a kit of the invention further
comprises a needle or syringe, preferably packaged in sterile form,
for injecting the complex, and/or a packaged alcohol pad.
Instructions are optionally included for administration of
compositions by a clinician or by the patient.
6. EXAMPLE
A Multifunctional Ubiquinol Oxidase of the External Cell Surface
and Sera
6.1. Materials and Methods
6.1.1. Measurement of Oxidation of Ubiquinol and of NADH
Oxidation
[0101] Ubiquinol oxidation was estimated based on increase in
absorbance at 410 nm for ubiquinone (FIG. 1). NADH oxidation was
determined from the decrease in A.sub.340 measured
spectrophotometrically as described (Brightman et al., 1992,
Biochim. Biophys. Acta 1105:109-117).
6.1.2. Superoxide generation by Cells and Response to UV
[0102] The generation of superoxide radical was determined by
assaying the rate of superoxide dismutase (SOD)inhibitable
cytochrome c reduction (Mayo and Curnette, 1990, Meth. Enzymol.
186:567-575 and Butler et al., 1982, J. Biol. Chem
257:10747-10750). The cytochrome c was from horse heart
mitochondria (type VI, Sigma) and was dissolved in PBSG buffer (see
below) to make a solution with a concentration of 1 mg/ml.
Air-saturated reaction mixtures of 100 ml cytochrome c stock
solution and 50 ml of a cell suspension (suspended in PBSG buffer)
to give a final concentration of .about.5.times.10.sup.6 cells/ml
were added to 2 ml of PBSG buffer (138 mM NaCl, 2.7 mM KCl, 8.1 mM
Na.sub.2HHPO.sub.4, pH 7.4 supplemented with 0.9 mM CaCl.sub.2, and
7.5 mM glucose and contained in plastic cuvettes. The formation of
reduced cytochrome c was measured in the presence and absence of
SOD (15 mg, Sigma) or capsaicin (2.5 mM) by comparing the
absorbance of the mixture at 550 nm-540 nm. Superoxide formation
was stimulated by using a handheld UV light (254 nm, 200
mw/cm.sup.2). The extent of cytochrome c reduction was monitored
spectrophotometrically at 550 nm every ten sec with gentle mixing
between readings. Data were analyzed from the slope of the change
in a 550 nm-540 mm before and after UV and then again after SOD or
capsaicin was added. Data were expressed as nmoles
O.sub.2.sup.-/10.sup.6 cells using a value of S.sub.M550
nm=19.1.times.10.sup.3 M.sup.-1 cm.sup.-1.
6.1.3. Cytochrome c Reduction by Sera and Inhibition by Ubiquinone
(Q.sub.10)
[0103] Cytochrome c reduction by sera was assayed as for cells
except that the samples were not mixed in between readings. The
Q.sub.10 was added as an ethanolic solution.
[0104] Sera were obtained from the patient population of St.
Elizabeth Hospital, Lafayette, Ind. and the resident population of
St. Anthony's Health Care, Lafayette, Ind. Confidentiality of
medical records was assured by assigning a number to each
sample.
6.2. Results
6.2.1. Characteristics of NOX
[0105] Characteristics of the surface quinol oxidase (FIG. 1) are
summarized in Table 2. The protein is of relatively low abundance
and specific activity but sufficient to catalyze a substantial flow
of electrons from cytosolic reduced pyridine nucleotide to
molecular oxygen. Molecular oxygen has been shown to represent a
physiological electron acceptor for the oxidase. However, under
certain conditions, protein disulfides also may function as
acceptors (Morr, 1994, J. of Bioenerg. and Biomemb.
26:421-433).
3TABLE 2 Properties of NOX-catalyzed oxidation of ubiquinol. pH
optimum 7.0 EC.sub.50 LY181984.sup.a inhibition 30 nM (competitive)
EC.sub.50 capsaicin.sup.b inhibition 1 nM (competitive) Cyanide
Resistant NOX-specific monoclonal antibodies Inhibited K.sub.m for
quinol 33 mM V.sub.max (nmoles/min/mg pro) 3 Oxygen consumption
(nmoles/min/mg/pro) 2.5
.sup.aN-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea
.sup.b8-methyl-N-vanillyl-6-noneamide
6.2.2. In Vitro Generation of Reactive Oxygen Species
[0106] Cultured cells were subjected to ultraviolet light to
perturbate electron flow to show that if the orderly two electron
flow to molecular oxygen or protein thiols was suitably
interrupted, a one electron process producing superoxide results.
Such production leads to formation of hydrogen peroxide and
possibly other oxidants such as hydroxide radical. These reactive
oxygen species could then be released into the environment to react
with neighboring cells and circulating molecules such as low
density lipoproteins (LDLs). Results indicate that based on
superoxide SOD-inhibitable reduction of cytochrome c, superoxide
was generated by all three cell lines tested (Table 3). The
SOD-inhibitable reduction of cytochrome c is assumed to be due at
least partially to the cell surface NADH oxidase based on drug
responsiveness. The HeLa (human cervical carcinoma) and BT-20
(human mammary adenocarcinoma) cells contain an activity form of
the ubiquinol oxidase that is drug responsive (Morr et al., 1995,
Proc. Natl. Acad. Sci. U.S.A. 92:1831-1835). Among the inhibitors
that are specific to the cancer activity form are putative quinone
site inhibitors such as capsaicin and the antitumor sulfonylurea
LY181984 (Morr et al., 1995, Proc. Natl. Acad. Sci. U.S.A.
92:1831-1835). The MCF-10A (noncancerous human mammary epithelia)
cells that lack the capsaicin-responsive NADH oxidase also lack the
capsaicin-responsive UV-induced generation of superoxide (Morr et
al., 1995, Proc. Natl. Acad. Sci. U.S.A. 92:1831-1835).
4TABLE 3 Superoxide production (reduction of cytochrome c) by cell
lines in response to UV irradiation and inhibition by superoxide
dismutase (SOD) and by capsaicin. Reduction of cytochrome c as a
measure of superoxide formation, nmoles/min/10.sup.6 cells After
UV.sup.2 Cell line.sup.1 Initial No Addition +SOD.sup.3
+Capsaicin.sup.4 HeLa S 0.8 .+-. 0.16 4.0 .+-. 1.0 1.1 0.8 BT-20
0.7 .+-. 0.2 5.1 .+-. 2.1 -0.1 -3.7 MCF 10A 1.5 .+-. 0.2 7.2 .+-.
0.1 -0.7 7.2 .sup.1HeLa S, human cervical carcinoma; BT-20, human
mammary adenocarcinoma; MCF 10A, human mammary epithelia
(non-cancer). .sup.210 minutes of 254 nm, 200 mw/cm.sup.2 .sup.315
mg (Sigma) .sup.42.5 mM added in DMSO. Rates were corrected for a
DMSO blank (0.1% final concentration)
6.23. Evidence for AR--NOX
[0107] A partially-purified preparation of the oxidase from HeLa
cells also responds to UV by generation of SOD- and
capsaicin-inhibited superoxide, suggesting that the effect of UV is
directly on the oxidase. It has previously been suggested that
NADPH oxidase may be a source of reactive oxygen species generated
by UV irradiation based on inhibition by diphenyliodinium (Gorman
et al., 1997, FEBS Lett. 404:27-33).
[0108] The Inventors have identified a serum form of AR--NOX that
is specific to sera from elderly subjects and low or absent from
sera of younger subjects (FIG. 2). Sera contains NADH oxidase
activities with properties similar if not identical to those of the
NADH oxidase found at the cell surface Worry et al., 1997, Archives
Biochem. Biophys. 342:224-230). The invention contemplates the
characterization and cloning of AR--NOX from the sera of aging
patients. The invention further contemplates large scale isolation
and purification of AR--NOX activity in the sera of subjects of
advanced age in a simple and non-invasive procedure for
side-by-side comparisons with sera of young adults. Such isolation
and purification methods are known in the at (Chueh et al., 1997,
Archives Biochem. Biophys. 342:3847).
[0109] Based on results of Table 3, the ability of sera to reduce
cytochrome c was measured. Sera of aged patients exhibited a much
more dramatic rate of cytocbrome c reduction than sera of young to
middle-aged patients (Table 4, FIG. 3). Sex differences, if any,
were negligible with sera from both male and female patients giving
similar responses.
5TABLE 4 Reduction of cytochrome c by sera comparing individuals
aged 21-46 years and individuals aged 76-98 years and response to
superoxide dismutase (SOD) and ubiquinone (Q.sub.10)
nmoles/min/ml/sera Patient age No Addition +15 mg SOD +45 mg
Q.sub.10 21-46 years (n = 16) 0.02 .+-. 0.1 0.02 .+-. 0.05 -- 76-98
years (n = 15) 1.5 .+-. 0.9 1.3 .+-. 0.8 -- 83-95 years (n = 5) 3.9
.+-. 1.6 -- 2.5 .+-. 1.4
[0110] Unlike the UV-induced changes with cells (Table 3, FIG. 3),
the reduction of cytochrome c by sera of aged patients occurred
spontaneously (no need for UV induction) and was only partially
inhibited by SOD. An interesting feature of the reduction of
cytochrome c by sera of aged patients was that the activity was
inhibited 36% on average by ubiquinone. The degree of inhibition by
0.1 mM ubiquinone varied from 6 to 90%.
[0111] NOX activities of cells, plasma membrane and sera (as well
as the purified protein) oscillate with a period of 24 minutes. The
oscillations are given by both the oxidation of NADH and by the
oxidation of ubiquinol or reduced vitamin K.sub.1 (Morr, 1998,
Plasma Membrane Redox Systems and their Role in Biological Stress
and Disease, Klewer Academic Publishers, Dordrecht, The
Netherlands, pp. 121-156). The oscillations have been observed for
several NADH oxidase activity forms such that the oscillations now
serve in the laboratory as a diagnostic feature to identify NOX
activity forms during their purification (Morr, 1998, Plasma
Membrane Redox Systems and their Role in Biological Stress and
Disease, Klewer Academic Publishers, Dordrecht, The Netherlands,
pp. 121-156).
[0112] Sera of healthy individuals normally exhibit an NADH oxidase
activity with a major period of 24 minutes (single arrows, FIG.
2A). There is, however, a second set of oscillations that conform
to a period of about 26 min (double arrows, FIG. 2A). The latter
oscillations are augmented in sera of aged patients (FIG. 2B). The
aging-related oscillatory activity represents a unique isoform
since it is resistant to inhibition by retinoic acid (10 mM)
whereas the isoform with a 26 minute period in sera of healthy
individuals appears to be inhibited by retinoic acid.
[0113] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described will
become apparent to those skilled in the art from the foregoing
description and accompanying figures. Such modifications are
intended to fall within the scope of the appended claims.
[0114] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
* * * * *