Note: Descriptions are shown in the official language in which they were submitted.
CA 02524551 2013-06-11
USE OF SELENIUM YEASTS IN THE TREATMENT OF ALZHEIMER'S
DISEASE
FIELD OF THE INVENTION
The present invention relates to compositions and methods for altering cell
function.
In particular, the present invention provides compositions comprising selenium
(e.g., Sel-
Plex6) and methods of using the same (e.g., as a therapeutic and/or
prophylactic treatment
for neurodegenerative disease). Additionally, the present invention
demonstrates that
specific forms of selenium (e.g., Sel-Plexe) possess the ability to alter
expression of genes
associated with disease and/or aging.
BACKGROUND OF THE INVENTION
Selenium is a trace element important for proper physiological function in
humans.
Selenium is ingested through the diet which can have a varying content of
selenium. For
example, in large parts of the world, crops with poor levels of selenium are
cultivated
because of low levels of selenium in the soil.
Selenium is incorporated into different organic molecules including, for
example,
amino acids such as 1-selenomethionine, selenocysteine, and selenocystine.
Thus,
selenium can be a component part of proteins, many of which are of structural
importance
to the body. Furthermore, selenium is an important ingredient in a number of
enzymes
which influence metabolism, reproduction, the prevention of cancer, and immune
defense
in humans (See, e.g., Rayman, M, Lancet 356:233-241 (2000)).
Multiple forms of selenium have been examined. These include inorganic
selenium
such as selenite, and organic sources, including selenium yeast. There is a
significant
difference between absorption and toxicity of inorganic and organic selenium,
the inorganic
compounds usually being absorbed and utilized less efficiently and also being
more toxic
than organic sources of selenium.
Multiple studies have attempted to reveal potential health benefits resulting
from the
ingestion of low levels of selenium. For example, low concentrations of an
inorganic form
of selenium, sodium selenate, have shown some potential health benefits (See,
e.g.,
Fumsinn et al., Int. J of Obesity and Related Metab. Dis., 19, 458-463
(1995)). However, at
elevated dosage levels, beneficial effects are reversed and dangerous toxicity
is manifested.
Research over the last two decades has suggested that selenium is effective in
the
reduction of cancer incidence when provided to animals at doses only 5- to 10-
fold above
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CA 02524551 2012-08-31
nutritional requirement (See, e.g., El-Bayoumy, The role of selenium in cancer
prevention,
Philadelphia, Lippincott, 1-15, 1991). Chemoprevention studies with selenium
in animal
model systems have indicated that this element is effective for most, if not
all of the organ
systems and is protective against the carcinogenic effects of a wide variety
of insults (See,
e.g., El-Bayoumy, The role of selenium in cancer prevention, Philadelphia,
Lippincott, 1-
15, 1991). Both epidemiological studies and supplementation trials have also
supported its
efficacy in lowering the incidence of cancers of the liver, colon, prostate
and lung (See,
e.g., Yu et al. Biol Trace Elem Res, 56: 117-124 (1997); Clark et al., J Am
Med Assoc,
276: 1957-1963 (1996); Yoshizawa etal., J Natl Cancer Inst, 90: 1219-1224,
(1998);
Brooks, et al., J Urol, 166: 2034-2038, (2001)). Other studies have
demonstrated no
beneficial effect for selenium reduction of cancers (See, e.g., Garland et
al., J. Am. Coll
Nutr., 12: 400-11 (1993); Ghadirian et al., Cancer Detect Prey, 24: 305-13
(2000)).
Heart disease has also been shown to be reduced in persons who consume certain
amounts of selenium in their diet. The levels of selenium in the blood stream
were
correlated with the degree of progression of cardiovascular disease with those
patients
having the lowest levels of selenium having the most extensive coronary artery
blockage
A need exists to identify new targets for selenium treatment that provide
beneficial
effects to a subject. Additionally, there is a need for information regarding
what forms of
selenium can and cannot be used for bringing about these effects. For example,
it would be
of great value to elucidate various ways in which different forms of selenium
(e.g., organic,
inorganic, or both) might be used to benefit certain systems (e.g., nervous,
endocrine, and
metabolic systems) of a subject (e.g., a human, bovine or other mammal).
Furthermore,
understanding how various forms of selenium differ in their ability to exert
effects on a
subject provides the ability to customize treatments for subjects suffering
from, or at risk
of, a disease or disorder that might be benefited by such treatment (e.g.,
specific forms of
selenium could be used independently or with other known agents to treat or
prevent
diseases or disorders). Identification of unwanted effects from the
consumption of certain
forms of selenium could also be identified and avoided.
SUMMARY OF THE INVENTION
The present invention relates to compositions and methods for altering cell
function.
In particular, the present invention provides compositions comprising selenium
(e.g., Sel-
PlexC) and methods of using the same (e.g., as a therapeutic and/or
prophylactic treatment
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CA 02524551 2012-08-31
for neurodegenerative disease). Additionally, the present invention
demonstrates that
specific forms of selenium (e.g., Sel-Plexe) possess the ability to alter
expression of genes
associated with disease and/or aging while other forms of selenium (e.g., free
selenomethionine) do not.
Accordingly, the present invention provides a method of treatment or
preventative for
Alzheimer's or reduction of signs or symptoms associated with Alzheimer's
disease or
prophylactically preventing or minimizing biological events associated with
the onset or
progression of Alzheimer's or reducing gene expression of genes correlated to
the onset or
progression of Alzheimer's disease comprising administering to a subject
(e.g., a subject
suffering from Alzheimer's disease, a subject with early-onset Alzheimer's
disease, a
subject having Alzheimer's disease, a subject displaying signs or symptoms or
pathology
indicative of Alzheimer's disease, a subject suspected of having Alzheimer's
disease, a
subject suspected of displaying signs or symptoms or pathology indicative of
Alzheimer's
disease, a subject at risk of Alzheimer's disease (e.g., a subject predisposed
(e.g., with a
family history or genetically (e.g., possessing an APO E variant) to
Alzheimer's disease,
etc.)), a subject at risk of displaying pathology indicative of Alzheimer's
disease, an animal
model of Alzheimer's disease, or a healthy subject wishing to reduce risk of
Alzheimer's
disease) a composition comprising selenium (e.g., organic selenium (e.g.,
selenized yeast
(e.g., Sel-PlexO))) under conditions such that the expression of a complement
gene is
reduced (e.g., in the cerebral cortex) in the subject or under conditions such
that one or
more signs or symptoms of Alzheimer's disease is reduced or eliminated or that
onset or
progression of Alzheimer's disease is delayed or prevented. In some
embodiments, the
treatment is prophylactic. In some embodiments, the complement gene expression
is age
related. In some embodiments, the complement gene is Clq, Clq alpha, Clq beta,
Clq
gamma, Clqr or other complement gene. In some embodiments, the prophylactic
treatment
prevents the onset of signs and symptoms of Alzheimer's disease in the
subject. In some
embodiments, the composition comprising selenium (e.g., organic selenium
(e.g., selenized
yeast (e.g., Sel-Plexg))) comprises one or more other forms of selenium. The
present
invention is not limited by the type of selenium co-administered. Indeed, a
variety of forms
of selenium are contemplated to be useful in co-administration including, but
not limited to,
selenomethionine, selenocysteine, a selenite compound, a selenate compound, or
derivatives, salts, or modifications thereof. In some embodiments, providing
selenium
(e.g., organic selenium (e.g., selenized yeast (e.g., Sel-Plext)))) and one or
more different
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CA 02524551 2012-08-31
forms of selenium provides an additive reduction in the expression of a
complement gene.
In some embodiments, providing selenium (e.g., organic selenium (e.g.,
selenized yeast
(e.g., Sel-Plexe))) and one or more different forms of selenium provides a
synergistic (e.g.,
more than additive) reduction in the expression of a complement gene. In some
embodiments, providing selenium (e.g., organic selenium (e.g., selenized yeast
(e.g., Sel-
Plex0))) and one or more different forms of selenium provides altered (e.g.,
reduced)
expression of more genes than are altered (e.g., reduced) with either form of
selenium
alone. In some embodiments, the composition comprising selenium (e.g., organic
selenium
(e.g., selenized yeast (e.g., Sel-Plext))) is co-administered with an
antioxidant. The
present invention is not limited by the antioxidant used. Indeed, a variety of
antioxidants
are contemplated to be useful for co-administration with selenium (e.g.,
organic selenium
(e.g., selenized yeast (e.g., Sel-Plexe))) including, but not limited to,
alkylated
diphenylamines, N-alkylated phenylenediamines, phenyl-a-naphthylamine,
alkylated
phenyl-a-naphthylamine, dimethyl quinolines, trimethyldihydroquinolines,
hindered
phenolics, alkylated hydroquinones, hydroxylated thiodiphenyl ethers,
alkylidenebisphenols, thiopropionates, metallic dithiocarbamates, 1,3,4-
dimercaptothiadiazole, an oil soluble copper compound, NAUGALUBE 438,
NAUGALUBE 438L, NAUGALUBE 640, NAUGALUBE 635, NAUGALUBE
680, NAUGALUBE AMS, NAUGALUBE APAN, Naugard PANA, NAUGALUBE
STMQ, NAUGALUBE 531, NAUGALUBE 431, NAUGALUBE BHT,
NAUGALUBE 403, NAUGALUBE 420, ascorbic acid, tocopherols, alpha-tocopherol,
a sulfhydryl compound, sodium metabisulfite, N-acetyl-cysteine, lipoic acid,
dihydrolipoic
acid, resveratrol, lactoferrin, ascorbic acid, ascorbyl palmitate, ascorbyl
polypeptide,
butylated hydroxytoluene, retinoids, retinol, retinyl palmitate, tocotrienols,
ubiquinone, a
flavonoid, an isoflavonoid, genistein, diadzein, resveratrol, grape seed,
green tea, pine bark,
propolis, IRGANOX , Antigene P, SUMILIZER GA-80, beta-carotene, lycopene,
vitamin C, vitamin E, and vitamin A. In some embodiments, the composition
comprising
selenium (e.g., organic selenium (e.g., selenized yeast (e.g., Sel-Plex0))) is
co-administered
with an Alzheimer's therapeutic. The present invention is not limited to any
particular
Alzheimer's therapeutic. Indeed, a variety of Alzheimer's therapeutics are
contemplated to
be useful in the present invention including, but not limited to, a NMDA
antagonist, an
AChE inhibitor, and a metal chelator. In some embodiments, the. NMDA
antagonist is
memantine. In some embodiments, the AChE inhibitor is tacrine, donepezil,
rivastigmine,
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CA 02524551 2012-08-31
or galantamine. In some embodiments, the metal chelator is clioquinol. In some
embodiments, the clioquinol chelates zinc and copper.
The present invention also provides a method of treating a subject having
Alzheimer's
disease comprising administering to the subject a composition comprising
selenium (e.g.,
organic selenium (e.g., selenized yeast (e.g., Sel-Plex8))) under conditions
such that the
expression of a gene (e.g., Clq, Clq alpha, Clq beta, Clq gamma, Clqr,
Cathepsin B,
Cathepsin D, Cathepsin Z, and Cathepsin 0, calsenilin, presenilin 1,
presenilin 2, nicastrin,
Apbbl/Fe65, Aplp 1, and/or Apbal) is altered; and testing the expression of
the gene. In
some embodiments, the composition comprising selenium comprises Sel-Plex . In
some
embodiments, testing the expression of the gene (e.g., presenilin 1 or
presenilin 2)
comprises use of an oligonucleotide probe. In some embodiments, testing the
expression of
a gene (e.g., presenilin 1 or presenilin 2) comprises use of PCR. In some
embodiments, the
PCR comprises RT-PCR. In some embodiments, testing is: before, during and/or
after
administration. In some embodiments, testing is for diagnostic uses. In some
embodiments, testing is used for research uses.
The present invention also provides a method of treatment for Alzheimer's
disease
comprising administering to a subject a composition comprising selenium (e.g.,
organic
selenium (e.g., selenized yeast (e.g., Sel-Plexe))) under conditions such that
the expression
of a cathepsin gene is reduced (e.g., in the cerebral cortex) in the subject.
In some
embodiments, the treatment is prophylactic. In some embodiments, the cathepsin
gene
expression is age related. In some embodiments, the cathepsin gene is
Cathepsin B,
Cathepsin D, Cathepsin Z, Cathepsin 0 or other cathepsin gene. In some
embodiments,
reducing the expression of a cathepsin gene reduces processing of amyloid
precursor
protein (APP) to amyloid 3-peptide. In some embodiments, reducing levels of
the amyloid
13-peptide reduces formation of Alzheimer's disease plaques in the brain of
the subject. In
some embodiments, the prophylactic treatment prevents the onset or progression
of signs
and symptoms of Alzheimer's disease in the subject.
The present invention also provides a method of treatment for Alzheimer's
disease
comprising administering to a subject a composition comprising selenium (e.g.,
organic
selenium (e.g., selenized yeast (e.g., Sel-Plext))) under conditions such that
the expression
of presenilin (e.g., presenilin 1 or presenilin 2) is reduced (e.g., in the
cerebral cortex) in the
subject. In some embodiments, the treatment is prophylactic. In some
embodiments, the
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CA 02524551 2012-08-31
expression of presenilin is age related. In some embodiments, reducing the
expression of
presenilin reduces processing of amyloid precursor protein (APP) to amyloid f3-
peptide. In
some embodiments, reducing levels of the amyloid 0-peptide reduces formation
of
Alzheimer's disease plaques in the brain of the subject. In some embodiments,
prophylactic
The present invention also provides a method of treatment for Alzheimer's
disease
comprising administering to a subject a composition comprising selenium (e.g.,
organic
selenium (e.g., selenized yeast (e.g., Sel-Plex8))) under conditions such that
the expression
The present invention also provides a method of treatment for Alzheimer's
disease
comprising administering to a subject a composition comprising selenium (e.g.,
organic
selenium (e.g., selenized yeast (e.g., Sel-Plexe))) under conditions such that
the expression
The present invention also provides a method of inhibiting the expression of a
gene
involved in processing amyloid precursor protein in a subject comprising
administering to
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Clqr, Cathepsin B, Cathepsin D, Cathepsin Z, and Cathepsin 0, presenilin 1,
presenilin 2,
nicastrin, calsenilin, Apbbl/Fe65, Aplp 1, and/or Apbal. In some embodiments,
the
composition comprising selenium is administered to the subject as a
prophylactic or
therapeutic treatment for neurodegenerative disease. Methods of the present
invention can
be used to treat a variety of subjects, including, but not limited to, a
subject at risk of
displaying pathology indicative of Alzheimer's disease and a subject having
Alzheimer's
disease. In some embodiments, the composition comprising selenium comprises
Sel-
Plex . In some embodiments, the composition comprising Sel-Plex comprises one
or
more other forms of selenium. In some embodiments, the composition comprising
selenium is co-administered with an Alzheimer's therapeutic. In some
embodiments,
administering the composition comprising selenium inhibits the onset of
Alzheimer's
disease signs and symptoms in the subject. In some embodiments, the
composition
comprising selenium is co-administered with an antioxidant.
The present invention also provides a method of inhibiting the expression of a
gene
involved in the generation of13-amyloid peptide in a subject comprising
administering to
the subject a composition comprising selenium (e.g., organic selenium (e.g.,
selenized yeast
(e.g., Sel-Plex ))) under conditions such that the expression of a gene
involved in the
generation of13-amyloid peptide is reduced. In some preferred embodiments, the
gene
involved in the generation of13-amyloid peptide is Cl q, Cl q alpha, Clq beta,
Clq gamma,
Clqr, Cathepsin B, Cathepsin D, Cathepsin Z, and Cathepsin 0, presenilin 1,
presenilin 2,
calsenilin, nicastrin, Apbbl/Fe65, Aplp 1, and/or Apbal. In some embodiments,
the
composition comprising selenium is administered to the subject as a
prophylactic or
therapeutic treatment for neurodegenerative disease. Methods of the present
invention can
be used to treat a variety of subjects, including, but not limited to, a
subject at risk of
displaying pathology indicative of Alzheimer's disease and a subject having
Alzheimer's
disease. In some embodiments, the composition comprising selenium comprises
Sel-
Plex . In some embodiments, the composition comprising Sel-Plex comprises one
or
more other forms of selenium. In some embodiments, the composition comprising
selenium is co-administered with an Alzheimer's therapeutic. In some
embodiments,
administering the composition comprising selenium inhibits the onset of
Alzheimer's
disease signs and symptoms in the subject. In some embodiments, the
composition
comprising selenium is co-administered with an antioxidant.
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CA 02524551 2012-08-31
The present invention also provides a composition comprising Sel-Plex and an
Alzheimer's therapeutic. In some embodiments, the Alzheimer's therapeutic is
selected
from the group consisting of a NMDA antagonist, an AChE inhibitor, and a metal
chelator.
In some embodiments, the NMDA antagonist is memantine. In some embodiments,
the
AChE inhibitor is tacrine, donepezil, rivastigmine, or galantamine. In some
embodiments,
the metal chelator is clioquinol.
The present invention also provides a composition comprising selenium, an
Alzheimer's therapeutic, and an antioxidant. In some embodiments, the
composition
comprising selenium comprises Sel-Plex . In some embodiments, the Alzheimer's
therapeutic is selected from the group consisting of a NMDA antagonist, an
AChE
inhibitor, and a metal chelator.
The present invention also provides a method of altering cognitive function or
reducing signs or symptoms associated with a decline in cognitive function or
prophylactically preventing or minimizing biological events associated with
the onset of a
decline in cognitive function or altering (e.g., enhancing or reducing) gene
expression of
genes correlated with an increase or decline in cognitive function in a
subject (e.g., a
subject suffering from a decline in cognitive function, a subject wishing to
enhance
cognitive function, a subject displaying signs or symptoms or pathology of a
decline in
cognitive function, a subject suspected of having a decline of cognitive
function, a subject
at risk for a decline in cognitive function (e.g., an elderly subject), or an
animal model of
cognitive function) comprising administering to the subject a composition
comprising
selenium (e.g., organic selenium (e.g., selenized yeast (e.g., Sel-Plex )))
under conditions
such that the expression of Lhx8 is enhanced in the subject or under
conditions such that
one or more signs or symptoms of a decline in cognitive function is reduced or
eliminated
or that onset or progression of a decline of cognitive function is delayed or
prevented. In
some embodiments, the altering cognitive function inhibits decline of
cognitive function of
the subject. In some embodiments, inhibiting decline of cognitive function in
the subject
comprises promoting development of basal forebrain cholinergic neurons. In
some
embodiments, inhibiting decline of cognitive function in the subject comprises
maintenance
of basal forebrain cholinergic neurons. In some embodiments, the composition
comprising
selenium (e.g., organic selenium (e.g., selenized yeast (e.g., Sel-Plex )))
comprises one or
more different forms of selenium. In some embodiments, the composition
comprising
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CA 02524551 2012-08-31
selenium (e.g., organic selenium (e.g., selenized yeast (e.g., Sel-Plex ))) is
co-administered
with an antioxidant.
The present invention further provides a method of altering cognitive function
in a
subject comprising administering to the subject a composition comprising
selenium (e.g.,
organic selenium (e.g., selenized yeast (e.g., Sel-Plex ))) under conditions
such that the
expression of TGF132 is enhanced in the subject. In some embodiments, altering
cognitive
function inhibits decline of cognitive function of the subject. In some
embodiments,
inhibiting decline of cognitive function in the subject comprises promoting
neuronal
proliferation in the subject. In some embodiments, the neuronal proliferation
occurs in the
cerebellum of the subject. In some embodiments, the composition comprising
selenium
(e.g., organic selenium (e.g., selenized yeast (e.g., Sel-Plex ))) comprises
one or more
other forms of selenium. In some embodiments, the composition comprising
selenium
(e.g., organic selenium (e.g., selenized yeast (e.g., Sel-Plex ))) is co-
administered with an
antioxidant.
The present invention also provides a prophylactic treatment for inhibiting
decline
of cognitive function in a subject comprising administering to a subject a
composition
comprising Sel-Plex . In some embodiments, the composition comprising selenium
(e.g.,
organic selenium (e.g., selenized yeast (e.g., Sel-Plex ))) is administered
under conditions
such that the expression of Lhx8 is enhanced in the subject. In some
embodiments,
enhanced expression of Lhx8 promotes development and/or maintenance of basal
forebrain
cholinergic neurons. In some embodiments, the composition comprising selenium
(e.g.,
organic selenium (e.g., selenized yeast (e.g., Sel-Plex ))) is administered
under conditions
such that the expression of TGF(32 is enhanced in the subject. In some
embodiments,
enhanced expression of TGF132 promotes neuronal proliferation in the subject.
In some
embodiments, the neuronal proliferation occurs in the cerebellum of the
subject. In some
embodiments, the composition comprising selenium (e.g., organic selenium
(e.g., selenized
yeast (e.g., Sel-Plex ))) comprises one or more other forms of selenium. In
some
embodiments, the composition comprising selenium (e.g., organic selenium
(e.g., selenized
yeast (e.g., Sel-Plex ))) is co-administered with an antioxidant. In some
embodiments, the
prophylactic treatment prevents (e.g., prevents the onset of, recurrence of,
and/or
ameliorates) signs and symptoms of Alzheimer's disease in the subject. In some
embodiments, the prophylactic treatment prevents (e.g., prevents the onset of,
recurrence
of, and/or ameliorates) signs and symptoms of multiple sclerosis in the
subject. In some
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CA 02524551 2012-08-31
embodiments, the prophylactic treatment prevents (e.g., prevents the onset of,
recurrence
of, and/or ameliorates) signs and symptoms of ALS in the subject. In some
embodiments,
the prophylactic treatment prevents (e.g., prevents the onset of, recurrence
of, and/or
ameliorates) signs and symptoms of Parkinson's disease in the subject. In some
embodiments, the prophylactic treatment prevents (e.g., prevents the onset of,
recurrence
of, and/or ameliorates) signs and symptoms of Huntington's disease in the
subject. In some
embodiments, the composition comprising selenium (e.g., organic selenium
(e.g., selenized
yeast (e.g., Sel-Plex ))) is administered under conditions such that the
expression of a
complement gene is reduced. Multiple complement genes have been demonstrated
to be
reduced using the compositions and methods of the present invention including,
but not
limited to, Clq, Clq alpha, Clq beta, Clq gamma and Clqr.
The present invention is not limited by the amount of selenium (e.g., organic
selenium (e.g., selenized yeast (e.g., Sel-Plex ))) administered to a subject.
Indeed a
variety of different doses are contemplated to be useful in the present
invention. In some
embodiments, the composition comprising selenium (e.g., organic selenium
(e.g., selenized
yeast (e.g., Sel-Plex ))) is administered to the subject so as to provide
between 25-800n
of selenium to the subject each day. In some embodiments, the composition
comprising
selenium (e.g., organic selenium (e.g., selenized yeast (e.g., Sel-Plex ))) is
administered to
the subject so as to provide between 200-400 pig of selenium to the subject
each day. In
other embodiments, the composition comprising selenium (e.g., organic selenium
(e.g.,
selenized yeast (e.g., Sel-Plex ))) is administered to the subject so as to
provide between
and 75 pg of selenium to the subject each day. In some embodiments, a
composition
comprising two or more different forms of selenium (e.g., selonmethionine, Sod-
sel and/or
Sel-Plex ) is administered to a subject so as to provide the subject between
25 and 5000
25 of selenium each day.
The present invention also provides a method of altering age associated
expression
of a gene (e.g., a complement or cathepsin gene) or reducing signs or symptoms
associated
with age or prophylactically preventing or minimizing biological events
associated with the
aging process (e.g., a decline in cognitive function) or altering (e.g.,
enhancing or reducing)
gene expression of genes correlated with an increase in age in a subject
(e.g., a subject older
than 16 years old, or a subject older than 25 years old, or preferably a
subject older than 40
years old, or more preferably a subject older than 50, or even more preferably
a subject
older than 60 years old, or a subject suffering from a decline in cognitive
function, or a
CA 02524551 2012-08-31
subject displaying signs or symptoms or pathology (e.g., decline in cognitive
function) of
the aging process, an animal model of aging or a subject wishing to prevent
the onset or
progression of the aging process) in a subject comprising administering to the
subject a
composition comprising selenium (e.g., organic selenium (e.g., selenized yeast
(e.g., Se!-
Plex8))) under conditions such that age associated gene expression (e.g.,
complement or
cathepsin genes) is reduced or under conditions such that one or more signs or
symptoms of
aging (e.g., a loss of cognitive function) is reduced or eliminated or that
the onset or
progression of the aging process is delayed or prevented. Many genes whose
expression is
altered (e.g., elevated) with age are contemplated to be altered (e.g.,
reduced) with
compositions and methods of the present invention including, but not limited
to,
complement genes (e.g., Clq, Clq alpha, Clq beta, Clq gamma, and Clqr),
cathepsin
genes (e.g., Cathepsin B, Cathepsin D, Cathepsin Z, and Cathepsin 0) junb and
homeobox
(Hox) transcription factor genes. In some embodiments, the composition
comprising
selenium (e.g., organic selenium (e.g., selenized yeast (e.g., Sel-Plex ))) is
administered to
the subject so as to provide 200 lig of selenium to the subject each day. In
some
embodiments, the composition comprising selenium (e.g., organic selenium
(e.g., selenized
yeast (e.g., Sel-Plex ))) is administered to the subject so as to provide
between 25 and 400
p.g of selenium to the subject each day. In some embodiments, the composition
comprising
selenium (e.g., organic selenium (e.g., selenized yeast (e.g., Sel-Plex )))
comprises one or
more different forms of selenium. In some embodiments, the one or more
different forms
of selenium comprises sodium-selenite. In some embodiments, the composition
comprising
selenium (e.g., organic selenium (e.g., selenized yeast (e.g., Sel-Plex ))) is
co-administered
with an Alzheimer's therapeutic. In some embodiments, administering the
composition
comprising selenium (e.g., organic selenium (e.g., selenized yeast (e.g., Sel-
Plex )))
inhibits (e.g., prevents the onset of, recurrence of, and/or ameliorates)
Alzheimer's disease
signs and symptoms in the subject. In some embodiments, the composition
comprising
selenium (e.g., organic selenium (e.g., selenized yeast (e.g., Sel-Plex ))) is
co-administered
with an antioxidant. In some embodiments, the composition comprising selenium
(e.g.,
organic selenium (e.g., selenized yeast (e.g., Sel-Plex ))) is administered to
the subject as a
prophylactic or therapeutic treatment for neurodegenerative disease.
In some embodiments, a composition comprising selenium (e.g., organic selenium
(e.g., selenized yeast (e.g., Sel-Plex ))) is administered to a subject in
combination with a
calorie restricted diet in order to prevent aging or the aging process (e.g.,
attenuate age-
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CA 02524551 2012-08-31
associated gene expression). In some preferred embodiments, the present
invention
provides a method of altering cognitive function (e.g., neuronal circuit
changes) associated
with age comprising administering to a subject a composition comprising
selenium (e.g.,
organic selenium (e.g., selenized yeast (e.g., Sel-Plexe))) under conditions
such that the
expression of Lhx8 is enhanced and/or elevated.
The present invention also provides a method of treatment or preventative for
diabetes or reduction of signs or symptoms associated with diabetes or
prophylactically
preventing or minimizing biological events associated with the onset or
progression of
diabetes or reducing gene expression of genes correlated to the onset or
progression of
diabetes comprising administering to a subject (e.g., a subject suffering from
diabetes, a
subject with type I or type II diabetes, a subject having diabetes, a subject
displaying signs
or symptoms or pathology indicative of diabetes, a subject suspected of having
diabetes, a
subjects suspected of displaying signs or symptoms or pathology indicative of
diabetes, a
subject at risk of diabetes (e.g., a subject predisposed (e.g., with a family
history of
diabetes, genetically, etc.)), a subject at risk of displaying pathology
indicative of diabetes,
an animal model of diabetes, or a healthy subject wishing to reduce risk of
diabetes) a
composition comprising selenium (e.g., organic selenium (e.g., selenized yeast
(e.g., Sel-
Plexe))) under conditions such that the expression of neurogenin-3 (Neurog3)
is reduced in
the subject or under conditions such that one or more signs or symptoms of
diabetes is
reduced or eliminated or that onset of progression of diabetes is delayed or
prevented. In
some embodiments, the treatment is prophylactic. The present invention
provides
compositions and methods for multiple types of diabetes. In some embodiments,
diabetes
treated with compositions and methods of the present invention is type I or
type II diabetes.
In some embodiments, the prophylactic treatment prevents the onset of signs
and symptoms
of diabetes in the subject. In some embodiments, the composition comprising
selenium
(e.g., organic selenium (e.g., selenized yeast (e.g., Sel-Plext))) comprises
one or more
different forms of selenium. In some embodiments, the composition comprising
selenium
(e.g., organic selenium (e.g., selenized yeast (e.g., Sel-PlexID))) is co-
administered with a
diabetes therapeutic. Multiple diabetes therapeutics find use with the
compositions and
methods of the present invention including, but not limited to, Vanadium,
metformin,
thiazolidinedione, TZD, intermediate-acting insulin, neutral protamine
Hagedorn, NPHõ a
long-acting insulin, glargine, Lantus, insulin, insulin detemir, Levemir,
Incretin mimetic,
Exenatide, Byetta, Sulfonylurea agent, chlorpropamide, tolbutamide,
tolazamide,
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CA 02524551 2012-08-31
acetohexamide, glyburide, glipizide, glimepiride, Meglitinides, Repaglinide,
Prandin,
Biguanides, Metformin, Glucophage, Alpha-glucosidase inhibitor, AGI, Acarbose,
Precose,
Miglitol, Glyset, thiazolidinedione, Pioglitazone, Actos, Rosiglitazone,
Avandia, Amylin
analog, Pramlintide acetate, and Symlin.
The present invention also provides a composition comprising selenium (e.g.,
organic selenium (e.g., selenized yeast (e.g., Sel-Plex ))) and a diabetes
therapeutic.
DESCRIPTION OF THE DRAWINGS
Figure 1 shows the body weight of mice receiving a selenium deficient diet (Se
def)
or a diet comprising selnomethionine (SeM), sodium-selenite (Sod-sel), or Sel-
Plex .
Figure 2 depicts a cartoon of the complement cascade.
Figure 3 shows that complement gene expression decreases after a subject is
treated
with a composition comprising selenium. (* indicates significant reduction,
p<0.01).
Figure 4 depicts a cartoon of peripheral efferent nerves.
DEFINITIONS
As used herein, the terms "peptide," "polypeptide" and "protein" all refer to
a
primary sequence of amino acids that are joined by covalent "peptide
linkages." In general,
a peptide consists of a few amino acids, typically from 2-50 amino acids, and
is shorter than
a protein. The term "polypeptide" encompasses peptides and proteins. In some
embodiments, the peptide, polypeptide or protein is synthetic, while in other
embodiments,
the peptide, polypeptide or protein are recombinant or naturally occurring. A
synthetic
peptide is a peptide that is produced by artificial means in vitro (i.e., was
not produced in
vivo).
The terms "sample" and "specimen" are used in their broadest sense and
encompass
samples or specimens obtained from any source. As used herein, the term
"sample" is used
to refer to biological samples obtained from animals (including humans), and
encompasses
fluids, solids, tissues, and gases. In some embodiments of this invention,
biological samples
include cerebrospinal fluid (CSF), serous fluid, urine, saliva, blood, and
blood products
such as plasma, serum and the like. However, these examples are not to be
construed as
limiting the types of samples that find use with the present invention.
As used herein, the terms "selenium-enriched yeast" and "selenized yeast"
refer to
any yeast (e.g., Saccharomyces cerevisiae) that is cultivated in a medium
containing
inorganic selenium salts. The present invention is not limited by the selenium
salt used.
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Indeed, a variety of selenium salts are contemplated to be useful in the
present invention
including, but not limited to, sodium selenite, sodium selenate, cobalt
selenite or cobalt
selenate. Free selenomethionine (e.g., not associated with a cell or yeast)
can also be used
as the selenium source for selenium enriched yeast as yeast does incorporate
this form of
selenium. During cultivation, because of the chemical similarity between
selenium and
sulfur, yeast incorporate selenium in place of sulfur in what are normally
sulfur-containing
organic compounds within the cell. A selenium-containing compound in such
yeast
preparations is selenomethionine which will be present in a form that is
incorporated into
polypeptides/proteins. The amount of total cellular selenium present in the
form of
selenomethionine in such preparations will vary, but can be between 10 and
100%, 20-60%,
50-75% and between 60 and 75%. The remainder of the organic selenium in
selenized
yeast preparations is predominantly made up of intermediates in the pathway
for
selenomethionine biosynthesis. These include, but are not limited to,
selenocysteine,
selenocystathionine, selenohomocysteine and seleno-adenosylselenomethionine.
The
amount of residual inorganic selenium salt in the finished product is
generally quite low
(e.g., <2%). However, the present invention is not limited by this percentage,
as
preparations that contain more (e.g., between 2 and 70%) or less (e.g.,
between 0.1 and 2%)
than this percentage are also encompassed by the invention.
As used herein, the term "Sel-Plexe" refers to a dried, nonviable selenium-
enriched
yeast (e.g., Sacchoromyces cerevisiae of accession number CNCM 1-3060,
Collection
Nationale De Cultures De Microorganismes (CNCM), Institut Pasteur, Paris,
France)
cultivated in a fed-batch fermentation that provides incremental amounts of
cane molasses
and selenium salts in a manner that minimizes the detrimental effects of
selenium salts on
the growth rate of the yeast and allows for optimal incorporation of inorganic
selenium into
cellular organic material. Residual inorganic selenium is eliminated (e.g.,
using a rigorous
washing process) and does not exceed 2% of the total selenium content.
As used herein, the term "organic selenium" refers to any organic compound
wherein selenium replaces sulfur. Thus, organic selenium can refer to any such
compound
biosynthesized by yeast, or it can refer to free organic seleno-compounds that
are
chemically synthesized. An example of the latter is free selenomethionine.
As used herein, the term "inorganic selenium" generally refers to any selenium
salt
(e.g., sodium selenite, sodium selenate, cobalt selenite and cobalt selenate).
There are also
a variety of other inorganic selenium sources (See e.g., those listed in the
Merck index).
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CA 02524551 2012-08-31
Selenized yeast may be generated using a source of inorganic selenium
including, but not
limited to, sodium selenite, sodium selenate, cobalt selenite, cobalt
selenate, selenic acid,
selenious acid, selenium bromide, selenium chloride, selenium hexafluoride,
selenium
oxide, selenium oxybromide, selenium oxychloride, selenium oxyfluoride,
selenium
sulfides, selenium tetrabromide, selenium tetrachloride and selenium
tetrafluoride.
As used herein, the term "13-amyloid protein" refers to a protein or peptide
proteolytically derived from the transmembrane amyloid precursor protein
(APP). 13-
amyloid proteins can form soluble, non-fibrillar oligomeric amyloid [3 protein
assembly
(e.g., oligomeric amyloid p protein assembly or oligomeric assembly) and
generally
comprise between 2-1213-amyloid proteins or peptides. 3-amyloid proteins can
also form
fibrillar assemblies that generally comprise more than 12 13-amyloid proteins
or peptides.
3-amyloid proteins (e.g., individually or as found in the structures described
above) are
involved in forming plaques, one of the characterisitic traits of Alzheimer's
disease.
As used herein, the term "oxidative stress" refers to the cytotoxic effects of
oxygen
radicals (e.g., superoxide anion (02), hydroxy radical (OH), and hydrogen
peroxide
(H202)), generated, for example, as byproducts of metabolic processes that
utilize
molecular oxygen (See e.g., Coyle et al., Science 262:689-695 (1993)).
As used herein, the terms "host," "subject" and "patient" refer to any animal,
including but not limited to, human and non-human animals (e.g., dogs, cats,
cows, horses,
sheep, poultry, fish, crustaceans, etc.) that is studied, analyzed, tested,
diagnosed or treated.
As used herein, the terms "host," "subject" and "patient" are used
interchangeably, unless
indicated otherwise.
As used herein, the terms "Alzheimer's disease" and "AD" refer to a
neurodegenerative disorder and encompasses familial Alzheimer's disease and
sporadic
Alzheimer's disease. The term "familial Alzheimer's disease" refers to
Alzheimer's disease
associated with genetic factors (i.e., demonstrates inheritance) while
"sporadic Alzheimer's
disease" refers to Alzheimer's disease that is not associated with prior
family history of the
disease. Symptoms indicative of Alzheimer's disease in human subjects
typically include,
but are not limited to, mild to severe dementia, progressive impairment of
memory (ranging
from mild forgetfulness to disorientation and severe memory loss), poor visuo-
spatial skills,
personality changes, poor impulse control, poor judgement, distrust of others,
increased
stubbornness, restlessness, poor planning ability, poor decision making, and
social
withdrawal. In severe cases, patients lose the ability to use language and
communicate, and
CA 02524551 2012-08-31
require assistance in personal hygiene, eating and dressing, and are
eventually bedridden.
Hallmark pathologies within brain tissue include extracellular neuritic p-
amyloid plaques,
neurofibrillary tangles, neurofibrillary degeneration, granulovascular
neuronal
degeneration, synaptic loss, and extensive neuronal cell death.
As used herein, the term "early-onset Alzheimer's disease" refers to the
classification used in Alzheimer's disease cases diagnosed as occurring before
the age of
65. As used herein, the term "late-onset Alzheimer's disease" refers to the
classification
used in Alzheimer's disease cases diagnosed as occurring after the age of 65.
As used herein, the terms "subject having Alzheimer's disease" or "subject
displaying signs or symptoms or pathology indicative of Alzheimer's disease"
or "subjects
suspected of displaying signs or symptoms or pathology indicative of
Alzheimer's disease"
refer to a subject that is identified as having or likely to have Alzheimer's
disease based on
known Alzheimer's signs, symptoms and pathology.
As used herein, the terms "subject at risk of displaying pathology indicative
of
Alzheimer's disease" and "subject at risk of Alzheimer's disease" refer to a
subject
identified as being at risk for developing Alzheimer's disease (e.g., due to
age or familial
inheritance pattern of Alzheimer's disease in the subject's family).
As used herein, the term "Alzheimer's therapeutic" refers to an agent used to
treat or
prevent Alzheimer's disease. Such agents include, but are not limited to,
small molecules,
drugs, antibodies, pharmaceuticals, and the like. For example, therepeutics
used to treat
Alzheimer's disease include, but are not limited to, NMDA antagonists (e.g.,
memantine),
and AChE inhibitors (e.g., tacrine (Cognex), donepezil (Aricept), rivastigmine
(Exelon),
and galantamine (galanthamine, Reminyl)).
As used herein, the term "lesion" refers to a wound or injury, or to a
pathologic
change in a tissue. For example, the13-amyloid plaque lesions observed in the
brains of
patients having Alzheimer's disease are considered the hallmark pathology
characteristic of
the disease.
As used herein, the terms "amyotrophic lateral sclerosis" and "ALS" refer to a
neurodegenerative disorder that is characterized as a devastating disorder of
the anterior
horn cells of the spinal cord and the motor cranial nuclei that leads to
progressive muscle
weakness and atrophy. Symptoms indicative of ALS in human subjects typically
include,
but are not limited to, mild to severe weakness of bulbar muscles or of single
or multiple
limb muscle groups (e.g., bilateral or symmetrical) limb weakness, weakness
and atrophy
16
CA 02524551 2012-08-31
of the intrinsic hand muscles that progresses to involve the forearms and
shoulder girdle
muscles and the lower extremities. Involvement of both upper and lower motor
neurons is
characteristic. Patients develop variable hyperreflexia, clonus, spasticity,
extensor plantar
responses, and limb or tongue fasciculations. Wallerian degeneration of
cortico spinal and
corticobulbar tracts may be demonstrated by MRI (high-intensity T2 lesions in
frontal
lobes) or in postmortem examination.
As used herein, the terms "subject having ALS" or "subject displaying signs or
symptoms or pathology indicative of ALS" or "subjects suspected of displaying
signs or
symptoms or pathology indicative of ALS" refer to a subject that is identified
as having or
likely to have ALS based on known ALS signs, symptoms and pathology.
As used herein, the terms "subject at risk of displaying pathology indicative
of
ALS" and "subject at risk of ALS" refer to a subject identified as being at
risk for
developing ALS (e.g., due to age or familial inheritance pattern of ALS in the
subject's
family).
As used herein, the term "ALS therapeutic" refers to an agent used to treat or
prevent ALS. Such agents include, but are not limited to, small molecules,
drugs,
antibodies, pharmaceuticals, and the like. For example, therepeutics used to
treat ALS
include, but are not limited to, Riluzole, Baclofen (Lioresal) and Tizanidine
(Zanaflex).
As used herein, the terms "Huntington's Disease" and "HD" refer to a
neurodegenerative disorder that is an adult-onset, autosomal dominant
inherited disorder
associated with cell loss within a specific subset of neurons in the basal
ganglia and cortex.
Characteristic features of HD include involuntary movements (e.g., chorea, a
state of
excessive, spontaneous movements, irregularly timed, randomly distributed, and
abrupt, is
a characteristic feature of HD), dementia, and behavioral changes.
Neuropathology in HD
occurs within the neostriatum, in which gross atrophy of the caudate nucleus
and putamen
is accompanied by selective neuronal loss and astrogliosis. Marked neuronal
loss also is
seen in deep layers of the cerebral cortex. Other regions, including the
globus pallidus,
thalamus, subthalamic nucleus, substantia nigra, and cerebellum, show varying
degrees of
atrophy depending on the pathologic grade.
As used herein, the terms "subject having HD" or "subject displaying signs or
symptoms or pathology indicative of HD" or "subjects suspected of displaying
signs or
symptoms or pathology indicative of HD" refer to a subject that is identified
as having or
likely to have HD based on known HD signs, symptoms and pathology.
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As used herein, the terms "subject at risk of displaying pathology indicative
of HD"
and "subject at risk of HD" refer to a subject identified as being at risk for
developing HD
(e.g., due to age or familial inheritance pattern of HD in the subject's
family).
As used herein, the term "HD therapeutic" refers to an agent used to treat or
prevent
HD. Such agents include, but are not limited to, small molecules, drugs,
antibodies,
pharmaceuticals, and the like. For example, therepeutics used to treat HD
include, but are
not limited to, anticonvulsant medications including, but not limited to
valproic acid (e.g.,
Depakote, Depakene, and Depacon) and benzodiazepines such as clonazepam (e.g.,
Klonopin), Antipsychotic medications (e.g., risperidone (e.g., Risperdal), and
haloperidol
(e.g., Haldol)), Rauwolfia alkoids (e.g., resperine), and antidepressants
(e.g., paroxetine
(e.g., Paxil)).
As used herein, the terms "Parkinson's disease" and "PD" refer to a
neurodegenerative disorder that is a progressive neurodegenerative disorder
associated with
a loss of doparninergic nigrostriatal neurons. Characteristic features of PD
include loss of
pigmented dopaminergic neurons in the substantia nigra and the presence of
Lewy bodies.
As used herein, the terms "subject having PD" or "subject displaying signs or
symptoms or pathology indicative of PD" or "subjects suspected of displaying
signs or
symptoms or pathology indicative of PD" refer to a subject that is identified
as having or
likely to have PD based on known PD signs, symptoms and pathology.
As used herein, the terms "subject at risk of displaying pathology indicative
of PD"
refer and "subject at risk of PD" refer to a subject identified as being at
risk for developing
PD (e.g., due to age or familial inheritance pattern of PD in the subject's
family).
As used herein, the term" PD therapeutic" refers to an agent used to treat or
prevent
PD. Such agents include, but are not limited to, small molecules, drugs,
antibodies,
pharmaceuticals, and the like. For example, therepeutics used to treat PD
include, but are
not limited to, dopamine prodrugs such as levadopa/PDI and levodopa/carbidopa
(e.g.,
Sinemet, Sinemet CR), dopamine agonsts such as apomorphine (e.g., Apokyn),
bromocriptine (e.g., Parlodel), pergolide (e.g., Permax), pramipexole (e.g.,
Mirapex), and
ropinirole (e.g., Requip), catechol-O-methyltransferase (COMT) inhibitors such
as
tolcapone (e.g., Tasmar), and entacapone (e.g., Comtan), anticholinergics such
as
trihexyphenidyl (e.g., Artane, Trihexy), and benztropine mesylate (e.g.,
Cogentin), MAO-B
inhibitors such as selegiline (e.g., Eldepry1), and amantadine (e.g.,
Symmetrel).
18
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As used herein, the terms "Multiple sclerosis" and "MS" refer to a
neurodegenerative disorder that is an inflammatory, demyelinating disease of
the central
nervous system (CNS). MS lesions, characterized by perivascular infiltration
of monocytes
and lymphocytes, appear as indurated areas in pathologic specimens; hence, the
term
"sclerosis in plaques." Characteristic features of MS include perivenular
infiltration of
lymphocytes and macrophages in the parenchyma of the brain, brain stem, optic
nerves, and
spinal cord, almost constant lesion formation and a progressive clinical
course leading to
physical disability.
As used herein, the terms "subject having MS" or "subject displaying signs or
symptoms or pathology indicative of MS" or "subjects suspected of displaying
signs or
symptoms or pathology indicative of MS" refer to a subject that is identified
as having or
likely to have MS based on known MS signs, symptoms and pathology.
As used herein, the terms "subject at risk of displaying pathology indicative
of MS"
and "subject at risk of MS" refer to a subject identified as being at risk for
developing MS.
As used herein, the term" MS therapeutic" refers to an agent used to treat or
prevent
MS. Such agents include, but are not limited to, small molecules, drugs,
antibodies,
pharmaceuticals, and the like. For example, therepeutics used to treat MS
include, but are
not limited to, immunomodulators (e.g., Interferon beta-la (Avonex),
Interferon beta-la
(Rebif), Interferon beta-lb (Betaseron), Glatiramer acetate (Copaxone), and
Natalizumab
(Tysabri)), corticosteroids (e.g., methylprednisolone), and
irrununosuppressors (e.g.,
Mitoxantrone (Novantrone), Cyclophosphamide (Cytoxan, Neosar), Azathioprine
(IMURAN), Methotrexate (Rheumatrex).
As used herein, the term "diabetes" refers to an autoimmune disease
characterized
by necrosis of pancreatic islet cells and a lack of insulin secretion. For
example, patients
with type 1 diabetes are dependent on insulin. Characteristics traits of
diabetes include
peripheral insulin resistance with an insulin-secretory defect that varies in
severity, and
complications that include hypoglycemia and hyperglycemia, increased risk of
infections,
microvascular complications (eg, retinopathy, nephropathy), neuropathic
complications,
and macrovascular disease.
As used herein, the terms "subject having diabetes " or "subject displaying
signs or
symptoms or pathology indicative of diabetes" or "subjects suspected of
displaying signs or
symptoms or pathology indicative of diabetes" refer to a subject that is
identified as having
or likely to have diabetes based on known diabetes signs, symptoms and
pathology.
19
CA 02524551 2012-08-31
As used herein, the term "subject at risk of displaying pathology indicative
of
diabetes" and "subject at risk of diabetes" refer to a subject identified as
being at risk for
developing diabetes (e.g., due to age, weight, race, or familial inheritance
pattern of
diabetes in the subject's family).
As used herein, the term "diabetes therapeutic" refers to an agent used to
treat or
prevent diabetes. Such agents include, but are not limited to, small
molecules, drugs,
antibodies, pharmaceuticals, and the like. For example, therepeutics used to
treat diabetes
include, but are not limited to, oral medication to increase insulin
sensitivity (eg,
metformin, a thiazolidinedione (TZD)), intermediate-acting insulin (eg,
neutral protamine
Hagedorn (NPH)), a long-acting insulin (eg, glargine (Lantus) insulin, insulin
detemir
(Levemir)), Incretin mimetics (e.g., Exenatide (Byetta)), Sulfonylurea agents
(e.g.,
chlorpropamide, tolbutamide, tolazamide, acetohexamide, glyburide, glipizide,
and
glimepiride), Meglitinides (e.g., Repaglinide (Prandin)), Biguanides (e.g.,
Metformin
(Glucophage)), Alpha-glucosidase inhibitors (AGIs) (e.g., Acarbose (Precose),
Miglitol
(Glyset)), thiazolidinediones (e.g., Pioglitazone (Actos), Rosiglitazone
(Avandia)), and
Amylin analogs (e.g., Pramlintide acetate (Symlin)).
As used herein, the terms "subject at risk of displaying pathology indicative
of
stroke" and "subject at risk of stroke" refer to a subject identified as being
at risk for
developing stroke (e.g., due to age, weight, race, or familial inheritance
pattern of stroke in
the subject's family).
As used herein, the term "cognitive function" generally refers to the ability
to think,
reason, concentrate, or remember. Accordingly, the term "decline in cognitive
function"
refers to the deterioration of lack of ability to think, reason, concentrate,
or remember.
As used herein, the term "antibody" (or "antibodies") refers to any
immunoglobulin
that binds specifically to an antigenic determinant, and specifically binds to
proteins
identical or structurally related to the antigenic determinant that stimulated
their production.
Thus, antibodies can be useful in assays to detect the antigen that stimulated
their
production. Monoclonal antibodies are derived from a single clone of B
lymphocytes (i.e.,
B cells), and are generally homogeneous in structure and antigen specificity.
Polyclonal
antibodies originate from many different clones of antibody-producing cells,
and thus are
heterogenous in their structure and epitope specificity, but all recognize the
same antigen.
In some embodiments, monoclonal and polyclonal antibodies are used as crude
preparations, while in preferred embodiments, these antibodies are purified.
For example, in
CA 02524551 2012-08-31
some embodiments, polyclonal antibodies contained in crude antiserum are used.
Also, it is
intended that the term "antibody" encompass any immunoglobulin (e.g., IgG,
IgM, IgA,
IgE, IgD, etc.) obtained from any source (e.g., humans, rodents, non-human
primates,
lagomorphs, caprines, bovines, equines, ovines, etc.).
As used herein, the terms "auto-antibody" or "auto-antibodies" refer to any
imrnunoglobulin that binds specifically to an antigen that is native to the
host organism that
produced the antibody (i.e., the antigen is directed against "self' antigens).
The presence of
auto-antibodies is referred to herein as "autoimmunity."
As used herein, the term "antigen" is used in reference to any substance that
is
capable of being recognized by an antibody. It is intended that this term
encompass any
antigen and "immunogen" (i.e., a substance that induces the formation of
antibodies). Thus,
in an immunogenic reaction, antibodies are produced in response to the
presence of an
antigen or portion of an antigen. The terms "antigen" and "immunogen" are used
to refer to
an individual macromolecule or to a homogeneous or heterogeneous population of
antigenic macromolecules. It is intended that the terms antigen and immunogen
encompass
protein molecules or portions of protein molecules, that contains one or more
epitopes. In
many cases, antigens are also inamunogens, thus the term "antigen" is often
used
interchangeably with the term "immunogen." In some preferred embodiments,
immunogenic substances are used as antigens in assays to detect the presence
of appropriate
antibodies in the serum of an immunized animal.
As used herein, the terms "antigen fragment" and "portion of an antigen" and
the
like are used in reference to a portion of an antigen. Antigen fragments or
portions typically
range in size, from a small percentage of the entire antigen to a large
percentage, but not
100%, of the antigen. However, in situations where "at least a portion" of an
antigen is
specified, it is contemplated that the entire antigen is also present (e.g.,
it is not intended
that the sample tested contain only a portion of an antigen). In some
embodiments, antigen
fragments and/or portions thereof, comprise an "epitope" recognized by an
antibody, while
in other embodiments these fragments and/or portions do not comprise an
epitope
recognized by an antibody. In addition, in some embodiments, antigen fragments
and/or
portions are not immunogenic, while in preferred embodiments, the antigen
fragments
and/or portions are immunogenic.
The terms "antigenic determinant" and "epitope" as used herein refer to that
portion
of an antigen that makes contact with a particular antibody variable region.
When a protein
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CA 02524551 2012-08-31
or fragment (or portion) of a protein is used to immunize a host animal,
numerous regions
of the protein are likely to induce the production of antibodies that bind
specifically to a
given region or three-dimensional structure on the protein (these regions
and/or structures
are referred to as "antigenic determinants"). In some settings, antigenic
determinants
compete with the intact antigen (i.e., the "immunogen" used to elicit the
immune response)
for binding to an antibody.
The terms "specific binding" and "specifically binding" when used in reference
to
the interaction between an antibody and an antigen describe an interaction
that is dependent
upon the presence of a particular structure (i.e., the antigenic determinant
or epitope) on the
antigen. In other words, the antibody recognizes and binds to a protein
structure unique to
the antigen, rather than binding to all proteins in general (i.e., non-
specific binding).
As used herein, the term "immunoassay" refers to any assay that uses at least
one
specific antibody for the detection or quantitation of an antigen.
Immunoassays include, but
are not limited to, Western blots, ELISAs, radio-immunoassays, and
immunofluorescence
assays.
The terms "Western blot," "Western imrnunoblot" "immunoblot" and "Western"
refer to the immunological analysis of protein(s), polypeptides or peptides
that have been
immobilized onto a membrane support. The proteins are first resolved by
polyacrylamide
gel electrophoresis (i.e., SDS-PAGE) to separate the proteins, followed by
transfer of the
protein from the gel to a solid support, such as nitrocellulose or a nylon
membrane. The
immobilized proteins are then exposed to an antibody having reactivity towards
an antigen
of interest. The binding of the antibody (i.e., the primary antibody) is
detected by use of a
secondary antibody that specifically binds the primary antibody. The secondary
antibody is
typically conjugated to an enzyme that permits visualization of the antigen-
antibody
complex by the production of a colored reaction product or catalyzes a
luminescent
enzymatic reaction (e.g., the ECL reagent, Amersham).
As used herein, the term "ELISA" refers to enzyme-linked immunosorbent assay
(or
EIA). Numerous ELISA methods and applications are known in the art, and are
described
in many references (See, e.g., Crowther, "Enzyme-Linked Immunosorbent Assay
(ELISA)," in Molecular Biomethods Handbook, Rapley et al. (eds.), pp. 595-617,
Humana
Press, Inc., Totowa, N.J. (1998); Harlow and Lane (eds.), Antibodies: A
Laboratory
Manual, Cold Spring Harbor Laboratory Press (1988); Ausubel et al. (eds.),
Current
CA 02524551 2012-08-31
Protocols in Molecular Biology, Ch. 11, John Wiley & Sons, Inc., New York
(1994)). In
addition, there are numerous commercially available ELISA test systems.
As used herein, the terms "reporter reagent," "reporter molecule," "detection
substrate" and "detection reagent" are used in reference to reagents that
permit the detection
and/or quantitation of an antibody bound to an antigen. For example, in some
embodiments,
the reporter reagent is a colorimetric substrate for an enzyme that has been
conjugated to an
antibody. Addition of a suitable substrate to the antibody-enzyme conjugate
results in the
production of a colorimetric or fluorimetric signal (e.g., following the
binding of the
conjugated antibody to the antigen of interest). Other reporter reagents
include, but are not
limited to, radioactive compounds. This defmition also encompasses the use of
biotin and
avidin-based compounds (e.g., including but not limited to neutravidin and
streptavidin) as
part of the detection system.
As used herein, the term "signal" is used generally in reference to any
detectable
process that indicates that a reaction has occurred, for example, binding of
antibody to
antigen. It is contemplated that signals in the form of radioactivity,
fluorimetric or
colorimetric products/reagents will all find use with the present invention.
In various
embodiments of the present invention, the signal is assessed qualitatively,
while in
alternative embodiments, the signal is assessed quantitatively.
As used herein, the term "solid support" is used in reference to any solid or
stationary material to which reagents such as antibodies, antigens, and other
test
components are attached. For example, in an ELISA method, the wells of
microtiter plates
provide solid supports. Other examples of solid supports include microscope
slides,
coverslips, beads, particles, cell culture flasks, as well as many other
suitable items.
As used herein, the term "characterizing tissue in a subject" refers to the
identification of one or more properties of a tissue sample. In some
embodiments, tissues
are characterized by the identification of the expression, or lack thereof, of
various genes
described in detail herein.
As used herein, the term "reagent(s) capable of specifically detecting gene
expression" refers to reagents capable of or sufficient to detect the
expression of various
genes described in detail herein (e.g., including, but not limited to,
Se1W,Sepnl, Se1R,
Sod2, Dio2, Glo 1, Phb, Lhx8, TGF-32, Neurog3, Spry2, Gstt2, Gsttl, Gsta3,
Gsta4, Gstml,
Gstm2, or Gstm3, Clq, Clq alpha, Clq beta, Clq gamma, CORS-26, cathepsin B,
cathepsin D, cathepsin Z, cathepsin 0, nicastrin, presenilin 1, presenilin 2,
calsenilin,
23
CA 02524551 2012-08-31
Apbbl/Fe65, Aplp 1, Apbal, Gstpl, Gstzl, Gstm7, Gadd45g1p, Gadd45b). Examples
of
suitable reagents include, but are not limited to, nucleic acid probes capable
of specifically
hybridizing to rnRNA or cDNA, and antibodies (e.g., monoclonal or polyclonal
antibodies).
As used herein, the term "effective amount" refers to the amount of a
composition
(e.g., comprising selenium ¨ e.g., Sel-Plext14) sufficient to effect
beneficial or desired
results. An effective amount can be administered in one or more
administrations,
applications or dosages and is not intended to be limited to a particular
formulation or
administration route.
As used herein, the terms "administration" and "administering" refer to the
act of
giving a drug, prodrug, or other agent, or therapeutic treatment (e.g.,
compositions of the
present invention) to a subject (e.g., a subject or in vivo, in vitro, or ex
vivo cells, tissues,
and organs). Exemplary routes of administration to the human body can be
through the
eyes (ophthalmic), mouth (oral), skin (topical or transdermal), nose (nasal),
lungs
(inhalant), oral mucosa (buccal), ear, rectal, vaginal, by injection (e.g.,
intravenously,
subcutaneously, intratumorally, intraperitoneally, etc.) and the like.
As used herein, the terms "co-administration" and "co-administering" refer to
the
administration of at least two agent(s) (e.g., composition comprising Sel-Plex
and one or
more other agents - e.g., an Alzheimer's disease therapeutic, or, a second
form of selenium)
or therapies to a subject. In some embodiments, the co-administration of two
or more
agents or therapies is concurrent. In other embodiments, a first agent/therapy
is
administered prior to a second agent/therapy. Those of skill in the art
understand that the
formulations and/or routes of administration of the various agents or
therapies used may
vary. The appropriate dosage for co-administration can be readily determined
by one
skilled in the art. In some embodiments, when agents or therapies are co-
administered, the
respective agents or therapies are administered at lower dosages than
appropriate for their
administration alone. Thus, co-administration is especially desirable in
embodiments
where the co-administration of the agents or therapies lowers the requisite
dosage of a
potentially harmful (e.g., toxic) agent(s), and/or when co-administration of
two or more
agents results in sensitization of a subject to beneficial effects of one of
the agents via co-
administration of the other agent.
As used herein, the term "treatment" or grammatical equivalents encompasses
the
improvement and/or reversal of the symptoms of disease (e.g.,
neurodegenerative disease).
A compound which causes an improvement in any parameter associated with
disease when
24
CA 02524551 2012-08-31
used in the screening methods of the instant invention may thereby be
identified as a
therapeutic compound. The term "treatment" refers to both therapeutic
treatment and
prophylactic or preventative measures. For example, those who may benefit from
treatment
with compositions and methods of the present invention include those already
with a
disease and/or disorder (e.g., neurodegenerative disease, diabetes or lack of
or loss of
cognitive function) as well as those in which a disease and/or disorder is to
be prevented
(e.g., using a prophylactic treatment of the present invention).
As used herein, the term "at risk for disease" refers to a subject (e.g., a
human) that
is predisposed to experiencing a particular disease. This predisposition may
be genetic (e.g.,
a particular genetic tendency to experience the disease, such as heritable
disorders), or due
to other factors (e.g., age, weight, environmental conditions, exposures to
detrimental
compounds present in the environment, etc.). Thus, it is not intended that the
present
invention be limited to any particular risk, nor is it intended that the
present invention be
limited to any particular disease.
As used herein, the term "suffering from disease" refers to a subject (e.g., a
human)
that is experiencing a particular disease. It is not intended that the present
invention be
limited to any particular signs or symptoms, nor disease. Thus, it is intended
that the
present invention encompass subjects that are experiencing any range of
disease (e.g., from
sub-clinical manifestation to full-blown disease) wherein the subject exhibits
at least some
of the indicia (e.g., signs and symptoms) associated with the particular
disease.
As used herein, the terms "disease" and "pathological condition" are used
interchangeably to describe a state, signs, and/or symptoms that are
associated with any
impairment of the normal state of a living animal or of any of its organs or
tissues that
interrupts or modifies the performance of normal functions, and may be a
response to
environmental factors (such as malnutrition, industrial hazards, or climate),
to specific
infective agents (such as worms, bacteria, or viruses), to inherent defect of
the organism
(such as various genetic anomalies, or to combinations of these and other
factors.
The term "compound" refers to any chemical entity, pharmaceutical, drug, and
the
like that can be used to treat or prevent a disease, illness, sickness, or
disorder of bodily
function. Compounds comprise both known and potential therapeutic compounds. A
compound can be determined to be therapeutic by screening using the screening
methods of
the present invention. A "known therapeutic compound" refers to a therapeutic
compound
that has been shown (e.g., through animal trials or prior experience with
administration to
CA 02524551 2012-08-31
humans) to be effective in such treatment. In other words, a known therapeutic
compound
is not limited to a compound efficacious in the treatment of disease (e.g.,
neurodegenerative
disease).
As used herein, the term "kit" is used in reference to a combination of
reagents and
other materials. It is contemplated that the kit may include reagents such as
nutrients and
drugs as well as administration means. It is not intended that the term "kit"
be limited to a
particular combination of reagents and/or other materials.
As used herein, the term "toxic" refers to any detrimental or harmful effects
on a
subject, a cell, or a tissue as compared to the same cell or tissue prior to
the administration
of the toxicant.
As used herein, the term "pharmaceutical composition" refers to the
combination of
an active agent (e.g., composition comprising Sel-Plexe) with a carrier, inert
or active,
making the composition especially suitable for diagnostic or therapeutic use
in vitro, in vivo
or ex vivo.
The terms "pharmaceutically acceptable" or "pharmacologically acceptable," as
used herein, refer to compositions that do not substantially produce adverse
reactions, e.g.,
toxic, allergic, or immunological reactions, when administered to a subject.
As used herein, the term "topically" refers to application of the compositions
of the
present invention to the surface of the skin and mucosal cells and tissues
(e.g., alveolar,
buccal, lingual, masticatory, or nasal mucosa, and other tissues and cells
that line hollow
organs or body cavities).
As used herein, the term "pharmaceutically acceptable carrier" refers to any
of the
standard pharmaceutical carriers including, but not limited to, phosphate
buffered saline
solution, water, emulsions (e.g., such as an oil/water or water/oil
emulsions), and various
types of wetting agents, any and all solvents, dispersion media, coatings,
sodium lauryl
sulfate, isotonic and absorption delaying agents, disintrigrants (e.g., potato
starch or sodium
starch glycolate), and the like. The compositions also can include stabilizers
and
preservatives. For examples of carriers, stabilizers and adjuvants. (See e.g.,
Martin,
Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa.
(1975)).
As used herein, the term "pharmaceutically acceptable salt" refers to any salt
(e.g.,
obtained by reaction with an acid or a base) of a compound of the present
invention that is
physiologically tolerated in the target subject (e.g., a mammalian subject,
and/or in vivo or
ex vivo, cells, tissues, or organs). "Salts" of the compounds of the present
invention may be
26
CA 02524551 2012-08-31
derived from inorganic or organic acids and bases. Examples of acids include,
but are not
limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric,
maleic,
phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic,
tartaric, acetic, citric,
methanesulfonic, ethanesulfonic, formic, benzoic, malonic, sulfonic,
naphthalene-2-
sulfonic, benzenesulfonic acid, and the like. Other acids, such as oxalic,
while not in
themselves pharmaceutically acceptable, may be employed in the preparation of
salts useful
as intermediates in obtaining the compounds of the invention and their
pharmaceutically
acceptable acid addition salts.
Examples of bases include, but are not limited to, alkali metal (e.g., sodium)
hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and
compounds
of formula NW4+, wherein W is Ci_4 alkyl, and the like.
Examples of salts include, but are not limited to: acetate, adipate, alginate,
aspartate,
benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate,
camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,
fumarate,
flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate,
chloride, bromide,
iodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-
naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate,
phenylpropionate,
picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate,
undecanoate, and the
like. Other examples of salts include anions of the compounds of the present
invention
compounded with a suitable cation such as Nat, NH4, and NW4+ (wherein W is a
C1-4 alkyl
group), and the like. For therapeutic use, salts of the compounds of the
present invention
are contemplated as being pharmaceutically acceptable. However, salts of acids
and bases
that are non-pharmaceutically acceptable may also find use, for example, in
the preparation
or purification of a pharmaceutically acceptable compound.
For therapeutic use, salts of the compounds of the present invention are
contemplated as being pharmaceutically acceptable. However, salts of acids and
bases that
are non-pharmaceutically acceptable may also find use, for example, in the
preparation or
purification of a pharmaceutically acceptable compound.
As used herein, the term "nucleic acid molecule" refers to any nucleic acid
containing molecule, including but not limited to, DNA or RNA. The term
encompasses
sequences that include any of the known base analogs of DNA and RNA including,
but not
limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine,
aziridinylcytosine,
pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-
bromouracil, 5-
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CA 02524551 2012-08-31
carboxymethylaminomethy1-2-thiouracil, 5-carboxymethylaminomethyluracil,
dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-
methylpseudouracil,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,
2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,
7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethy1-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-
thiocytosine, 5-methyl-
2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic
acid
methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine,
and
2,6-diaminopurine.
The term "gene" refers to a nucleic acid (e.g., DNA) sequence that comprises
coding sequences necessary for the production of a polypeptide, precursor, or
RNA (e.g.,
rRNA, tRNA). The polypeptide can be encoded by a full length coding sequence
or by any
portion of the coding sequence so long as the desired activity or functional
properties (e.g.,
enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.)
of the full-
length or fragment are retained. The term also encompasses the coding region
of a
structural gene and the sequences located adjacent to the coding region on
both the 5' and 3'
ends for a distance of about 1 kb or more on either end such that the gene
corresponds to
the length of the full-length mRNA. Sequences located 5' of the coding region
and present
on the mRNA are referred to as 5' non-translated sequences. Sequences located
3' or
downstream of the coding region and present on the mRNA are referred to as 3'
non-
translated sequences. The term "gene" encompasses both cDNA and genomic forms
of a
gene. A genomic form or clone of a gene contains the coding region interrupted
with non-
coding sequences termed "introns" or "intervening regions" or "intervening
sequences."
Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA);
introns may
contain regulatory elements such as enhancers. Introns are removed or "spliced
out" from
the nuclear or primary transcript; introns therefore are absent in the
messenger RNA
(mRNA) transcript. The mRNA functions during translation to specify the
sequence or
order of amino acids in a nascent polypeptide.
As used herein, the terms "gene expression" and "expression" refer to the
process of
converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA,
tRNA, or
snRNA) through "transcription" of the gene (i.e., via the enzymatic action of
an RNA
28
CA 02524551 2012-08-31
polymerase), and for protein encoding genes, into protein through
"translation" of mRNA.
Gene expression can be regulated at many stages in the process. "Up-
regulation" or
"activation" refer to regulation that increases and/or enhances the production
of gene
expression products (e.g., RNA or protein), while "down-regulation" or
"repression" refer
to regulation that decrease production. Molecules (e.g., transcription
factors) that are
involved in up-regulation or down-regulation are often called "activators" and
"repressors,"
respectively.
In addition to containing introns, genomic forms of a gene may also include
sequences located on both the 5' and 3' end of the sequences that are present
on the RNA
transcript. These sequences are referred to as "flanking" sequences or regions
(these
flanking sequences are located 5' or 3' to the non-translated sequences
present on the
mRNA transcript). The 5' flanking region may contain regulatory sequences such
as
promoters and enhancers that control or influence the transcription of the
gene. The 3'
flanking region may contain sequences that direct the termination of
transcription, post-
transcriptional cleavage and polyadenylation.
The term "wild-type" refers to a gene or gene product isolated from a
naturally
occurring source. A wild-type gene is that which is most frequently observed
in a
population and is thus arbitrarily designed the "normal" or "wild-type" form
of the gene. In
contrast, the term "modified" or "mutant" refers to a gene or gene product
that displays
modifications in sequence and or functional properties (i.e., altered
characteristics) when
compared to the wild-type gene or gene product. It is noted that naturally
occurring
mutants can be isolated; these are identified by the fact that they have
altered characteristics
(including altered nucleic acid sequences) when compared to the wild-type gene
or gene
product.
As used herein, the terms "nucleic acid molecule encoding," "DNA sequence
encoding," and "DNA encoding" refer to the order or sequence of
deoxyribonucleotides
along a strand of deoxyribonucleic acid. The order of these
deoxyribonucleotides
determines the order of amino acids along the polypeptide (protein) chain. The
DNA
sequence thus codes for the amino acid sequence.
As used herein, the terms "an oligonucleotide having a nucleotide sequence
encoding a gene" and "polynucleotide having a nucleotide sequence encoding a
gene,"
means a nucleic acid sequence comprising the coding region of a gene or in
other words the
nucleic acid sequence that encodes a gene product. The coding region may be
present in a
29
CA 02524551 2012-08-31
cDNA, genomic DNA or RNA form. When present in a DNA form, the oligonucleotide
or
polynucleotide may be single-stranded (i.e., the sense strand) or double-
stranded. Suitable
control elements such as enhancers/promoters, splice junctions,
polyadenylation signals,
etc. may be placed in close proximity to the coding region of the gene if
needed to permit
proper initiation of transcription and/or correct processing of the primary
RNA transcript.
Alternatively, the coding region utilized in the expression vectors of the
present invention
may contain endogenous enhancers/promoters, splice junctions, intervening
sequences,
polyadenylation signals, etc. or a combination of both endogenous and
exogenous control
elements.
As used herein, the term "oligonucleotide," refers to a short length of single-
stranded polynucleotide chain. Oligonucleotides are typically less than 200
residues long
(e.g., between 15 and 100), however, as used herein, the term is also intended
to encompass
longer polynucleotide chains. Oligonucleotides are often referred to by their
length. For
example a 24 residue oligonucleotide is referred to as a "24-mer".
Oligonucleotides can
form secondary and tertiary structures by self-hybridizing or by hybridizing
to other
polynucleotides. Such structures can include, but are not limited to,
duplexes, hairpins,
cruciforms, bends, and triplexes.
As used herein, the terms "complementary" or "complementarity" are used in
reference to polynucleotides (i.e., a sequence of nucleotides) related by the
base-pairing
rules. For example, for the sequence "5'-A-G-T-3'," is complementary to the
sequence "3'-
T-C-A-5'." Complementarity may be "partial," in which only some of the nucleic
acids'
bases are matched according to the base pairing rules. Or, there may be
"complete" or
"total" complementarity between the nucleic acids. The degree of
complementarity
between nucleic acid strands has significant effects on the efficiency and
strength of
hybridization between nucleic acid strands. This is of particular importance
in
amplification reactions, as well as detection methods that depend upon binding
between
nucleic acids.
The term "homology" refers to a degree of complementarity. There may be
partial
homology or complete homology (i.e., identity). A partially complementary
sequence is a
nucleic acid molecule that at least partially inhibits a completely
complementary nucleic
acid molecule from hybridizing to a target nucleic acid is "substantially
homologous." The
inhibition of hybridization of the completely complementary sequence to the
target
sequence may be examined using a hybridization assay (Southern or Northern
blot, solution
CA 02524551 2012-08-31
hybridization and the like) under conditions of low stringency. A
substantially homologous
sequence or probe will compete for and inhibit the binding (i.e., the
hybridization) of a
completely homologous nucleic acid molecule to a target under conditions of
low
stringency. This is not to say that conditions of low stringency are such that
non-specific
binding is permitted; low stringency conditions require that the binding of
two sequences to
one another be a specific (i.e., selective) interaction. The absence of non-
specific binding
may be tested by the use of a second target that is substantially non-
complementary (e.g.,
less than about 30% identity); in the absence of non-specific binding the
probe will not
hybridize to the second non-complementary target.
When used in reference to a double-stranded nucleic acid sequence such as a
cDNA
or genomic clone, the term "substantially homologous" refers to any probe that
can
hybridize to either or both strands of the double-stranded nucleic acid
sequence under
conditions of low stringency as described above.
A gene may produce multiple RNA species that are generated by differential
splicing of the primary RNA transcript. cDNAs that are splice variants of the
same gene
will contain regions of sequence identity or complete homology (representing
the presence
of the same exon or portion of the same exon on both cDNAs) and regions of
complete
non-identity (for example, representing the presence of exon "A" on cDNA 1
wherein
cDNA 2 contains exon "B" instead). Because the two cDNAs contain regions of
sequence
identity they will both hybridize to a probe derived from the entire gene or
portions of the
gene containing sequences found on both cDNAs; the two splice variants are
therefore
substantially homologous to such a probe and to each other.
When used in reference to a single-stranded nucleic acid sequence, the term
"substantially homologous" refers to any probe that can hybridize (i.e., it is
the complement
of) the single-stranded nucleic acid sequence under conditions of low
stringency as
described above.
As used herein, the term "hybridization" is used in reference to the pairing
of
complementary nucleic acids. Hybridization and the strength of hybridization
(i.e., the
strength of the association between the nucleic acids) is impacted by such
factors as the
degree of complementary between the nucleic acids, stringency of the
conditions involved,
the Tm of the formed hybrid, and the G:C ratio within the nucleic acids. A
single molecule
that contains pairing of complementary nucleic acids within its structure is
said to be "self-
hybridized."
31
CA 02524551 2012-08-31
As used herein, the term "Tm" is used in reference to the "melting
temperature."
The melting temperature is the temperature at which a population of double-
stranded
nucleic acid molecules becomes half dissociated into single strands. The
equation for
calculating the Tm of nucleic acids is well known in the art. As indicated by
standard
references, a simple estimate of the Tm value may be calculated by the
equation: Tm =
81.5 + 0.41(% G + C), when a nucleic acid is in aqueous solution at 1 M NaCl
(See e.g.,
Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid
Hybridization
(1985)). Other references include more sophisticated computations that take
structural as
well as sequence characteristics into account for the calculation of Tm.
As used herein the term "stringency" is used in reference to the conditions of
temperature, ionic strength, and the presence of other compounds such as
organic solvents,
under which nucleic acid hybridizations are conducted. Under "low stringency
conditions"
a nucleic acid sequence of interest will hybridize to its exact complement,
sequences with
single base mismatches, closely related sequences (e.g., sequences with 90% or
greater
homology), and sequences having only partial homology (e.g., sequences with 50-
90%
homology). Under 'medium stringency conditions," a nucleic acid sequence of
interest will
hybridize only to its exact complement, sequences with single base mismatches,
and closely
relation sequences (e.g., 90% or greater homology). Under "high stringency
conditions," a
nucleic acid sequence of interest will hybridize only to its exact complement,
and
(depending on conditions such a temperature) sequences with single base
mismatches. In
other words, under conditions of high stringency the temperature can be raised
so as to
exclude hybridization to sequences with single base mismatches.
"High stringency conditions" when used in reference to nucleic acid
hybridization
comprise conditions equivalent to binding or hybridization at 42 C in a
solution consisting
of 5X SSPE (43.8 g/lNaC1, 6.9 g/1 NaH2P041120 and 1.85 g/1 EDTA, pH adjusted
to 7.4
with NaOH), 0.5% SDS, 5X Denhardt's reagent and 100 ptg/m1 denatured salmon
sperm
DNA followed by washing in a solution comprising 0.1X SSPE, 1.0% SDS at 42 C
when a
probe of about 500 nucleotides in length is employed.
"Medium stringency conditions" when used in reference to nucleic acid
hybridization comprise conditions equivalent to binding or hybridization at 42
C in a
solution consisting of 5X SSPE (43.8 g/1 NaC1, 6.9 g/1 NaH2P041-120 and 1.85
g/1 EDTA,
pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt's reagent and 100 z/m1
32
CA 02524551 2012-08-31
denatured salmon sperm DNA followed by washing in a solution comprising 1.0X
SSPE,
1.0% SDS at 42 C when a probe of about 500 nucleotides in length is employed.
"Low stringency conditions" comprise conditions equivalent to binding or
hybridization at 42 C in a solution consisting of 5X SSPE (43.8 g/1 NaC1, 6.9
g/1
NaH2PO4.1-120 and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5X
Denhardt's reagent (50X Denhardt's contains per 500 ml: 5 g Ficoll (Type 400,
Pharamcia),
5 g BSA (Fraction V; Sigma)) and 10014/m1 denatured salmon sperm DNA followed
by
washing in a solution comprising 5X SSPE, 0.1% SDS at 42 C when a probe of
about 500
nucleotides in length is employed.
The art knows well that numerous equivalent conditions may be employed to
comprise low stringency conditions; factors such as the length and nature
(DNA, RNA,
base composition) of the probe and nature of the target (DNA, RNA, base
composition,
present in solution or immobilized, etc.) and the concentration of the salts
and other
components (e.g., the presence or absence of formamide, dextran sulfate,
polyethylene
glycol) are considered and the hybridization solution may be varied to
generate conditions
of low stringency hybridization different from, but equivalent to, the above
listed
conditions. In addition, the art knows conditions that promote hybridization
under
conditions of high stringency (e.g., increasing the temperature of the
hybridization and/or
wash steps, the use of formamide in the hybridization solution, etc.) (see
definition above
for "stringency").
As used herein, the term "primer" refers to an oligonucleotide, whether
occurring
naturally as in a purified restriction digest or produced synthetically, that
is capable of
acting as a point of initiation of synthesis when placed under conditions in
which synthesis
of a primer extension product that is complementary to a nucleic acid strand
is induced,
(i.e., in the presence of nucleotides and an inducing agent such as DNA
polymerase and at a
suitable temperature and pH). The primer is preferably single stranded for
maximum
efficiency in amplification, but may alternatively be double stranded. If
double stranded,
the primer is first treated to separate its strands before being used to
prepare extension
products. Preferably, the primer is an oligodeoxyribonucleotide. The primer
must be
sufficiently long to prime the synthesis of extension products in the presence
of the
inducing agent. The exact lengths of the primers will depend on many factors,
including
temperature, source of primer and the use of the method.
33
CA 02524551 2012-08-31
As used herein, the term "probe" refers to an oligonucleotide (i.e., a
sequence of
nucleotides), whether occurring naturally as in a purified restriction digest
or produced
synthetically, recombinantly or by PCR amplification, that is capable of
hybridizing to
another oligonucleotide of interest. A probe may be single-stranded or double-
stranded.
Probes are useful in the detection, identification and isolation of particular
gene sequences.
It is contemplated that any probe used in the present invention will be
labeled with any
"reporter molecule," so that is detectable in any detection system, including,
but not limited
to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays),
fluorescent,
radioactive, and luminescent systems. It is not intended that the present
invention be
limited to any particular detection system or label.
The term "isolated" when used in relation to a nucleic acid, as in "an
isolated
oligonucleotide" or "isolated polynucleotide" refers to a nucleic acid
sequence that is
identified and separated from at least one component or contaminant with which
it is
ordinarily associated in its natural source. Isolated nucleic acid is present
in a form or
setting that is different from that in which it is found in nature. In
contrast, non-isolated
nucleic acids are nucleic acids such as DNA and RNA found in the state they
exist in
nature. For example, a given DNA sequence (e.g., a gene) is found on the host
cell
chromosome in proximity to neighboring genes; RNA sequences, such as a
specific mRNA
sequence encoding a specific protein, are found in the cell as a mixture with
numerous
other mRNAs that encode a multitude of proteins. However, isolated nucleic
acid encoding
a given protein includes, by way of example, such nucleic acid in cells
ordinarily
expressing the given protein where the nucleic acid is in a chromosomal
location different
from that of natural cells, or is otherwise flanked by a different nucleic
acid sequence than
that found in nature. The isolated nucleic acid, oligonucleotide, or
polynucleotide may be
present in single-stranded or double-stranded form. When an isolated nucleic
acid,
oligonucleotide or polynucleotide is to be utilized to express a protein, the
oligonucleotide
or polynucleotide will contain at a minimum the sense or coding strand (i.e.,
the
oligonucleotide or polynucleotide may be single-stranded), but may contain
both the sense
and anti-sense strands (i.e., the oligonucleotide or polynucleotide may be
double-stranded).
As used herein, the term "purified" or "to purify" refers to the removal of
components (e.g., contaminants) from a sample. For example, antibodies are
purified by
removal of contaminating non-immunoglobulin proteins; they are also purified
by the
removal of immunoglobulin that does not bind to the target molecule. The
removal of non-
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CA 02524551 2012-08-31
immunoglobulin proteins and/or the removal of immunoglobulins that do not bind
to the
target molecule results in an increase in the percent of target-reactive in-n-
nunoglobulins in
the sample. In another example, recombinant polypeptides are expressed in
bacterial host
cells and the polypeptides are purified by the removal of host cell proteins;
the percent of
recombinant polypeptides is thereby increased in the sample.
As used herein, the term "vector" is used in reference to nucleic acid
molecules that
transfer DNA segment(s) from one cell to another. The term "vehicle" is
sometimes used
interchangeably with "vector." Vectors are often derived from plasmids,
bacteriophages, or
plant or animal viruses.
The term "expression vector" as used herein refers to a recombinant DNA
molecule
containing a desired coding sequence and appropriate nucleic acid sequences
necessary for
the expression of the operably linked coding sequence in a particular host
organism.
Nucleic acid sequences necessary for expression in prokaryotes usually include
a promoter,
an operator (optional), and a ribosome binding site, often along with other
sequences.
Eukaryotic cells are known to utilize promoters, enhancers, and termination
and
polyadenylation signals.
The term "transfection" as used herein refers to the introduction of foreign
DNA
into eukaryotic cells. Transfection may be accomplished by a variety of means
known to
the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-
mediated
transfection, polybrene-mediated transfection, electroporation,
microinjection, liposome
fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.
The term "stable transfection" or "stably transfected" refers to the
introduction and
integration of foreign DNA into the genome of the transfected cell. The term
"stable
transfectant" refers to a cell that has stably integrated foreign DNA into the
genomic DNA.
The term "transient transfection" or "transiently transfected" refers to the
introduction of foreign DNA into a cell where the foreign DNA fails to
integrate into the
genome of the transfected cell. The foreign DNA persists in the nucleus of the
transfected
cell for several days. During this time the foreign DNA is subject to the
regulatory controls
that govern the expression of endogenous genes in the chromosomes. The term
"transient
transfectant" refers to cells that have taken up foreign DNA but have failed
to integrate this
DNA.
As used herein, the term "selectable marker" refers to the use of a gene that
encodes
an enzymatic activity that confers the ability to grow in medium lacking what
would
CA 02524551 2012-08-31
otherwise be an essential nutrient (e.g. the HIS3 gene in yeast cells); in
addition, a
selectable marker may confer resistance to an antibiotic or drug upon the cell
in which the
selectable marker is expressed. Selectable markers may be "dominant"; a
dominant
selectable marker encodes an enzymatic activity that can be detected in any
eukaryotic cell
line. Examples of dominant selectable markers include the bacterial
aminoglycoside 3'
phosphotransferase gene (also referred to as the neo gene) that confers
resistance to the
drug G418 in mammalian cells, the bacterial hygromycin G phosphotransferase
(hyg) gene
that confers resistance to the antibiotic hygromycin and the bacterial
xanthine-guanine
phosphoribosyl transferase gene (also referred to as the gpt gene) that
confers the ability to
grow in the presence of mycophenolic acid. Other selectable markers are not
dominant in
that their use must be in conjunction with a cell line that lacks the relevant
enzyme activity.
Examples of non-dominant selectable markers include the thymidine kinase (tk)
gene that is
used in conjunction with tk cell lines, the CAD gene that is used in
conjunction with
CAD-deficient cells and the mammalian hypoxanthine-guanine phosphoribosyl
transferase
(hprt) gene that is used in conjunction with hprt cell lines. A review of the
use of
selectable markers in mammalian cell lines is provided in Sambrook, J. et al.,
Molecular
Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press,
New York
(1989) pp.16.9-16.15.
As used herein, the term "cell culture" refers to any in vitro culture of
cells.
Included within this term are continuous cell lines (e.g., with an immortal
phenotype),
primary cell cultures, transformed cell lines, finite cell lines (e.g., non-
transformed cells),
and any other cell population maintained in vitro.
As used, the term "eukaryote" refers to organisms distinguishable from
"prokaryotes." It is intended that the term encompass all organisms with cells
that exhibit
the usual characteristics of eukaryotes, such as the presence of a true
nucleus bounded by a
nuclear membrane, within which lie the chromosomes, the presence of membrane-
bound
organelles, and other characteristics commonly observed in eukaryotic
organisms. Thus,
the term includes, but is not limited to such organisms as fungi, protozoa,
and animals (e.g.,
humans).
As used herein, the term "in vitro" refers to an artificial environment and to
processes or reactions that occur within an artificial environment. In vitro
environments
can consist of, but are not limited to, test tubes and cell culture. The term
"in vivo" refers to
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CA 02524551 2012-08-31
the natural environment (e.g., an animal or a cell) and to processes or
reaction that occur
within a natural environment.
The terms "test compound" and "candidate compound" refer to any chemical
entity,
pharmaceutical, drug, and the like that is a candidate for use to treat or
prevent a disease,
illness, sickness, or disorder of bodily function (e.g., Alzheimer's disease,
ALS, Parkinson's
disease, etc.). Test compounds comprise both known and potential therapeutic
compounds.
A test compound can be determined to be therapeutic by screening using the
screening
methods of the present invention.
As used herein, the term "sample" is used in its broadest sense. In one sense,
it is
meant to include a specimen or culture obtained from any source, as well as
biological and
environmental samples. Biological samples may be obtained from animals
(including
humans) and encompass fluids, solids, tissues, and gases. Biological samples
include blood
products, such as plasma, serum and the like. Environmental samples include
environmental material such as surface matter, soil, water, crystals and
industrial samples.
Such examples are not however to be construed as limiting the sample types
applicable to
the present invention.
The term "RNA interference" or "RNAi" refers to the silencing or decreasing of
gene expression by siRNAs. It is the process of sequence-specific, post-
transcriptional gene
silencing in animals and plants, initiated by siRNA that is homologous in its
duplex region
to the sequence of the silenced gene. The gene may be endogenous or exogenous
to the
organism, present integrated into a chromosome or present in a transfection
vector that is
not integrated into the genome. The expression of the gene is either
completely or partially
inhibited. RNAi may also be considered to inhibit the function of a target
RNA; the
function of the target RNA may be complete or partial.
The term "siRNAs" refers to short interfering RNAs. In some embodiments,
siRNAs comprise a duplex, or double-stranded region, of about 18-25
nucleotides long;
often siRNAs contain from about two to four unpaired nucleotides at the 3' end
of each
strand. At least one strand of the duplex or double-stranded region of a siRNA
is
substantially homologous to or substantially complementary to a target RNA
molecule. The
strand complementary to a target RNA molecule is the "antisense strand;" the
strand
homologous to the target RNA molecule is the "sense strand," and is also
complementary to
the siRNA antisense strand. siRNAs may also contain additional sequences; non-
limiting
examples of such sequences include linking sequences, or loops, as well as
stem and other
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CA 02524551 2012-08-31
folded structures. siRNAs appear to function as key intermediaries in
triggering RNA
interference in invertebrates and in vertebrates, and in triggering sequence-
specific RNA
degradation during posthanscriptional gene silencing in plants.
The term "target RNA molecule" refers to an RNA molecule to which at least one
strand of the short double-stranded region of an siRNA is homologous or
complementary.
Typically, when such homology or complementary is about 100%, the siRNA is
able to
silence or inhibit expression of the target RNA molecule. Although it is
believed that
processed mRNA is a target of siRNA, the present invention is not limited to
any particular
hypothesis, and such hypotheses are not necessary to practice the present
invention. Thus, it
is contemplated that other RNA molecules may also be targets of siRNA. Such
targets
include unprocessed mRNA, ribosomal RNA, and viral RNA genomes.
DETAILED DESCRIPTION OF THE INVENTION
Selenium is a trace element involved in regulating aspects of the antioxidant
defense
mechanism in all living tissues by interacting with the body's glutathione
(GSH) and its
major Se-containing antioxidant enzymes, glutathione peroxidase (GPX) and
thioredoxin
reductase (See, e.g., Goehring et al., J. Anim. Sci. 59, 725-732 (1984);
Gerloff et al., J. Anim.
Sci. 70, 3934-3940 (1992)). Glutathione and GPX have the capacity to protect
the integrity
of unsaturated bonds of membrane phospholipids by extinguishing free radical
attacks
capable of initiating and propagating lipid oxidation (See, e.g., Meister and
Anderson,
Atmu. Rev. Biochem. 52, 711-760 (1983); Deleve and Kaplowitz, Pharm. Ther. 52,
287-
305 (1991); Palmer and Paulson, Nutr. Rev. 55, 353-361 (1997)).
Selenium has also been associated with reduced cancer risk in several
epidemiologic
studies (See, e.g., Salonen et al., Am. J. Epidemiol. 120: 342-349 (1984);
Willett et al.,
Lancet 2: 130-134 (1983); Virtamo et al., Cancer 60: 145-148 (1987)). Various
selenium
compounds of natural and synthetic origin have been shown to inhibit tumor
development
in animal studies in a wide range of dosages (See, e.g., Ip,. J. Nutr. 128:
1845-1854
(1998)). Although most animal studies have employed pharmacologic doses of
selenium
(>2 mg/kg) in cancer chemoprevention (See, e.g., Ip,. J. Nutr. 128: 1845-1854
(1998)),
selenium deficiency has also been shown to enhance mammary (See, e.g., Ip and
Daniel,
Cancer Res. 45: 61-65 (1985)) and UVB-induced skin carcinogenesis (See, e.g.,
Pence et
al., 102: 759-761 (1994)).
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CA 02524551 2012-08-31
A recent double-blind, randomized cancer prevention trial in humans involving
physiologic doses (0.2 mg) of selenium demonstrated a reduction in incidence
of lung,
prostate and intestinal cancers (See, e.g., Clark etal., J. Am. Med. Assoc.
276:1957-1963
(1996)).
Despite decades of research in the mechanisms of action of selenium, little to
nothing is known regarding other potential targets of selenium (e.g., genes or
regulatory
pathways) and beneficial effects that could be provided to a subject through
administration
of selenium. Also lacking is information regarding what forms of selenium
(e.g., organic,
inorganic, or both) can and cannot be used for bringing about these effects.
Thus, it would
be of great value to elucidate various ways in which different forms of
selenium could be
used to benefit certain systems (e.g., nervous, endocrine, and metabolic
systems) of a
subject (e.g., a human, bovine or other mammal). Furthermore, understanding
how various
forms of selenium differ in their ability to exert effects on a subject
provides the ability to
customize treatments for subjects suffering from, or at risk of, a disease or
disorder that
might be benefited by such treatment (e.g., specific forms of selenium could
be used
independently or with other known agents to treat or prevent diseases or
disorders).
Accordingly, the present invention demonstrates how specific forms of selenium
(e.g., selenomethionine (SeM), Sodium-selenite (Sod-se!), and Sel-Plexe) may
be used to
benefit a subject. In particular, the present invention demonstrates that
compositions and
methods of the present invention can be used to stabilize or increase the
general health and
cognitive function of a subject. For example, as described in detail herein,
the present
invention provides compositions and methods that alter cellular function
thereby providing
beneficial effects to multiples systems of a subject including, but not
limited to, the
neurological system, the nervous system, the endocrine system, the metabolic
system, and
the immune system. The present invention also provides methods of using
selenium with
other agents for treating or preventing disease. Thus, the present invention
provides
compositions comprising selenium (e.g., Sel-PlexC) and methods of using the
same as a
therapeutic and/or prophylactic treatment (e.g., for neurodegenerative
disease, for
enhancing cognitive function, or retarding age-associated gene expression).
The following
examples are provided in order to illustrate multiple ways that compositions
and methods of
the present invention may be utilized in order to provide a beneficial effect
to a subject, and
are not meant to be construed as limiting the scope of the invention.
39
CA 02524551 2012-08-31
I. Neurodegenerative diseases.
In some embodiments, the present invention provides the administration of
selenium, independently or in combination with one or more other agents, for
the
prophylactic or therapeutic treatment of neurodegenerative disease. In
preferred
embodiments, the present invention provides a prophylactic treatment
comprising
administering a composition comprising selenium to a subject at risk of
developing a
neurodegenerative disease, thereby preventing the neurodegenerative disease.
In other
preferred embodiments, the present invention provides a method of treating or
preventing a
neurodegenerative disease comprising providing to a subject an agent used for
treatment or
prevention of a neurodegenerative disease in combination with a composition
comprising
selenium. The present invention demonstrates for the first time that certain
forms of
selenium may be compatible with the above mentioned methods, while others
forms of
selenium may not. Thus, in some embodiments, the present invention provides
one or more
forms of selenium (e.g., Sel-Plex and/or Sod-sel) that is biologically
available and is
administered alone or co-administered with an agent used for treatment or
prevention of a
neurodegenerative disease, wherein the form of selenium chosen functions to
treat or
prevent the neurodegenerative disease.
The form of selenium administered to a subject will depend on the target
(e.g., gene)
sought to be treated. As demonstrated by the present invention, the presence
and level of
beneficial effect attained varies depending on the form of selenium used (See
Examples 2-
10). In preferred embodiments, selenium is provided in the form of Sel-Plex .
In other
embodiments, selenium is provided as sodium-selenite. In still other
embodiments,
selenium is provided as selenomethionine or selenium enriched yeast. In some
embodiments, selenium is provided as selenocysteine or a selenate compound. In
some
embodiments, selenium may be chemically linked to an agent (e.g., an agent
used for
treating neurodegenerative disease) to form a selenium-agent derivative.
Once the desired form of selenium is chosen, it can be administered alone or
in
combination with one or more agents used for the prevention or treatment of a
neurodegenerative disease. The agent may be one approved by a regulatory
authority for
such a treatment (e.g., the US Food and Drug Administration (FDA) or the
European
Medicines Evaluation Agency (EMEA)). Compositions and methods of the present
invention are contemplated to be useful for the treatment of a variety of
neurodegenerative
diseases including, but not limited to, Alzheimer's disease, amyotrophic
lateral sclerosis,
CA 02524551 2012-08-31
Parkinson's disease, Huntington's disease, multiple sclerosis, spinocerebellar
ataxias,
Friedreich's ataxia, and myotonic dystrophy.
A. Alzheimer's Disease
In some embodiments of the present invention, compositions and methods of the
present invention are utilized in the treatment of Alzheimer disease.
Alzheimer disease
(AD) is a common cause of dementia, which is an acquired cognitive and
behavioral
impairment of sufficient severity to markedly interfere with social and
occupational
functioning.
AD affects approximately 5 million people in the United States and more than
30
million people worldwide. A larger number of individuals have decreased levels
of
cognitive impairment (eg, minimal cognitive impairment), which frequently
evolves into a
full-blown dementia, thereby increasing the number of affected persons. The
prevalence of
AD is expected to substantially increase in this century because it
preferentially affects the
elderly, who constitute the fastest growing age group in many, especially
industrialized,
countries. Statistical projections indicate that the number of persons
affected by the
disorder in the United States will nearly triple by the year 2050.
AD is also a major public health problem from the economic perspective. In the
United States, the cost of caring for patients with AD was more than $110
billion per year
in the early 1990s, and the average yearly cost per patient is about $45,000.
Because
methods for assessing the economic effects of neurodegenerative disorders are
still in their
infancy, these figures are likely underestimates.
The anatomic pathology of AD includes neurofibrillary tangles (NFTs); senile
plaques (SPs) at the microscopic level; and cerebrocortical atrophy, which
predominantly
involves the association regions and particularly the medial aspect of the
temporal lobe.
Although NFTs and SPs are characteristic of AD, they are not pathognomonic. In
fact
many other neurodegenerative conditions distinct from AD are characterized by
NFTs (eg,
progressive supranuclear palsy, dementia pugilistica) or SPs (eg, normal
aging). Therefore,
the mere presence of these lesions is not sufficient to diagnose AD. These
lesions must be
present in sufficient numbers and in a characteristic topographic distribution
to fulfill the
current histopathologic criteria for AD.
In addition to NFTs and SPs, many other lesions of AD have been recognized
since
Alzheimer's original papers were published. These include (1) the
granulovacuolar
41
CA 02524551 2012-08-31
degeneration of Shimkowicz; (2) the neuropil threads of Braak et al; and (3)
neuronal loss
and synaptic degeneration, which are thought to ultimately mediate the
cognitive and
behavioral manifestations of the disorder.
AD is the most common neurodegenerative disorder worldwide. In AD, neurons of
the hippocampus and cerebral cortex are selectively lost. Brains of
individuals with AD
manifest two characteristic lesions: extracellular amyloid (or senile) plaques
and
intracellular neurofibrillary tangles of hyperphosphorylated tau protein (See,
e.g., Selkoe,
Nature 426, 900-904 (2003)). Amyloid plaques contain small, toxic cleavage
products
(denoted as A1340 and A[342) of the amyloid precursor protein (APP). The apoE4
(apolipoprotein E4) genotype is a powerful risk factor for developing AD, and
it may
possibly affect 13-amyloid protein (A13) deposition and neurofibrillary tangle
formation
(See, e.g., Roses, Curr. Opin. Neurol. 4, 265-270 (1996)). Mutations in three
genes that
are inherited in an autosomal dominant fashion have been linked to rare
familial, early-
onset forms of AD. These genes include those encoding APP, presenilin 1 (PS1)
and
presenilin 2 (PS2). One common event in both familial and sporadic types of AD
is the
increased production and accumulation of the toxic [3-amyloid protein. This
observation
has led to the 'amyloid cascade hypothesis' that excessive Al3 production is
the primary
cause of the disease.
APP is a type I membrane protein and contains a large extracellular region, a
transmembrane helix and a short cytoplasmic tail. Toxic Ai13 originates from
regulated
intramembrane proteolysis of APP by a complex of secretases. The first
cleavage of APP is
mediated by [3- or a-secretase, releasing most of the extracellular portion of
APP as two
fragments, APPs-a and APPs-13, leaving behind the C-terminal membrane bound
fragment.
This portion of APP is then cleaved by a large protein complex, y-secretase,
at several sites
including amino acid (aa) 711 (Ab40) and at least three additional subsites at
aa713 (A1342),
aa714 (A1343) and aa720 (A1349). Several mutations in APP, such as the Swedish
mutation,
cluster at the y-secretase cleavage sites; these mutations result in increased
amounts of Al3
peptide and protofibril formation (See, e.g., Singleton et al., Hum. Mol.
Genet. 13 (Spec.
no. 1), R123¨R126 (2004)).
The precise composition of the y-secretase complex is still under debate, but
presenilin 1 (PS1), presenilin 2 (PS2), nicastrin, Aph-1 and Pen-2 appear to
be required
(See, e.g., Haas and Steiner, Trends Cell Biol. 12, 556-562 (2002); Edbauer et
al., Nat. Cell
Biol. 5, 486-488 (2003); Haass, EMBO J. 23, 483-488 (2004)). PS1 is a
transmembrane
42
CA 02524551 2012-08-31
domain aspartyl protease that cleaves its substrates in the membrane-spanning
region. PS1
is probably responsible for the generation of AP fragments. An additional
protein involved
is calsenilin. Calsenilin is over-expressed (e.g., up-regulated) in neurons
and astrocytes in
an Alzheimer's diseased brain (See, e.g., Jin et al., Neuroreport 16, 451-455
(2005)).
Overexpression of calsenilin enhances g-secretase activity, demonstrating that
calsenilin is
a regulatory factor for g-secretase (See, e.g., Jo et al., Neurosci Lett, 378,
59-64 (2005)).
More than 100 missense mutations in PS1 and PS2 have been identified in rare
familial, early-onset AD (See, e.g., Hutton et al., Essays Biochem. 33, 117-
131(1998)).
Experiments in culture and transgenic mice reveal that these mutations result
in increased
AP production (See, e.g., Scheuner. etal., Nat. Med. 2, 864-870 (1996);
Borchelt et al.,
Neuron 19, 939-945 (1997)). Conversely, mice lacking PS1 have decreased A1340
and
AP42 production (See, e.g., De Strooper, etal., Nature 391, 387-390 (1998);
Naruse, et
al., 21, 1213-1221 (1998)), suggesting that PS1 has a pivotal role in y-
secretase activity. C-
terminal cleavage of APP by caspase enzymes may also be required for toxicity
(See, e.g.,
Lu et al., Nat. Med. 6, 385-386 (2000)).
Several mechanisms have been proposed regarding how AP does its damage. One
view suggests that AP protofibrils activate microglia, inciting an
inflammatory response
and release of neurotoxic cytokines. Nonsteroidal anti-inflammatory drugs
(NSAIDs)
including ibuprofen delay the onset of AD (See, e.g., Stewart et al.,
Neurology 48, 626-632
(1997)). Additionally, NSAIDs reduce the production of A342 (See, e.g., Weggen
et al.,
Nature 414, 212-216 (2001)).
In a second view, AP protofibrils trigger excessive release of excitatory
amino acids
like glutamate from glial cells that may injure nearby neurons by
excitotoxicity.
Overactivation of glutamate receptors of the N-methyl-D-aspartate (NMDA)
subtype results
in increased intracellular Ca2+, which activates neuronal nitric oxide
synthase and
consequently generates nitric oxide (NO). When generated in excess, NO
combines with
superoxide anion (02:), forming the highly reactive and neurotoxic product
peroxynitrite
(ON00), which leads to further oxidative and nitrosative stress in part via
mitochondrial
injury. In fact, positive phase III human trials of the uncompetitive NMDA
receptor channel
blocker, memantine, led to its recent approval for the treatment of AD (See,
e.g., Lipton,
Nature 428, 473 (2004)).
Cholinergic transmission and synaptic density are considerably decreased in AD
patients. The mechanism for synaptic damage is unknown, but diffusible
oligomeric forms
43
CA 02524551 2012-08-31
of A13 may be important. Synaptic dysfunction probably contributes to memory
loss and
cognitive deficits in AD. In fact, APP transgenic mice manifest cellular,
biochemical and
electrophysiological evidence of synaptic deficits before A13 deposition,
including reduced
excitatory postsynaptic potentials and long term potentiation regarded as a
correlate of
learning and memory (See, e.g., Chapman et al., Nat. Neurosci. 2,271-276
(1999)).
Inhibition of y-secretase decreases oligomeric A13 and LTP deficits (See,
e.g., Walsh et al.,
Nature 416,535-539 (2002)).
A13 may also mediate harmful effects by binding redox-reactive metals, which
in
turn release free radicals (See, e.g., Bush etal., J. Biol. Chem. 268,16109-
16112 (1993);
Bush et al.,Science 265,1464-1467 (1994); Lovell et al., J. Neurol. Sci.
158,47-52
(1998); Dong et al., Biochemistry 42,2768-2773 (2003); Opazo et al., J. Biol.
Chem. 277,
40302-40308 (2002); Bush et al., Alzheimer Dis. Assoc. Disord. 17,147-150
(2003); 36
Huang et al., Biochemistry 38,7609-7616 (1999)). Chelation of zinc and copper
provides
neuroprotective effects (See, e.g., Bush, Aging 23,1031-1038 (2002)). For
example,
clioquinol (CQ), an antibiotic that also chelates zinc and copper and crosses
the blood-brain
barrier, decreases brain A13 deposition and improves learning in mutant APP
transgenic
mice (See, e.g., Cherny et al., Neuron 30,665-676 (2001)).
In the US the lifetime risk of AD is estimated to be 1:4-1:2. More than 14% of
individuals older than 65 years have AD, and the prevalence increases to at
least 40% in
individuals older than 80 years. Prevalences similar to those in the United
States have been
reported in industrialized nations. Countries experiencing rapid increases in
the elderly
segments of their population have rates approaching those in the United
States.
AD affects both men and women. Many studies indicate that the risk of AI:30 is
significantly higher in women than in men. Some authorities have postulated
that this
difference is due to the loss of the neurotrophic effect of estrogen in
postmenopausal
women. Other factors may also influence this relative difference.
AD is generally diagnosed by cognitive symptoms. In assessing AD, brain MRIs
or
CT scans show diffuse cortical and/or cerebral atrophy. These studies are also
used to rule
out other CNS disease. EEG and Tau protein tests are also used to confirm
diagnosis and
rule out other diseases that cause dementia.
The mainstay of AD therapy is the use of centrally acting cholinesterase
inhibitors
to palliate the depletion of ACh in the cerebral cortex and hippocampus.
Because the
clinical manifestations of AD are believed to be partly due to a loss of the
cholinergic
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CA 02524551 2012-08-31
innervation to the cerebral cortex, compounds have been developed to palliate
the
cholinergic defect by interfering with the degradation of ACh by AChE, the
synaptic (or
specific) form of cholinesterase. Some of the more recently available
compounds are
substances that inhibit also the nonsynaptic (or nonspecific) cholinesterases,
which are
frequently called BuChE.
AChE inhibitors approved by the FDA for use in the early and intermediate
stages
of AD are tacrine (Cognex), donepezil (Aricept), rivastigmine (Exelon), and
galantamine
(galanthamine, Reminyl). Among these, only tacrine and rivastigmine also
inhibit BuChE.
This may be important for their therapeutic efficacy because BuChE levels
increase during
the course of AD and are present in some AD lesions, including senile plaques.
At present,
tacrine, is used seldom if at all because it has been superseded by other
treatments.
An increasing number of clinical studies demonstrate that cholinesterase
inhibition
can have modest but detectable effects, such as improvement in cognitive
performance, as
measured by tools such as the Alzheimer's Disease Assessment Scale-cognitive
subscale
(ADAS-cog). More recent evidence indicates that ChEIs may also alleviate the
noncognitive manifestations of AD. For example, they can ameliorate behavioral
manifestations, as assessed by using tools such as the Neuropsychiatric
Inventory, and they
may improve the performance of activities of daily living, as evaluated by
using the
Progressive Deterioration Scale.
In general, the benefits are temporary because ChEIs do not address the
underlying
cause of the degeneration of cholinergic neurons, which continues during the
disease.
Although the increasingly large family of ChEIs was originally expected to
help in only the
early and intermediate stages of AD, results indicate that (1) they improve
cognitive
performance in advanced stages; (2) they significantly improve behavioral
manifestations
(eg, wandering, agitation, socially inappropriate behavior associated with
advanced stages);
and (3) they help in patients with presumed vascular components added to
dementia due to
AD, as well as in patients with the DLB, which often co-occurs or overlaps
with AD (Lewy
body variant of AD).
The ChEIs share a common profile of adverse effects, the most frequent of
which
are nausea, vomiting, diarrhea, and dizziness. These are typically dose
related and can be
mitigated with slow uptitration to the desired maintenance dose. Use of drugs
whose
absorption peaks are blunted by food (eg, rivastigmine) can further mitigate
adverse effects
and improve the tolerability of ChEI treatment.
CA 02524551 2012-08-31
NMDA antagonists are the newest class of agents indicated for the treatment of
AD.
As of October 2003, the only approved drug in this class is memantine. These
agents may
be used alone or combined with AChE inhibitors. Accordingly, in some
embodiments,
compositions and methods of the present invention are used in combination with
the above
described agents for the therapeutic and/or prophylactic treatment of AD.
In preferred embodiments, the present invention provides a method of treating
an
Alzheimer's disease patient comprising administering to the Alzheimer's
disease patient a
composition comprising selenium (e.g., Sel-Plexe) under conditions such that
symptoms
(e.g., described above) of Alzheimer's disease in the patient are reduced.
Although an
understanding of the mechanism is not necessary to practice the present
invention and the
present invention is not limited to any particular mechanism of action,
administering a
composition comprising selenium (e.g., Sel-PlexS) to an Alzheimer's subject
reduces
symptoms associated with Alzheimer's through reducing the expression of genes
that
encode proteins involved in processing amyloid precursor protein (APP) (e.g.,
Nicastrin,
presenilin 1, presenilin 2, calsenilin, Cathepsin B, Cathepsin D, Cathepsin Z,
or Cathepsin
0) (See Example 10). In other preferred embodiments, the expression of genes
involved in
the generation of beta amyloid peptide (e.g., Apbbl, Aplp 1, and Apbal) are
aletered (e.g.,
reduced) using compositions and methods of the present invention. In some
preferred
embodiments, compositions and methods of the present invention are used as a
prophylactic
treatment in order to prevent Alzheimer's disease. In some embodiments,
compositions and
methods of the present invention are used in combination with other known
therapeutic
treatments (e.g., those described above) for the treatment Alzheimer's
disease. In still other
embodiments, the present invention provides a method of prophylactic and/or
therapeutic
treatment for Alzheimer's disease comprising co-administering to a subject a
composition
comprising selenium (e.g., Sel-Plexe), an Alzheimer's therapeutic and one or
more anti-
oxidants. In other embodiments, compositions and methods of the present
invention are
used to prevent and/or treat neurodegeneration associated with Alzheimer's
disease
comprising inhibiting expression of genes that encode proteins involved in
processing
amyloid precursor protein, genes involved in the generation of 3-amyloid
peptide, and
complement associated genes (e.g., Nicastrin, presenilin 1, presenilin 2,
calsenilin,
Cathepsin B, Cathepsin D, Cathepsin Z, Cathepsin 0, Apbbl, Aplp 1, Apba 1 ;
Clq, Clq
alpha, Clq beta, Clq gamma, and Clqr, See Example 10). Thus, in some
embodiments,
the present invention provides a method of inhibiting the expression of genes
involved in
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CA 02524551 2012-08-31
processing amyloid precursor protein in a subject comprising providing to the
subject a
composition comprising selenium (e.g., Sel-Plex ) under conditions such that
the
expression of genes involved in processing amyloid precursor protein are
reduced. In some
embodiments, the present invention provides a method of inhibiting the
expression of genes
involved in the generation of P-amyloid peptide in a subject comprising
providing to the
subject a composition comprising selenium under conditions such that the
expression of
genes involved in the generation of -amyloid peptide are reduced. In some
embodiments,
the present invention provides a method of inhibiting the expression of
complement genes
(e.g., Clq, Clq alpha, Clq beta, Clq gamma, and Clqr ) (See Example 10) in a
subject
comprising providing to the subject a composition comprising selenium under
conditions
such that the expression of complement genes are reduced. In preferred
embodiments, the
composition comprising selenium comprises Sel-Plex . The composition
comprising Sel-
Plex may also comprise other forms of selenium, for example, Sod-sel, thereby
enhancing
downregulation of expression of the above mentioned genes.
B. Amyotrophic Lateral Sclerosis (ALS).
In some embodiments of the present invention, compositions and methods of the
present invention are utilized in the prophylactic or therapeutic treatment of
amyotrophic
lateral sclerosis (ALS). ALS is a devastating disorder of the anterior horn
cells of the spinal
cord and the motor cranial nuclei that leads to progressive muscle weakness
and atrophy.
The frequency of ALS in the United States is approximately 5 cases per 100,000
population. ALS leads to death within a decade. In most cases, death occurs
within 5
years. Some patients with familial, juvenile-onset ALS have been reported to
survive for
longer periods (2-3 decades). In the United States, ALS affects whites more
often than
nonwhites; the white-to-nonwhite ratio is 1.6:1. The ratio of ALS-affected
males to
females is 1.5:1. Onset occurs in the fourth to seventh decades of life.
ALS involves degeneration of motor neurons, resulting in progressive muscle
wasting and weakness, culminating in paralysis, respiratory failure and death.
Perhaps 10%
of cases are familial, and of those, about 2-3% are caused by mutations in the
gene
encoding Cu/Zn superoxide dismutase-1 (SOD1), producing a toxic gain of
function rather
than loss of (catalytic) function (See, e.g., Rosen et al., Nature 362, 59-62
(1993)). The
precise pathogenic mechanism is not clear, but implicated in motor neuron
dysfunction and
death are protein misfolding and aggregation, defective axonal transport,
mitochondrial
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CA 02524551 2012-08-31
dysfunction and excitotoxicity via faulty glutamate reuptake into glial cells.
Recent
structural evidence suggests that some Cu/Zn SOD] mutations result in
destabilization of
normal dimers of the enzyme and foster aggregation, forming amyloid or pores
depending
on the conditions, not unlike familial amyloid polyneuropathy (See, e.g.,
Hough et al., Proc.
Natl. Acad. Sci. USA 101, 5976-5981 (2004); Koo et al., Proc. Natl. Acad. Sci.
USA 96,
9989-9990 (1999)). Stabilization of dimers has therefore been proposed as a
therapeutic
intervention (See, e.g., Ray and Lansbury, Proc. Natl. Acad. Sci. USA 101,
5701-5702
(2004)).
A recent report on sporadic ALS (representing the vast majority of cases)
revealed
abnormal RNA editing in GluR2 subunits of glutamate receptors, producing
increased Ca2+
entry into neurons (See, e.g., Kawahara et al.,Nature 427, 801 (2004); Lipton,
Nat. Med. 10,
347 (2004)). This mechanism may contribute to neuronal demise, suggesting
possible
therapeutic targets, such as counteracting overly active calcium-permeable
glutamate
receptors or compensating for potentially dysfunctional RNA-editing enzymes.
Patients may have weakness of bulbar muscles or of single or multiple limb
muscle
groups. Presentation is not always bilateral or symmetrical. A predominantly
bulbar form
usually leads to more rapid deterioration and death. Limb weakness is
predominantly
distal. Weakness and atrophy of the intrinsic hand muscles are prominent.
Weakness
progresses to involve the forearms and shoulder girdle muscles and the lower
extremities.
Involvement of both upper and lower motor neurons is characteristic. Patients
develop variable hyperreflexia, clonus, spasticity, extensor plantar
responses, and limb or
tongue fasciculations. Wallerian degeneration of corticospinal and
corticobulbar tracts may
be demonstrated by MRI (high-intensity T2 lesions in frontal lobes) or in
postmortem
examination. Extraocular muscles and bladder and anal sphincter muscles
typically are
spared.
Nearly 10% of ALS cases are familial; the disease is transmitted in an
autosomal
dominant fashion. The copper/zinc SOD1 gene is mutated in 10-20% of these
familial
cases. Although the primary mechanism of SOD1-mediated neural injury is
currently
unknown, apoptosis, excitotoxicity, and oxidative stress are thought to play
major roles in
pathogenesis. Sporadic ALS shares clinical features with familial ALS.
However, no
SOD1 mutations or polymorphisms have been identified in these patients. Common
pathways of disease pathogenesis may play a role, with different molecular
abnormalities
that lead to similar phenotypes.
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CA 02524551 2012-08-31
Knockout mice for SOD1 exhibit typical progressive muscle atrophy and weakness
with selective damage to motor neurons that closely resembles human ALS. There
appears
to be a causal relationship between mutant SOD1 secretion and neural toxicity
(e.g., the
mutant protein is not secreted). However, infustion of wild-type SOD in an ALS
rat model
significantly delays disease onset (See, e.g., J. Neurosci, 25, 108-117
(2005)).
Additionally, it has been shown that a copper (Cu) chaperone is required for
efficient
loading of Cu into SOD (See, e.g., Nat. Neurosci, 5, 301-307 (2002)). Thus,
the ability to
maintain normal levels of wild-type SOD or to enhance expression or function
of the same
may provide a beneficial therapeutic effect for ALS subjects.
Furthermore, it has been shown that regressive numbers of basal forebrain
cholinergic neurons appear in several areas of the brain of ALS subjects (See,
e.g.,
Neurochem Int. 46, 357-368, (2005)). Thus, the ability to upregulate genes
involved in
basal forebrain cholinergic neuron growth and/or maintenance may provide
beneficial
effects for a subject with ALS.
Although an inflammatory process may be present, new evidence points toward
multiple mechanisms that promote neuronal cell death in the CNS as the
underlying basis
for ALS. The recent demonstration of superoxide dismutase 1 (SOD1) mutations
in human
familial ALS and in murine ALS models supports the view that oxidative stress,
mitochondrial dysfunction, and excitotoxicity pathways may be involved in the
process of
neuronal cell death.
ALS begins insidiously as weakness, atrophy, or fasciculations in 1 or more
limbs.
The manifestations are usually distal but gradually progress to involve the
more proximal
muscles. Fasciculations and atrophy of the tongue may be noted. Respiratory
insufficiency
is usually a late event. Physical examination reveals weakness and atrophy of
the intrinsic
hand muscles, hyperreflexia with extensor plantar responses, and clonus. Thigh
fasciculations are common. Hyperreflexia can be variable and in some cases may
be
absent. Sensory involvement, if any, is minimal. Patients may present with an
inability to
write due to weakness. Gait function may be preserved.
Needle EMG and nerve conduction studies are the tests of choice for confirming
the
diagnosis of ALS. The confirmation of ALS is facilitated by demonstrating
diffuse
denervation signs, decreased amplitude of compound muscle action potentials,
and normal
conduction velocities. However, for a more detailed confirmation of ALS, more
strict
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CA 02524551 2012-08-31
electrophysiologic criteria have been developed by a subcommittee of the World
Federation
of Neurology and are referred to as the "El Escorial" criteria for motor
neuron disease.
Riluzole is the only medication that has shown treatment efficacy for ALS.
Riluzole is thought to counteract the excitatory amino acid (glutaminergic)
pathways, but
its exact mechanism of action in ALS is unknown. That it prolongs tracheostomy-
free
survival compared to placebo has been shown in 2 randomized trials. No
statistically
significant difference in mortality rates was revealed at the end of these
studies, however.
In other clinical trials, creatine, human recombinant IGF-1, and ciliary
neurotrophic factor
(CNTF) also have shown promise.
Antispastic agents are also used to relieve spastic ity and muscle spasms in
patients
with symptoms of limb stiffness. Examples include Baclofen (Lioresal)
andTizanidine
(Zanaflex). In some embodiments, Sel-Plex is used in combination with the
above
described agents.
Accordingly, in some embodiments, compositions and methods of the present
invention are used in combination with other known therapeutic treatments
(e.g., those
described above) for the treatment ALS. Although an understanding of the
mechanism is
not necessary to practice the present invention and the present invention is
not limited to
any particular mechanism of action, administering a composition comprising
selenium to a
subject suspected of having ALS enhances the expression of genes (e.g., SOD
genes,Lhx8,
TGF[3-2 or other genes described herein) in the subject thereby treating ALS.
In some
embodiments, the present invention provides a method of enhancing the
expression of SOD
genes (e.g., SOD1 and SOD2) in a subject comprising providing to the subject a
composition comprising selenium (e.g., Sel-Plex ) under conditions such that
the
expression of SOD genes are enhanced. In preferred embodiments, the
composition
comprising selenium comprises Sel-Plex (See, Example 4). The composition
comprising
Sel-Plex may also comprise other forms of selenium, for example, Sod-sel,
thereby
enhancing the downregulation of expression of SOD genes.
C. Parkinsons' Disease
In some embodiments, compositions and methods of the present invention (e.g.,
Sel-Plex ) are utilized for the prophylactic and therapeutic treatment of
Parkinson's
disease. Parkinson's disease (hereinafter, "PD") is a progressive
neurodegenerative
disorder associated with a loss of dopaminergic nigrostriatal neurons. PD is
recognized as
CA 02524551 2012-08-31
a common neurological disorder, affecting approximately 1% of individuals
older than 60
years. Clinical features include resting tremor, rigidity, bradykinesia, and
postural
instability. The symptoms of PD are caused by selective and progressive
degeneration of
pigmented dopaminergic (DA) neurons in the substantia nigra pars compacta.
Neuropathologic findings in PD include a loss of pigmented dopaminergic
neurons
in the substantia nigra and the presence of Lewy bodies. The loss of
dopaminergic neurons
occurs most prominently in the ventral lateral substantia nigra. Approximately
60-80% of
dopaminergic neurons are lost before clinical symptoms of PD emerge. Lewy
bodies are
concentric, eosinophilic, cytoplasmic inclusions with peripheral halos and
dense cores. The
presence of Lewy bodies within pigmented neurons of the substantia nigra is
characteristic,
but not pathognomonic, of idiopathic PD. Lewy bodies also are found in the
cortex, nucleus
basalis, locus ceruleus, intermediolateral column of the spinal cord, and
other areas. Lewy
bodies are not specific to PD, as they are found in some cases of atypical
parkinsonism,
Hallervorden-Spatz disease, and other disorders. Incidental Lewy bodies are
found at
postmortem in patients without clinical signs of parkinsonism. The prevalence
of
incidental Lewy bodies increases with age. Incidental Lewy bodies have been
hypothesized
to represent the presymptomatic phase of PD. Alpha-synuclein is a structural
component of
Lewy bodies. Lewy bodies stain for alpha-synuclein and most also stain for
ubiquitin.
The basal ganglia motor circuit modulates cortical output necessary for normal
movement. Signals from the cerebral cortex are processed through the basal
ganglia-
thalamocortical motor circuit and return to the same area via a feedback
pathway. Output
from the motor circuit is directed through the internal segment of the globus
pallidus (GPi)
and the substantia nigra pars reticulata (SNr). This inhibitory output is
directed to the
thalamocortical pathway and suppresses movement.
Two pathways exist within the basal ganglia circuit; they are referred to as
the direct
and indirect pathways. In the direct pathway, outflow from the striatum
directly inhibits
GPi and SNr. The indirect pathway comprises inhibitory connections between the
striatum
and the external segment of the globus pallidus (GPe) and the GPe and the
subthalamic
nucleus (STN). The subthalamic nucleus exerts an excitatory influence on the
GPi and SNr.
The GPi/SNr sends inhibitory output to the ventral lateral (VL) nucleus of the
thalamus.
Striatal neurons containing D1 receptors constitute the direct pathway and
project to the
GPi/SNr. Striatal neurons containing D2 receptors are part of the indirect
pathway and
project to the GPe.
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CA 02524551 2012-08-31
Dopamine is released from nigrostriatal (SNc) neurons to activate the direct
pathway and inhibit the indirect pathway. In PD, decreased striatal dopamine
causes
increased inhibitory output from the GPi/SNr. This increased inhibition of the
thalamocortical pathway suppresses movement. Via the direct pathway, decreased
striatal
dopamine stimulation causes decreased inhibition of the GPi/SNr. Via the
indirect pathway,
decreased dopamine inhibition causes increased inhibition of the GPe,
resulting in
disinhibition of the STN. Increased STN output increases GPi/SNr inhibitory
output to the
thalamus.
Rare hereditary forms of PD have provided insight into the molecular pathways
of
this disorder (See, e.g., Hardy et al., Lancet Neurol. 2, 221-228 (2003)).
Mutations in at
least four genes have been linked to PD, including a-synuclein (PARK]), parkin
(PARK2),
DJ-1 (PARK7), and PTEN (phosphatase and tensin homolog deleted on chromosome
10)-
induced kinase 1 (PINK] , also known as PARK6)( See, e.g., Polymeropoulos, et
al,
Science 276, 2045-2047 (1997); Kitada et al.,Nature 392, 605-608 (1998);
Bonifati et al.,
Science 299, 256-259 (2003); Valente et al., Science 304, 1158-1160 (2004)).
Parkin is an
E3 ligase, catalyzing the addition of ubiquitin to specific substrates that
targets them for
degradation by the ubiquitin-proteasome system (UPS). Parkin is a target of
oxidative and
nitrosative stress in sporadic PD. Cysteine residues in the RING domains are
sensitive to
nitrosative and oxidative modifications, which alter protein function. New
findings indicate
that parkin's E3 ligase activity is modified by NO, thus linking environmental
stress to a
molecular abnormality and a clinical phenotype similar to that seen in
hereditary forms of
PD (See, e.g., Chung et al, Science 304, 1328-1331 (2004)).
In some embodiments, compositions of the present invention (e.g., Sel-PlexS)
are
administered with other therapeutic interventions for treating PD. The present
invention is
not limited to particular therapeutic interventions useful in treating PD. In
some
embodiments, compositions of the present invention are administered along with
surgical
intervention in the treatment of PD. Surgical interventions useful in the
treatment of PD
include, but are not limited to, stereotactic surgery (e.g., thalamotomy,
thalamic deep brain
stimulation, pallidotomy, pallidal stimulation, subthalamotomy, subthalamic
stimulation,
and neuronal transplantation). In some embodiments, compositions of the
present invention
are administered along with dopamine prodrugs in the treatment of PD. Dopamine
prodrugs useful in treating PD include, but are not limited to, levadopa/PDI
and
levodopa/carbidopa (e.g., Sinemet, Sinemet CR). Current treatments, such as
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CA 02524551 2012-08-31
administration of L-DOPA to produce dopamine, are only symptomatic and do not
stop or
delay the progressive loss of neurons. In fact, some studies have suggested
that oxidative
injury via dopamine may lead to further neuronal damage (See, e.g., Xu et al.,
Nat. Med. 8,
600-606 (2002)). Thus, in some embodiments, compositions of the present
invention are
administered with a PD therapeutic agent (e.g., L-DOPA) and an anti-oxidant.
Anti-
oxidants suitable for co-administration are described herein.
In some embodiments, compositions comprising selenium of the present invention
are administered along with dopamine agonists in the treatment of PD. Dopamine
agonsts
useful in treating PD include, but are not limited to, apomorphine (e.g.,
Apokyn),
bromocriptine (e.g., Parlodel), pergolide (e.g., Permax), pramipexole (e.g.,
Mirapex), and
ropinirole (e.g., Requip). In some embodiments, the compounds of the present
invention
are administered with catechol-O-methyltransferase (COMT) inhibitors in the
treatment of
PD. COMT inhibitors useful in the treatment of PD include, but are not limited
to,
tolcapone (e.g., Tasmar), and entacapone (e.g., Comtan). In some embodiments,
the
compounds of the present invention are administered along with
anticholinergics in the
treatment of PD. Anticholinergics useful in the treatment of PD include, but
are not limited
to, trihexyphenidyl (e.g., Artane, Trihexy), and benztropine mesylate (e.g.,
Cogentin). In
some embodiments, the compounds of the present invention are administered
along with
MAO-B inhibitors in the treatment of PD. MAO-B inhibitors useful in the
treatment of PD
include, but are not limited to, selegiline (e.g., Eldepryl). In some
embodiments, the
compounds of the present invention are administered along with amantadine
(e.g.,
Symmetrel) in the treatment of PD.
D. Huntington's Disease
In some embodiments, compositions and methods of the present invention (e.g.,
Sel-Plex ) are utilized for the prophylactic and therapeutic treatment of
Huntington's
Disease. Huntington disease (hereinafter, "HD") is an autosomal dominant
inherited
neurodegenerative disorder affecting 1 in 10,000 individuals. It is caused by
an insertion of
multiple CAG repeats in the huntingtin gene. This results in an N-terminal
polyglutamine
(polyQ) expansion of the large protein huntingtin (Htt), similar to other
polyQ-related
neurodegenerative disorders. Disease severity depends on the length of the
polyQ stretch,
with repeats greater than 40 clearly linked to HD. The polyQ expansion is
thought to
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CA 02524551 2012-08-31
confer a toxic gain of function with selective loss of neurons in the striatum
and cerebral
cortex.
Characteristic features of HD include involuntary movements, dementia, and
behavioral changes. Neuropathology in HD occurs within the neostriatum, in
which gross
atrophy of the caudate nucleus and putamen is accompanied by selective
neuronal loss and
astrogliosis. Marked neuronal loss also is seen in deep layers of the cerebral
cortex. Other
regions, including the globus pallidus, thalamus, subthalamic nucleus,
substantia nigra, and
cerebellum, show varying degrees of atrophy depending on the pathologic grade.
The function of huntingtin is not known. Normally, it is located in the
cytoplasm.
The association of huntingtin with the cytoplasmic surface of a variety of
organelles,
including transport vesicles, synaptic vesicles, microtubules, and
mitochondria, raises the
possibility of the occurrence of normal cellular interactions that might be
relevant to
neurodegeneration. N-terminal fragments of mutant huntingtin accumulate and
form
inclusions in the cell nucleus in the brains of patients with HD, as well as
in various animal
and cell models of HD.
Patients with HD have a mixed pattern of neurological and psychiatric
abnormalities. Chorea, a state of excessive, spontaneous movements,
irregularly timed,
randomly distributed, and abrupt, is a characteristic feature of HD. Severity
of chorea may
vary from restlessness with mild intermittent exaggeration of gesture and
expression,
fidgeting movements of the hands, and unstable dancelike gait to a continuous
flow of
disabling violent movements. Additional clinical features of HD include, for
example,
bradykinesia, akinesia, dystonia, eye movement abnormalities, dementia,
depression, and
other psychiatric manifestations.
Calpains are proteases that have a key role in Huntington proteolysis and
disease
pathology. Calpain family members, including Calpain-5, have increased levels
or are
activated in HD tissue culture and transgenic mouse models (See, e.g., J Biol
Chem, 279,
20211-20220 (2004)).
Compositions and methods of the present invention were analyzed to determine
if
they were capable of altering the expression levels of calpain genes.
Compositions
comprising various forms of selenium (e.g., SeM, Sod-sel, and Sel-PlexS) were
administered to subjects and the expression levels of calpain genes monitored.
The
expression level of calpain-5 was altered by treatment in the following ways:
free
selenomethionine (SM) +1.32*, Sod-sel ¨1.07, Sel-Plex ¨1.44*.
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CA 02524551 2012-08-31
In some embodiments, compositions comprising selenium of the present invention
(e.g., Sel-Plex ) are used for treating HD. In preferred embodiments, the
present invention
provides a method of treating a subject with HD comprising administering to
the subject a
composition comprising selenium under conditions such that the expression of a
calpain
gene is reduced. In some embodiments, the calpain gene is calpain-5. In some
embodiments, compositions of the present invention are administered with other
therapeutic agents for treating HD. The present invention is not limited to
particular
therapeutic agents useful in treating HD. In some embodiments, the
compositions
comprising selenium are administered with anticonvulsant medication in the
treatment of
HD. Anticonvulsant medication useful in treating HD include, but are not
limited to,
valproic acid (e.g., Depakote, Depakene, and Depacon) and benzodiazepines such
as
clonazepam (e.g., Klonopin). In some embodiments, the compositions comprising
selenium are administered with antipsychotic medication in the treatment of
HD.
Antipsychotic medication useful in treating HD include, but are not limited
to, risperidone
(e.g., Risperdal), and haloperidol (e.g., Haldol In some embodiments, the
compositions
comprising selenium are administered with rauwolfia alkaloids in the treatment
of HD.
Rauwolfia alkoids useful in treating HD include, but are not limited to,
resperine. In some
embodiments, the compositions comprising selenium are administered with
antidepressants
in the treatment of HD. Antidepressants useful in treating HD include, but are
not limited
to, paroxetine (e.g., Paxil).
E. Multiple sclerosis
In some embodiments, compositions and methods of the present invention are
utilized in the treatment of multiple sclerosis. Multiple sclerosis (MS) is an
inflammatory,
demyelinating disease of the central nervous system (CNS). MS lesions,
characterized by
perivascular infiltration of monocytes and lymphocytes, appear as indurated
areas in
pathologic specimens; hence, the term "sclerosis in plaques."
MS is a dynamic disease, with almost constant lesion formation and a
progressive
clinical course leading to physical disability. For every 8-10 new lesions
detected on
magnetic resonance imaging (MRI), only one clinical manifestation typically
can be
demonstrated. Patients with relapsing remitting MS have an average of 20 new
lesions per
year and one or two clinical exacerbations.
CA 02524551 2012-08-31
With the advent of MRI, the ability to confirm the diagnosis of MS has
improved
dramatically. MiRI characteristically shows lesions of high T2 signal
intensity of variable
location in the white matter of the brain, brain stem, optic nerves, or spinal
cord. In typical
cases, the lesions tend to occur in periventricular areas and may occur in the
corpus
callosum. Newer MRI techniques (e.g., magnetization transfer, fluid attenuated
inversion
recovery (FLAIR), MR spectroscopy (MRS)) promise to yield important
information
regarding MS heterogeneity, prognosis, and treatment effects.
Despite intensive efforts at finding the source of the disease, no etiologic
agent for
MS has been identified. The disease presumably can be exacerbated by hormonal
changes
during the postpartum period. Some argue that MS could be a heterogeneous
disorder
triggered by several different environmental agents. In fact, only 1 of every
4 MS attacks is
associated with a viral infection.
The disease can present in different forms, such as primary progressive,
relapsing
remitting, relapsing progressive, and secondary progressive phenotypes.
Genetic
susceptibility factors may play a role, as the disease is more common in
Caucasian
populations living in northern latitudes. This susceptibility may be part of a
complex and
heterogeneous group of factors that have an impact, along with environmental
factors, on
the initiation and maintenance of disease. In addition, migration to high-risk
areas before
age 15 years is known to increase the risk of developing MS, lending further
support to the
environmental factor hypothesis.
MS is characterized by perivenular infiltration of lymphocytes and macrophages
in
the parenchyma of the brain, brain stem, optic nerves, and spinal cord.
Expression of
adhesion molecules on the surface seems to underlie the ability of these
inflammatory cells
to penetrate the blood-brain barrier. The elevated immunoglobulin G (IgG)
level in the
cerebrospinal fluid (CSF), which can be demonstrated by an oligoclonal band
pattern on
electrophoresis, suggests an important humoral (ie, B cell activation)
component to MS. In
fact, variable degrees of antibody-producing plasma cell infiltration have
been
demonstrated in MS lesions (see Image 1). Molecular studies of the white
matter plaque
tissue have shown that interleulcin (IL)-12, a potent pro-inflammatory
substance, is
expressed at high levels in early formed lesions.
In the US, MS has a prevalence of nearly 350,000 cases in the United States
alone.
Every year, approximately 10,000 persons are newly diagnosed with MS. More
than 1
million worldwide are affected. MS causes considerable disability in the
working age
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CA 02524551 2012-08-31
group. People with MS usually die of complications rather than of MS itself,
including
recurrent infections (especially in bedridden patients). Patients with MS have
an average
life expectancy 7 years shorter than that of the general population.
MS presents more often in populations of northern European ancestry. Whether
disease severity also may be accounted for by racial differences is
controversial. The
concordance rate for MS is 20-40% among monozygotic twins, suggesting the
presence of
predisposing genetic factors of non-Mendelian inheritance.
MS affects females more than males (1.6-2:1), but the basis for this
difference is
unknown. This ratio is even higher (3:1) among patients in whom onset of MS is
before
age 15 years or after age 50 years, suggesting a hormonal component to the
disease process.
Males have a greater tendency to develop primary progressive MS, while females
tend to
experience more relapses. MS most commonly afflicts people between the ages of
18 and
50 years, but any age group can be affected.
C4d-immunoreacive complement-activated oligodendrocytes (C4d-CA0s)have
been described in MS (See, e.g., Schwab and McGeer, Experimental Neurology,
174, 81-88
(2002)). C4d-CAOs were reported to delineate miniature MS plaques of 300 ¨ 500
pm. In
large MS lesions, immunoreactive fibers corresponding to the Clq ¨ C9
components of the
complement cascade were identified indicating that complete activation of the
complement
cascade is present with MS lesions. Association of C4d-CAOs with areas of
demyelination
demonstrated a direct attack on oligodendroglial cells by the early complement
components
as an initiating event in MS. Furthermore, incomplete complement activation
idicated that
this step may be reversible (See, e.g., Schwab and McGeer, Experimental
Neurology, 174,
81-88 (2002)).
Drug therapy seeks to delay progression to disability, reduce relapse rate,
increase
the number of relapse-free patients, and increase the time to first relapse as
well as decrease
MRI lesion burden, atrophy, and "Tl holes," or presence of new lesions.
Accordingly, in some embodiments, compositions comprising selenium of the
present
invention (e.g., Sel-PlexC) are used for treating and/or preventing MS. In
preferred
embodiments, the present invention provides a method of treating a subject
with MS
comprising administering to the subject a composition comprising selenium
under
conditions such that the expression of a complement gene is reduced. In some
embodiments, the complement gene is comprises Clq, Clq alpha, Clq beta, Clq
gamma
and/or Clqr. In some embodiments, compositions of the present invention are
administered
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CA 02524551 2012-08-31
with other therapeutic agents for treating MS. The present invention is not
limited to
particular therapeutic agents useful in treating MS. In some embodiments, the
compositions comprising selenium are administered with immunomodulators (e.g.,
Interferon beta-1a (Avonex), Interferon beta-1a (Rebif), Interferon beta-lb
(Betaseron),
Glatiramer acetate (Copaxone), and Natalizumab (Tysabri)), which delay
progression to
disability and reduce the number of new MS lesions by MRI; corticosteroids
(e.g.,
methylprednisolone), which are used to reduce acute inflammation and expedite
recovery
from acute exacerbations of MS; and immunosuppressors (e.g., Mitoxantrone
(Novantrone), Cyclophosphamide (Cytoxan, Neosar), Azathioprine (IMURAN),
Methotrexate (Rheumatrex), which are used to suppress immune reactions.
Additional
drugs may be used to treat common secondary conditions such as depression,
spasticity,
tonic spasms, fatigue, urinary dysfunction, and erectile dysfunction. In some
embodiments,
compositions comprising selenium are used in combination with the above
described
agents.
Cognitive function
The central nervous system consists of the brain and the spinal cord. All
other
nerves in the body comprise the peripheral nervous system. Efferent nerves
carry messages
from the central nervous system to all parts of the body (the periphery).
Afferent nerves
carry information such as pain intensity from the periphery to the central
nervous system.
There are two types of efferent nerves: somatic, which go to skeletal muscles,
and
autonomic, which go to smooth muscles, glands and the heart. Messages in the
form of
electrical activity are conducted along the nerve fibers or axons. Between the
terminus of
the axon and the muscle or gland that the nerve controls (innervates), there
is a gap called
the synapse or synaptic cleft. When the conducted electrical impulse (action
potential)
reaches the nerve terminus, it provokes the release of chemicals called
neurotransmitters.
These chemicals diffuse across the synaptic cleft and react with a specialized
structure
(receptor) on the postjunctional membrane. The receptor is then said to be
activated or
excited, and its activation triggers a series of chemical events resulting
ultimately in a
biological response such as muscle contraction. The processes involving
neurotransmitter
release, diffusion and receptor activation are referred to collectively as
transmission. There
are many types of transmission, and they are named for the specific
neurotransmitter
involved. Thus, cholinergic transmission involves the release of the
neurotransmitter,
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CA 02524551 2012-08-31
acetylcholine, and its activation of the postsynaptic receptor. Things that
bind to and
activate receptors are called agonists. Thus, acetylcholine is the endogenous
agonist for all
cholinergic receptors.
After leaving the central nervous system, somatic nerves to skeletal muscles
have
only one synapse, namely, that between the nerve terminus and the muscle it
innervates.
The neurotransmitter at that synapse is acetylcholine. Thus, this myo-(for
muscle)-neural
junction is one site of cholinergic transmission. The postjunctional receptor
is called the
motor end plate. Autonomic nerves, in contrast to somatic nerves, have an
additional
synapse between the central nervous system and the innervated structure (end
organ).
These synapses are in structures called ganglia, and these are nerve-to-nerve
junctions
instead of nerve-to-end organ junctions. Like somatic nerves, however,
autonomic nerves
also have a final nerve-to-end organ synapse. The neurotransmitter in
autonomic ganglia is
also acetylcholine; hence, this represents another site of cholinergic
transmission. The
motor end plate and the ganglionic receptors can also be activated by
exogenously added
nicotine. Thus, nicotine is an agonist for this particular subfamily of
cholinergic receptors
which are called nicotinic, cholinergic receptors.
There are two anatomically and functionally distinct divisions of the
autonomic
nervous system: the sympathetic division and the parasympathetic division. The
preganglionic fibers of the two divisions are functionally identical, and they
innervate
nicotinic, cholinergic receptors in ganglia to initiate action potentials in
the postganglionic
fibers. Thus, all ganglia are created pretty much equal. Only the
postganglionic fibers of
the parasympathetic division, however, are cholinergic. The postganglionic
fibers of the
sympathetic division generally, but not always, secrete norepinephrine. The
cholinergic
receptors innervated by the postganglionic fibers of the parasympathetic
division of the
autonomic nervous system can also be activated by exogenously added muscarine,
an
agonist found in small amounts in the poisonous mushroom, Amanita muscaria.
These
constitute a second subset of cholinergic receptors which are called
muscarinic, cholinergic
receptors.
Although the receptors in ganglia and the motor end plate both respond to
nicotine,
they actually constitute two distinct subgroups of nicotinic receptors. Each
of the three
families of cholinergic receptors can be blocked by specific receptor
antagonists to prevent
their activation by endogenous acetylcholine or added agonists. Thus, specific
blockers are
known for cholinergic, muscarinic receptors innervated by postganglionic
fibers of the
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CA 02524551 2012-08-31
parasympathetic division of the autonomic nervous system, for cholinergic,
nicotinic
receptors in both sympathetic and parasympathetic ganglia, and for cholinergic
nicotinic
receptors at the myoneural junction (motor end plates) of the somatic nervous
system.
When these receptors are blocked, the on-going biological activity associated
with their
normal and continuous activation is lost. For example, blockade of the motor
end plate
leads to generalized, flaccid paralysis.
There are some anomalous fibers in the sympathetic division of the autonomic
nervous system. For example, the sympathetic postganglionic nerves that go to
sweat
glands are cholinergic instead of adrenergic, like most other sympathetic
fibers, and they
innervate mucarinic receptors. The sympathetic nerve to the adrenal gland
innervates a
receptor that is nicotinic like all autonomic ganglia, but there is no
postganglionic fiber.
The gland itself is analogous to a postganglionic sympathetic fiber, but,
instead of secreting
a neurotransmitter, it secretes epinephrine and norepinephrine into the blood
stream, where
they function as hormones. These hormones activate adrenergic receptors
throughout the
body. Nicotinic and muscarinic receptors in the central nervous system are
incompletely
understood.
Cholinergic drugs are medications that produce the same effects as the
parasympathetic nervous system. Cholinergic drugs produce the same effects as
acetylcholine. Acetylcholine is the most common neurohormone of the
parasympathetic
nervous system, the part of the peripheral nervous system responsible for the
every day
work of the body. While the sympathetic nervous system acts during times of
excitation,
the parasympathetic system deals with everyday activities such as salivation,
digestion, and
muscle relaxation.
The cholinergic drugs may be used in several ways. The cholinergic muscle
stimulants are used to diagnose and treat myathenia gravis, a disease that
causes severe
muscle weakness. This class of drugs includes ambenonium chloride (Mytelase),
edrophonium chloride (Tensilon), neostigmine (Prostigmine), and piridogstimina
(Mestin0n). These drugs are also widely used in surgery, both to reduce the
risk of urinary
retention, and to reverse the effects of the muscle relaxant drugs that are
used in surgery.
Cholinergic drugs are also used in control of glaucoma, a disease that is
caused by
increased pressure inside the eye. The most common drugs used for this purpose
are
demecarium (Humorsol) and echthiophate (Phospholine iodide).
CA 02524551 2012-08-31
Cholinergic drugs usually act in one of two ways. Some directly mimic the
effect of
acetylcholine, while others block the effects of acetylcholinesterase.
Acetylcholinesterase
is an enzyme that destroys naturally occurring acetylcholine. By blocking the
enzyme, the
naturally occurring acetylcholine has a longer action. Cholinergic drugs are
available only
by prescription. They may be available as eye drops, capsules, tablets, or
injections.
Cognitive function has been demonstrated to decline or deteriorate in several
diseases, such as MS, Alzheimer's disease, Parkinson's disease, Huntington's
disease, ALS,
among others.
In MS, for example, the cognitive function most likely to be affected appears
to be
memory. Other cognitive functions frequently affected in subjects with
neurodegenerative
disease include speed of information processing, executive functions (planning
and
prioritizing), visuospatial functions (impairment in visual perception and
constructional
abilities), abstract reasoning and problem-solving, and attention and
concentration-
especially sustained attention and ability to divide attention between
separate tasks. One of
the most vexing cognitive deficits seen in MS is word-fmding difficulty-the
experience of
having a word on the tip of your tongue but not being able to remember it.
The first signs of cognitive dysfunction or decline may be subtle. The person
may
have difficulty in finding the right words to say, or trouble remembering what
to do on the
job or during daily routines at home. Decisions that once were easy now
demonstrate poor
judgment. Often, the family becomes aware of the problem first, noticing
changes in
behavior or personal habits. Cognitive dysfunction can have an impact on role
performance
at home and at work. Cognitive function can also be affected by aging or
medications.
Substantial biologic evidence supports the importance of estrogen to cognitive
function. Estrogen receptors have been identified throughout the brain, and
appear
particularly concentrated in the basal forebrain. The basal forebrain is of
special interest
since it is the major source of cholinergic innervation to the hippocampus.
The cholinergic
system is a neurotransmitter system important for regulation of memory and
learning, while
the hippocampus is the primary region of the brain mediating cognitive
function. In
experiments using animal models and cell lines, several mechanisms have been
identified
whereby estrogen may influence cognitive function.
Basal forebrain cholinergic neurons (BFCNs) are involved in cognitive
functions
such as learning and memory and are affected in several neurodegenerative
diseases, such
as Alzheimer's disease (AD). The LIM homeobox protein 8 gene (Lhx8), is
important for
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CA 02524551 2012-08-31
the proper development and maintenance of BFCNs (See, e.g., Mori et al., Eur.
J.
Neurosci., 19, 3129 (2004)).
Mice with a null mutation in the Lhx8 gene are deficient in the development of
forebrain cholinergic neurons (Zhao et al.õ Proc. Nat. Acad. Sci 100: 9005
(2003)). Lhx8
mutants lacked the nucleus basalis, a major source of the cholinergic input to
the cerebral
cortex.
Using compositions and methods of the present invention, the observed
expression
level of Lhx8 was not significantly different between Se-deficient subjects
and that of
subjects receiving certain forms of selenium (e.g., SeM or Sod-sel). However,
when
subjects were administered (e.g., received a dietary supplement) a composition
comprising
Sel-Plex , the expression of Lhx8 was upregulated 12.9-fold (p<0.01) (See
Example 5).
Thus, in some preferred embodiments, the present invention provides a method
of
maintaining and/or stabilizing neurologic function (e.g., cholinergic neuron
growth and
function associated with cognitive function) in a subject comprising
administering to the
subject a composition comprising Sel-Plex . In preferred embodiments, Sel-Plex
is
administered to the subject under conditions such that the expression of Lhx8
is enhanced.
In some preferred embodiments, compositions and methods of the present
invention are
used as a prophylactic treatment in order to prevent the loss of cognitive
function.
Although an understanding of the mechanism is not necessary to practice the
present
invention and the present invention is not limited to any particular mechanism
of action,
preventing loss of cognitive function may occur due to promoting development
of basal
forebrain cholinergic neurons or simply the maintenance (e.g., lack of
apoptosis) of basal
forebrain cholinergic neurons. In some embodiments, a composition comprising
Sel-Plex
is administered to a subject suspected of having myasthenia gravis for the
treatment of
myathenia gravis. In some embodiments, a composition comprising selenium
(e.g., Sel-
Plex ) is co-administered in combination with other known therapeutic
treatments (e.g.,
those described above) for the treatment of myasthenia gravis. In still other
embodiments,
the present invention provides a method of prophylactic and/or therapeutic
treatment for
myathenia gravis comprising co-administering to a subject a composition
comprising
selenium (e.g., Sel-Plex ) and a cholinergic muscle stimulant.
The product of another gene, transforming growth factor beta 2 (TGF-132), is
known
to increase neuronal proliferation in the developing cerebellum (See, e.g.,
Elvers et al.,
Mechanisms of Development, 122, 587 (2004)). Furthermore, it has been shown
that TGF-
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CA 02524551 2012-08-31
(32 is a growth and survival factor for granule cell precursors in the
cerebellum and that
antibody-mediated neutralization of endogenous TGF-I32 represses proliferation
of
cerebellar granule cell precursors and induces neurodegeneration. It has also
been
demonstrated that knocking out (e.g., deleting) TGF-132 is a lethal phenotype
with TGF-I32
deficient mice developing a range of defects and dying before development of
the
cerebellum occurs (See, e.g., Sanford et al., Development, 124, 2659 (1997)).
The expression level of TGF-132 was not altered, compared to controls, in
subjects
administered certain forms of selenium (e.g., SeM or Sod-Sel). However, when
subjects
were administered (e.g., received a dietary supplement) a composition
comprising Sel-
Plex , the expression TGF-13 was upregulated 2.4-fold (See Example 5). Thus,
in some
embodiments, the present invention provides a method of increasing cerebellum
function in
a subject comprising administering to the subject a composition comprising Sel-
Plex .
Although an understanding of the mechanism is not necessary to practice the
present
invention and the present invention is not limited to any particular mechanism
of action,
administering a composition comprising selenium (e.g., a daily dietary
supplement
comprising Sel-Plex ) to a subject increases neuronal activity (e.g.,
increases neuronal
proliferation) and/or inhibits neurodegeneration. In some preferred
embodiments, the
present invention provides a method of maintaining and/or stabilizing
neurologic function
(e.g., cholinergic neuron growth and function) in a subject comprising
administering to the
subject a composition comprising Sel-Plex under conditions such that the
expression of
TGF-I3 is enhanced. In some preferred embodiments, compositions comprising Sel-
Plex
are used as a prophylactic treatment in order to prevent the loss of cognitive
function (e.g.,
by upregulation of genes such as Lhx8 and TGF-(3 that enhance neurologic
function).
III. Retardation of age-associated gene expression
Aging of the brain leads to impairments in cognitive function and motor
skills, and
is a major risk factor for several common neurological disorders such as
Alzheimer disease
(AD) and Parkinson disease (PD). Recent studies suggest that normal brain
aging is
associated with subtle morphological and functional alterations in specific
neuronal circuits,
as opposed to large-scale neuronal loss (See, e.g., Morrison and Hof, Science
278, 412-419
(1997)). In fact, aging of the central nervous system in diverse mammalian
species shares
many features, such as atrophy of pyramidal neurons, synaptic atrophy,
decrease of striatal
dopamine receptors, accumulation of fluorescent pigments, cytoskeletal
abnormalities, and
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CA 02524551 2012-08-31
reactive astrocytes and microglia (See, e.g., Finch and Roth, in Basic
Neurochemistry (eds
Seigel, G., Agranoff, J.B., Albers, W.R.W., Fisher, S.K. & Uhler, M.D.) 613-
633
(Lippincott-Raven, Philadelphia, 1999).
Postulated mechanisms of CNS aging include instability of nuclear and
mitochondrial genomes (See, e.g., Gaubatz, in Molecular Basis of Aging (ed.
Macieira-
Coelho, A.) 71-182 (CRC Press, Boca Raton, 1995)), neuroendocrine dysfunction
(See,
e.g., McEwen, Front. Neuroendocrinol. 20, 49-70 (1998)), production of
reactive oxygen
species (See, e.g., Sohal and Weindruch, Science 273, 59-63 (1996)), altered
calcium
metabolism (See, e.g., Disterhoft et al., Hypothesis of Aging and Dementia
(New York
Academy of Sciences Press, New York, 1994), and inflammation-mediated neuronal
damage (See, e.g., Blumenthal, J. Gerontol. Biol. Sci. Med. Sci. 52, Bl¨B9
(1997)).
Caloric restriction, the only intervention shown to slow the intrinsic rate of
aging in
mammals (See, e.g., Weindruch and Walford, The Retardation of Aging and
Disease by
Dietary Restriction (C.C. Thomas, Springfield, Illinois, 1988), retards age-
related declines
in psychomotor and spatial memory tasks (See, e.g., Ingram et al., J.
Gerontol. 42, 78-81
(1987)), reduces the age-associated loss of dendritic spines (See, e.g., Moroi-
Fetters et al.,
Neurobiol. Aging 10, 317-322 (1989)) and reduces neuronal degeneration in
models of PD
(See, e.g., Duan and Mattson, J. Neurosci. Res. 57, 195-206 (1999)).
Brain aging has been characterized at the molecular level (e.g., through gene-
expression profiling of the aging neocortex and cerebellum in mice (See, e.g.,
Lee et al.,
Nature Genetics, 25, 294-297 (2000)). Aged mice display expression of genes
indicative of
an inflammatory response, oxidative stress and reduced neurotrophic support.
At the
transcriptional level, brain aging in mice displays parallels with human
neurodegenerative
disorders (See, e.g., Lee et al., Nature Genetics, 25, 294-297 (2000)).
In aged mice, a concerted induction of the complement cascade genes C4, Clqa,
Clqb and Clqc, was observed (See, e.g., Lee et al., Nature Genetics, 25, 294-
297 (2000)).
As described elsewhere herein, these genes are part of the humoral immune
system
involved in inflammation and cytolysis. Production of complement proteins in
the brain,
which leads to the generation of proinflammatory peptide fragments,
contributes to
neuronal damage associated with stroke (See, e.g., Huang et al., Science 285,
595-599
(1999)) and has been observed in the striatum of aged rats (See, e.g.,
Pasinetti et al.,
Synapse 31, 278-284 (1999)).
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CA 02524551 2012-08-31
Additionally, a coordinated induction of the genes encoding cathepsins D, S,
and Z
were observed in aged mice (See, e.g., Lee et al., Nature Genetics, 25, 294-
297 (2000)).
Cathepsins are major components of the lysosomal proteolytic system.
Cathepsins have
been implicated in the processing of amyloid precursor protein (APP) to
amyloid 13-
peptides and are induced in the brains of Alzheimer's disease patients (See,
e.g. Lemere et
al., Am. J. Pathol. 146, 848-860 (1995)).
Aging is well known to be associated with increased oxidant generation (See,
e.g.,
Peinado et al., Anat Rec, 247, 420 (1997)). For example, highly reactive
oxygen species
(ROS) promote a wide spectrum of cell damage, including DNA damage, lipid
peroxidation, alteration of intracellular redox balance and inactivation of
enzymes. A key
host mechanism in the defense against ROS is performed by the family of
Glutathione-S-
Transferases (GSTs) that protect against the by-products of oxidative stress
through a
variety of reactions (See, e.g., Hayes et al., Annu. Rev. Phramacol. Toxicol.,
45, 51,
(2004)).
Accordingly, in some preferred embodiments, the present invention provides a
method of retarding age related expression of complement associated genes
(e.g., Clq, Clq
alpha, Clq beta, Clq gamma, or Clqr) in a subject comprising administering to
the subject
a composition comprising Sel-Plex (e.g., a dietary supplement comprising Sel-
Plexe)
under conditions such that complement associated gene expression is reduced
(See
Example 10). In some embodiments, the present invention provides a
prophylactic or
therapeutic treatment for stroke comprising administering to a subject at risk
of stroke (e.g.,
an elderly person) a composition comprising selenium (e.g., Sel-Plext) under
conditions
such that the expression of complement genes (e.g., Clq, Clq alpha, Clq beta,
Clq gamma,
or C1qr) are reduced. In other preferred embodiments, the present invention
provides a
method of retarding age related expression of cathepsin gene expression (e.g.,
Cathepsin D,
Cathepsin S, or Cathepsin Z) in a subject comprising administering to the
subject a
composition comprising Sel-Plexe (e.g., a dietary supplement comprising Sel-
Plex8)
under conditions such that cathepsin gene expression is reduced (See Example
10). In
some embodiments, the present invention provides a method of treating an
Alzheimer's
disease patient comprising administering to the Alzheimer's disease patient a
composition
comprising selenium (e.g., Sel-Plex8) under conditions such that symptoms of
Alzheimer's
disease in the patient are reduced. Although an understanding of the mechanism
is not
necessary to practice the present invention and the present invention is not
limited to any
CA 02524551 2012-08-31
particular mechanism of action, administering a composition comprising
selenium (e.g.,
Sel-Plex ) to an Alzheimer's subject reduces symptoms associated with
Alzheimer's
through reducing the expression of cathepsin genes (e.g., Cathepsin D,
Cathepsin S, or
Cathepsin Z). In some embodiments, compositions and methods of the present
invention
are used as a prophylactic treatment in order to prevent age-associated gene
expression. In
some embodiments, a composition comprising selenium (e.g., Sel-Plex ) is
administered
to a subject in combination with a calorie restricted diet in order to prevent
aging (e.g.,
attenuate age-associated gene expression). In some preferred embodiments, the
present
invention provides a method of altering neuronal circuit changes (e.g.,
described above)
associated with age comprising administering to a subject a composition
comprising
selenium (e.g., Sel-Plex ) under conditions such that the expression of Lhx8
is enhanced
and/or elevated (See Example 6). Although an understanding of the mechanism is
not
necessary to practice the present invention and the present invention is not
limited to any
particular mechanism of action, enhanced and/or elevated expression of Lhx8
stimulates the
proper development and/or works to maintain levels basal forebrain cholinergic
neurons
(BFCNs).
It has further been shown that there is a significant up-regulation of certain
transcription factors in response to a calorie restricted diet, itself
providing age retardation
(See, e.g., Lee et al., Nature Genetics, 25, 294-297 (2000)). For example,
homeobox (Hox)
transcription factors were upregulated, which are proposed to be involved in
neural
development. Using compositions and methods of the present invention, it was
demonstrated that several Hox transcriptions factors were upregulated in a
subject
administered a composition comprising selenium (e.g., Sel-Plex ), Although an
understanding of the mechanism is not necessary to practice the present
invention and the
present invention is not limited to any particular mechanism of action,
enhanced and/or
elevated expression of Hox factors functions to maintain normal neural
activity in an aging
subject.
IV. The Endocrine System and Diabetes
In some embodiments of the present invention, compositions and methods of the
present invention are utilized in the treatment of diabetes. Diabetes mellitus
is a chronic
disease that requires long-term medical attention both to limit the
development of its
devastating complications and to manage them when they do occur. It is a
66
CA 02524551 2012-08-31
disproportionately expensive disease; patients diagnosed with diabetes
accounted for 6.2%
of the US population in 2002, or 18.2 million people. In that year, the per
capita cost of
healthcare for people with diabetes was $13,243 for people with diabetes and
$2560 for
people without diabetes.
The 2 basic types of diabetes mellitus are type 1 and type 2. Type 1 diabetes
is an
autoitrunune disease characterized by necrosis of pancreatic islet cells and a
complete lack
of insulin secretion. Patients with type 1 diabetes are dependent on insulin.
Complications
are similar to those described below for type 2 diabetes. The only treatment
is insulin
injections.
Type 2 diabetes mellitus was once called adult-onset diabetes. Now, because
the
epidemic of obesity and inactivity in children, type 2 diabetes is occurring
at younger and
younger ages. Although type 2 diabetes typically affects individuals older
than 40 years, it
has been diagnosed in children as young as 2 years of age who have a family
history of
diabetes.
Type 2 diabetes is characterized by peripheral insulin resistance with an
insulin-
secretory defect that varies in severity. For type 2 diabetes to develop, both
defects must
exist: All overweight individuals have insulin resistance, but only those with
an inability to
increase beta-cell production of insulin develop diabetes. In the progression
from normal
glucose tolerance to abnormal glucose tolerance, postprandial glucose levels
first increase.
are considered to require insulin but not to depend on insulin. Moreover,
patients with type
Maturity-onset diabetes of the young (MODY) is a form of type 2 diabetes that
affects many generations in the same family with an onset in individuals
younger than 25
years. Several types exist. Some of the genes responsible can be detected by
using
Gestational diabetes mellitus (GDM) is defined as any degree of glucose
intolerance
with onset or first recognition during pregnancy. GDM is a complication in
approximately
4% of all pregnancies in the United States, though the rates may be 1-14%
depending on
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CA 02524551 2012-08-31
the population studied. Untreated GDM can lead to fetal macrosomia,
hypoglycemia,
hypocalcemia, and hyperbilirubinemia. In addition, mothers with GDM have
increased
rates of cesarean delivery and chronic hypertension. To screen for GDM, a 50-g
glucose
screening test should be done at 24-28 weeks of gestation. This is followed by
a 100-g, 3-
hour oral glucose tolerance test if the patient's plasma glucose concentration
at 1 hour after
screening is greater than >140 mg/dL.
Approximately 13 million people in the United States have a diagnosis of
diabetes,
and diabetes is undiagnosed in another 5 million. Approximately 10% have type
1
diabetes, and the rest have type 2.
The morbidity and mortality associated with diabetes are related to the short-
and
long-term complications. Complications include hypoglycemia and hyperglycemia,
increased risk of infections, microvascular complications (eg, retinopathy,
nephropathy),
neuropathic complications, and macrovascular disease.
Diabetes is the major cause of blindness in adults aged 20-74 years, as well
as the
leading cause of nontraumatic lower-extremity amputation and end-stage renal
disease
(ESRD).
Type 2 diabetes mellitus is more prevalent among Hispanics, Native Americans,
African Americans, and Asians/Pacific Islanders than in non-Hispanic whites.
The
incidence is essentially equal in women and men in all populations. Type 2
diabetes is
becoming increasingly common because people are living longer, and the
prevalence of
diabetes increases with age. It is also seen more frequently now than before
in young
people, in association with the rising prevalence of childhood obesity.
Although type 2
diabetes still occurs most commonly in adults aged 40 years or older, though
the incidence
of disease is increasing more rapidly in adolescents and young adults than in
other age
groups.
Neurogenin 3 (Neurog3) is a key transcription factor in the differentiation of
the
endocrine pancreas. Neurog3 is an important part of the activation pathway for
insulin
gene expression and helps to ameliorate glucose tolerance (See, e.g., Watada,
Endocrine
Journal, 51, 255 (2004)). It is thought that lower than normal levels (e.g.,
under-
expression) of Neurog 3 plays a role in certain types of Diabetes (See, e.g.,
Lee et al.,
Genes Dev. 16: 1488 (2002)).
Fingerstick glucose test is appropriate in the diagnosis for virtually all
patients with
diabetes. In patients who present with symptoms of uncontrolled diabetes (eg,
polyuria,
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CA 02524551 2012-08-31
polydipsia, nocturia, fatigue, weight loss) with a confirmatory random plasma
glucose level
of >200 mg/dL, diabetes can be diagnosed.
In asymptomatic patients whose random serum glucose level suggests diabetes, a
fasting plasma glucose (FPG) concentration should be measured. The oral
glucose
tolerance test no longer is recommended for the routine diagnosis of diabetes.
An FPG
level of >126 mg/dL on 2 separate occasions is diagnostic for diabetes. An FPG
level of
110-125 mg/dL is considered impaired IFG. An FPG level of <110 mg/dL is
considered
normal glucose tolerance, though blood glucose levels above >90 mg/dL may be
associated
with an increased risk for the metabolic syndrome if other features are
present.
Islet-cell autoantibodies are present in early type 1 but not type 2 diabetes.
Measurements
of these autoantibodies within 6 months of diagnosis can help differentiate
type 1 and type
2 diabetes.
Most diabetic patients have type 2 diabetes, and most of those are
asymptomatic at
diagnosis. Initial treatment for these patients is a trial of medical
nutrition therapy (MNT,
diet therapy). Therefore, if an asymptomatic patient is incidentally found to
have an
elevated blood glucose level in the ED, the patient's primary physician can
perform follow-
up. Patients with mild symptoms of poorly controlled and previously
undiagnosed diabetes
can usually be treated as an outpatient, often with the initiation of a low
dose of a
sulfonylurea agent or metformin.
The treatment of markedly symptomatic patients with newly discovered type 2
diabetes and glucose levels >400 mg/dL is controversial. If close follow-up
can be
arranged, maximal doses of a sulfonylurea agent can be started, and they can
be treated as
outpatients. Patients generally feel better in 1-2 days, and in a week, their
blood glucose
levels are markedly lower. Their sulfonylurea dose can be tapered as they
comply with
MNT; in some, diabetes can be controlled with diet alone. Patients who cannot
drink
adequate amounts of fluid, those with serious coexisting medical conditions
(eg, myocardial
infarction (MI), systemic infection), and those without reliable follow-up
should generally
be hospitalized to start therapy.
The goal of oral antidiabetic therapy is to lower blood glucose levels to near-
normal
(preprandial levels of 90-130 mg/dL or 80-140 mg/dL and HbA 1 C levels <7%)
and to
maintain them in this range for the patient's lifetime. Patients with no or
mild symptoms
should initially be treated with MNT (diet therapy), and MNT should be
encouraged
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CA 02524551 2012-08-31
throughout treatment. Drugs are started when a patient presents with moderate-
to-marked
symptoms of diabetes.
Treatment of type 2 diabetes is aimed at lowering insulin resistance and
increasing
function of beta cells. In many patients, beta-cell dysfunction worsens over
time,
necessitating exogenous insulin. Because patients with type 2 diabetes have
both insulin
resistance and beta-cell dysfunction, oral medication to increase insulin
sensitivity (eg,
metformin, a thiazolidinedione (TZD)) is often given with an intermediate-
acting insulin
(eg, neutral protamine Hagedorn (NPH)) at bedtime or a long-acting insulin
(eg, glargine
(Lantus) insulin, insulin detemir (Levemir)) given in the morning or evening.
An insulin
secretagogue, such as a sulfonylurea agent, can also be given to increase
preprandial insulin
secretion.
Drugs include Incretin mimetics (e.g., Exenatide (Byetta)) which mimic glucose-
dependent insulin secretion, suppresses elevated glucagon secretion, and
delays gastric
emptying, Sulfonylurea agents (e.g., chlorpropamide, tolbutamide, tolazamide,
acetohexamide, glyburide, glipizide, and glimepiride) that reduce glucose by
increasing
insulin secretion from pancreatic beta cells in patients with residual beta
cell function,
Meglitinides (e.g., Repaglinide (Prandin)) that are short-acting insulin
secretagogues,
Biguanides (e.g., Metformin (Glucophage)) that increase sensitivity of insulin
by
decreasing hepatic gluconeogenesis (primary effect) and increasing peripheral
insulin
sensitivity (secondary effect), Alpha-glucosidase inhibitors (AGIs) (e.g.,
Acarbose
(Precose), Miglitol (Glyset)) that inhibit action of alpha-glucosidase
(carbohydrate
digestion), delaying and attenuating postprandial blood glucose peaks,
thiazolidinediones
(e.g., Pioglitazone (Actos), Rosiglitazone (Avandia)) that increase peripheral
insulin
sensitivity by increasing transcription of nuclear proteins that help increase
uptake of
glucose, probably with effects on free fatty acid levels, Amylin analogs
(e.g., Pramlintide
acetate (Symlin)) that have endogenous amylin effects by delaying gastric
emptying,
decreasing postprandial glucagon release, and modulate appetite. In some
embodiments,
Sel-Plex is used in combination with the above described agents.
Using compositions and methods of the present invention, it was determined
that
Neurog3 expression was significantly upregulated 1.7-fold in subjects
administered a
composition comprising Sel-Plex , whereas subjects that were administered SeM
or Sod-
sel treatments displayed no significant alteration of Neurog3 expression (See
Example 6).
CA 02524551 2012-08-31
Thus, in some preferred embodiments, the present invention provides a method
of
treating a subject (e.g., a subject with diabetes) comprising administering to
the subject a
composition comprising Sel-Plex under conditions such that the expression of
Neurog3 is
altered (e.g., enhanced) in the subject. Although an understanding of the
mechanism is not
necessary to practice the present invention and the present invention is not
limited to any
particular mechanism of action, administering to a subject with diabetes a
composition
comprising Sel-Plex ameliorates glucose tolerance in the subject via up-
regulating the
expression of Neurog3 expression. In some embodiments, the present invention
provides a
method of treating a subject with diabetes comprising administrating to the
subject a
composition comprising Sel-Plex with one or more other agents (e.g.,
vanadium, or those
described above). In some embodiments, the present invention provides a method
of
enhancing the expression of Neurog3 in a subject comprising providing to the
subject a
composition comprising selenium under conditions such that the expression of
Neurog3 is
enhanced. In preferred embodiments, the composition comprising selenium
comprises Sel-
Plex . The composition comprising Sel-Plex may also comprise other forms of
selenium, for example, Sod-sel.
V. Compositions and formulations comprising selenium
Nutritional selenium levels have been established by the FDA (See 21 C.F.R.
101.9(c)(8)(iv), January 1994). Humans and animals can safely metabolize
limited
amounts of both inorganic and organic forms of selenium and can convert non-
methylated
selenium to mono-ordi-or trimethylated derivatives, of which the
monomethylated
derivatives are most toxic. (See, e.g., Bedwal, R. S., etal., Medical
Hypotheses, 41 (2):150-
159 (August 1993)). The FDA has adopted Reference Daily Intakes (RDIs) of 70
micrograms for selenium. Selenium dosage of 600 micrograms per day has been
reported
as safe. (See, e.g., Ferris G. M. Lloyd, et al., App. Chin. Biochem.,26:83-88
(1989)). At
about this dosage, normal activity of the enzyme glutathione reductase safely
converts
selenogluthatione to hydrogen selenide in the liver and erythrocytes and is
ultimately
excreted. Thus, at such lower dosages, the body is able to safely metabolize
and excrete
selenium that is present in a free metallic form. However, as with many trace
elements
(e.g., selenium), at higher dosage levels or concentrations the beneficial
effects are reversed
and dangerous toxicity is manifested. (See, e.g., Furnsinn, C. et al.,
Internat'l J. of Obesity
and Related Metab. Dis., 19(7):458-463 (1995)).
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CA 02524551 2012-08-31
Therefore, the administration of selenium in the natural form involves a
scientific
and medical trade-off because, when administered in relatively low
concentrations,
selenium provides beneficial health effects, however, at higher
concentrations, selenium
exhibits dramatic toxicity such that the potential health benefits are lost
and toxicity
becomes the primary concern.
As described above, the present invention demonstrates for the first time that
certain
forms of selenium (e.g., Sel-Plex ) are capable of providing beneficial
effects to a subject
that other forms of selenium (e.g., selenomethionine) do not. The present
invention
contemplates the use of multiple forms of selenium. The source of selenium may
be a
synthetic or natural source, and the selenium may be organic or inorganic.
Evidence has
shown that organic forms of selenium (e.g., selenomethionine and selenium
enriched yeast)
may be less toxic and better absorbed than inorganic forms (See, e.g., Mahan,
Proceedings
of the 15th Annual Symposium Nottingham University Press, Nottingham, UK, pp.
523-
535 (1999)). As described herein, and depending on the target sought to be
treated in a
subject (e.g., gene expression involved in a neurodegenerative or other
disease), multiple
forms of selenium may be used independently or in combination with one
another. Natural
sources of selenium include, but are not limited to, selenium enriched (e.g.,
selenized)
yeast. The yeast strain used is not limiting.
In certain preferred embodiments of the present invention, Sel-Plex (Alltech,
Lexington, KY) is the selenium form of choice for formulations and
compositions. In some
embodiments, compositions comprising Sel-Plex provide a more biologically
available
form of selenium compared to other forms of selenium (See Example 9). However,
other
forms of selenium may also find use in the present invention including
derivative or
modifications of Sel-Plex or other forms of selenium enriched yeast,
selenomethionine,
selenocysteine, a selenite compound, a selenate compound, or derivatives,
salts, or
modifications thereof Thus, in some preferred embodiments, each of these forms
of
selenium may be used as a component of a formulation. Alternatively, each of
the above
described forms of selenium may be linked (e.g., chemically or physically) to
a drug or
therapeutic (e.g., an Alzheimer's therapeutic) to form a selenium-drug
derivative.
Additionally, compositions and formulations are not limited to one form or
selenium.
Indeed, a composition or formulation may comprise multiple forms of selenium
(e.g., Sel-
Plex and Sod-sel).
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CA 02524551 2012-08-31
Other forms of selenium that fmd use in various embodiments of the present
invention are described in U.S. Pat. Nos. 6,911,550 6,197,295, 5,221,545, 6
and 6,576,233,
and U.S. Pat. App. Nos. 20010043925, 20050069594, and 20050089530.
Accordingly, the present invention provides pharmaceutical compositions which
may comprise one or more forms of selenium, alone or in combination with at
least one
other agent, such as a stabilizing compound, or Alzheimer's therapeutic, and
may be
administered in any sterile, biocompatible pharmaceutical carrier, including,
but not limited
to, saline, buffered saline, dextrose, and water.
The methods of the present invention find use in treating (e.g.,
prophylacticly or
therapeutically) diseases or altering physiological states. Selenium (e.g.,
Sel-PlexS) can be
administered to a subject (e.g., a patient) intravenously in a
pharmaceutically acceptable
carrier such as physiological saline. Standard methods for intracellular
delivery of
compounds can be used (e.g., delivery via liposome). Such methods are well
known to
those of ordinary skill in the art. The formulations of this invention are
useful for
parenteral administration, such as intravenous, subcutaneous, intramuscular,
and
intraperitoneal.
As is well known in the medical arts, dosages for any one subject may depend
upon
many factors, including the patient's size, body surface area, age, the
particular compound
to be administered, sex, time and route of administration, general health, and
interaction
with other drugs being concurrently administered.
Accordingly, in some embodiments of the present invention, compositions and/or
formulations comprising selenium can be administered to a subject alone, or in
combination
with other forms of selenium, drugs, small molecules, or in pharmaceutical
compositions
where it is mixed with excipient(s) or other pharmaceutically acceptable
carriers. In one
embodiment of the present invention, the pharmaceutically acceptable carrier
is
pharmaceutically inert. In another embodiment of the present invention,
compositions
comprising selenium may be administered alone to individuals subject to or
suffering from
a disease or condition (e.g., Alzheimer's disease, Parkinson's disease,
diabetes, etc.).
Compositions comprising selenium (e.g., Sel-Plex alone or in combination with
one or
more other forms of selenium) may be added to a nutritional drink or food
(e.g., ENSURE,
POWERBAR, or the like), a multi-vitamin, nutritional products, food products,
etc. for
daily consumption.
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CA 02524551 2012-08-31
Depending on the target sought to be altered by treatment (e.g., gene
expression
associated with aging), these pharmaceutical compositions may be formulated
and
administered systemically or locally. Techniques for formulation and
administration may
be found in the latest edition of "Remington's Pharmaceutical Sciences" (Mack
Publishing
Co, Easton Pa.). Suitable routes may, for example, include oral or
transmucosal
administration; as well as parenteral delivery, including intramuscular,
subcutaneous,
intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal,
or intranasal
administration.
For injection, the pharmaceutical compositions of the invention may be
formulated
in aqueous solutions, preferably in physiologically compatible buffers such as
Hanks'
solution, Ringer's solution, or physiologically buffered saline. For tissue or
cellular
administration, penetrants appropriate to the particular barrier to be
permeated are used in
the formulation. Such penetrants are generally known in the art.
In other embodiments, the pharmaceutical compositions of the present invention
can
be formulated using pharmaceutically acceptable carriers well known in the art
in dosages
suitable for oral administration. Such carriers enable the pharmaceutical
compositions to be
formulated as tablets, pills, capsules, liquids, gels, syrups, slurries,
suspensions and the like,
for oral or nasal ingestion by a patient to be treated.
Pharmaceutical compositions suitable for use in the present invention include
compositions wherein the active ingredients are contained in an effective
amount to achieve
the intended purpose. For example, an effective amount of the pharmaceutical
agent may
be that amount that alters the expression of a specific gene (e.g., Lhx8,
presenilin 1,
presenilin 2, or Apbbl). Determination of effective amounts is well within the
capability of
those skilled in the art, especially in light of the disclosure provided
herein.
In addition to the active ingredients these pharmaceutical compositions may
contain
suitable pharmaceutically acceptable carriers comprising excipients and
auxiliaries which
facilitate processing of the active compounds into preparations which can be
used
pharmaceutically. The preparations formulated for oral administration may be
in the form
of tablets, dragees, capsules, or solutions.
The pharmaceutical compositions of the present invention may be manufactured
in a
manner that is itself known (e.g., by means of conventional mixing,
dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping or
lyophilizing
processes).
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CA 02524551 2012-08-31
Pharmaceutical formulations for parenteral administration include aqueous
solutions
of the active compounds in water-soluble form. Additionally, suspensions of
the active
compounds may be prepared as appropriate oily injection suspensions. Suitable
lipophilic
solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty
acid esters, such
as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions
may contain
substances which increase the viscosity of the suspension, such as sodium
carboxymethyl
cellulose, sorbitol, or dextran. Optionally, the suspension may also contain
suitable
stabilizers or agents which increase the solubility of the compounds to allow
for the
preparation of highly concentrated solutions.
Pharmaceutical preparations for oral use can be obtained by combining the
active
compounds with solid excipient, optionally grinding a resulting mixture, and
processing the
mixture of granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee
cores. Suitable excipients are carbohydrate or protein fillers such as sugars,
including
lactose, sucrose, maimitol, or sorbitol; starch from corn, wheat, rice,
potato, etc; cellulose
such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium
carboxymethylcellulose; and gums including arabic and tragacanth; and proteins
such as
gelatin and collagen. If desired, disintegrating or solubilizing agents may be
added, such as
the cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof
such as sodium
alginate.
Dragee cores are provided with suitable coatings such as concentrated sugar
solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone,
carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable
organic
solvents or solvent mixtures. Dyestuffs or pigments may be added to the
tablets or dragee
coatings for product identification or to characterize the quantity of active
compound, (i.e.,
dosage).
Pharmaceutical preparations which can be used orally include push-fit capsules
made of gelatin, as well as soft, sealed capsules made of gelatin and a
coating such as
glycerol or sorbitol. The push-fit capsules can contain the active ingredients
mixed with a
filler or binders such as lactose or starches, lubricants such as talc or
magnesium stearate,
and, optionally, stabilizers. In soft capsules, the active compounds may be
dissolved or
suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid
polyethylene
glycol with or without stabilizers.
CA 02524551 2012-08-31
Compositions comprising a compound of the invention formulated in a
pharmaceutical acceptable carrier may be prepared, placed in an appropriate
container, and
labeled for treatment of an indicated condition. For compositions or
formulations
comprising selenium, conditions indicated on the label may include treatment
of condition
related to prophylactic or therapeutic treatment of neurodegenerative disease
or cognitive
function.
The pharmaceutical composition may be provided as a salt and can be formed
with
many acids, including but not limited to hydrochloric, sulfuric, acetic,
lactic, tartaric, malic,
succinic, etc. Salts tend to be more soluble in aqueous or other protonic
solvents that are
the corresponding free base forms. In other cases, the preferred preparation
may be a
lyophilized powder in 1 inM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol
at a pH
range of 4.5 to 5.5 that is combined with buffer prior to use.
For any compound used in the methods of the invention, the therapeutically
effective dose can be estimated initially from cell culture assays. Then,
preferably, dosage
can be formulated in animal models (particularly murine models) to achieve a
desirable
circulating concentration range.
A therapeutically effective dose refers to that amount of which ameliorates or
prevents symptoms of a disease state or condition (e.g., through altering gene
expression)
Toxicity and therapeutic efficacy of such compounds can be determined by
standard
pharmaceutical procedures in cell cultures or experimental animals, e.g., for
determining
the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose
therapeutically
effective in 50% of the population). The dose ratio between toxic and
therapeutic effects is
the therapeutic index, and it can be expressed as the ratio LD50/ED50.
Compounds which
exhibit large therapeutic indices are preferred. The data obtained from these
cell culture
assays and additional animal studies can be used in formulating a range of
dosage for
human use. The dosage of such compounds lies preferably within a range of
circulating
concentrations that include the ED50with little or no toxicity. The dosage
varies within this
range depending upon the dosage form employed, sensitivity of the patient, and
the route of
administration.
The exact dosage may be chosen by a subject or by a physician in view of the
patient to be treated. Dosage and administration are adjusted to provide
sufficient levels of
the active moiety or to maintain the desired effect (e.g., alteration of gene
expression in a
subject). Additional factors that may be taken into account include the
severity of the
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CA 02524551 2012-08-31
disease state; age, weight, and gender of the patient; diet, time and
frequency of
administration, drug combination(s), reaction sensitivities, and
tolerance/response to
therapy. Long acting pharmaceutical compositions might be administered every 3
to 4 days,
every week, or once every two weeks depending on half-life and clearance rate
of the
particular formulation.
In some embodiments, selenium (e.g., organic selenium (e.g., selenized yeast
(e.g.,
Sel-Plex ))) is administered at a daily dose of between 25 and 800 lig per day
(e.g., Sel-
Plex is administered to a subject in such a way so as to provide between 25
and 800 lig of
selenium to the subject each day). In preferred embodiments, the selenium
(e.g., organic
selenium (e.g., selenized yeast (e.g., Sel-Plex ))) is administered at a daily
dose of between
200 and 500 lig per day. In other preferred embodiments, selenium is
administered at a
daily dose of between 200 and 400 lig per day. Doses outside of 25 and 800 14
may be
used. In some embodiments, a single dose of selenium (e.g., organic selenium
(e.g.,
selenized yeast (e.g., Sel-Plex ))) is administered once daily. In other
embodiments, 2, 3,
4, or more doses may be administered each day (e.g., once in the morning and
once at
night, or once every 4 to 6 hours). For example, in some embodiments, selenium
is
adminitstered to a subject in three separate, more than three separate, two
separate, or less
than two separate doses. In some preferred embodiments, the daily dose is
administered in
a time release capsule. In some preferred embodiments, the daily dose is
between 25-75 lig
of selenium. In other preferred embodiments, the daily dose is 200 g of
selenium (e.g.,
organic selenium (e.g., selenized yeast (e.g., Sel-Plex ))).
The pharmaceutical compositions of the present invention may be administered
in a
number of ways depending upon whether local or systemic treatment is desired
and upon
the area to be treated. Administration may be topical (including ophthalmic
and to mucous
membranes including vaginal and rectal delivery), pulmonary (e.g., by
inhalation or
insufflation of powders or aerosols, including by nebulizer; intratracheal,
intranasal,
epidermal and transdermal), oral or parenteral. Parenteral administration
includes
intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular
injection or
infusion; or intracranial, e.g., intrathecal or intraventricular,
administration. Compositions
and formulations comprising selenium are believed to be particularly useful
for oral
administration.
Pharmaceutical compositions and formulations for topical administration may
include transdermal patches, ointments, lotions, creams, gels, drops,
suppositories, sprays,
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CA 02524551 2012-08-31
liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or
oily bases,
thickeners and the like may be necessary or desirable.
Compositions and formulations for oral administration include powders or
granules,
suspensions or solutions in water or non-aqueous media, capsules, sachets or
tablets.
Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or
binders may be
desirable.
Compositions and formulations for parenteral, intrathecal or intraventricular
administration may include sterile aqueous solutions that may also contain
buffers, diluents
and other suitable additives such as, but not limited to, penetration
enhancers, carrier
compounds and other pharmaceutically acceptable carriers or excipients.
Thus, in some embodiments, pharmaceutical compositions of the present
invention
include, but are not limited to, solutions, emulsions, and liposome-containing
formulations.
These compositions may be generated from a variety of components that include,
but are
not limited to, preformed liquids, self-emulsifying solids and self-
emulsifying semisolids.
The pharmaceutical formulations of the present invention, which may
conveniently
be presented in unit dosage form, may be prepared according to conventional
techniques
well known in the pharmaceutical industry. Such techniques include the step of
bringing
into association the active ingredients with the pharmaceutical carrier(s) or
excipient(s). In
general the formulations are prepared by uniformly and intimately bringing
into association
the active ingredients with liquid carriers or finely divided solid carriers
or both, and then,
if necessary, shaping the product.
Thus, in some embodiments, the compositions of the present invention may be
formulated into any of many possible dosage forms such as, but not limited to,
tablets,
capsules, liquid syrups, soft gels, suppositories, and enemas. The
compositions of the
present invention may also be formulated as suspensions in aqueous, non-
aqueous or mixed
media. Aqueous suspensions may further contain substances that increase the
viscosity of
the suspension including, for example, sodium carboxymethylcellulose, sorbitol
and/or
dextran. The suspension may also contain stabilizers.
In one embodiment of the present invention the pharmaceutical compositions may
be formulated and used as foams. Pharmaceutical foams include formulations
such as, but
not limited to, emulsions, microemulsions, creams, jellies and liposomes.
While basically
similar in nature these formulations vary in the components and the
consistency of the final
product.
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CA 02524551 2012-08-31
The compositions of the present invention may additionally contain other
adjunct
components conventionally found in pharmaceutical compositions. Thus, for
example, the
compositions may contain additional, compatible, pharmaceutically-active
materials such
as, for example, antipruritics, astringents, local anesthetics or anti-
inflammatory agents, or
may contain additional materials useful in physically formulating various
dosage forms of
the compositions of the present invention, such as dyes, flavoring agents,
preservatives,
antioxidants, opacifiers, thickening agents and stabilizers. However, such
materials, when
added, should not unduly interfere with the biological activities of the
components of the
compositions of the present invention. The formulations can be sterilized and,
if desired,
mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers,
wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers, colorings,
flavorings and/or
aromatic substances and the like which do not deleteriously interact with the
nucleic acid(s)
of the formulation.
In some embodiments, the invention provide pharmaceutical compositions
containing (a) one or more forms of selenium (e.g., Sel-Plex and/or Sod-sel)
and (b) one
or more other agents (e.g., Alzheimer's therapeutic). Examples of such
Alzheimer's
therapeutic agents are described above. In some embodiments, two or more
combined
agents (e.g., Alzheimer's therapeutics) may be used together or sequentially.
The present invention also includes methods involving co-administration of
compounds comprising selenium described herein with one or more additional
active agents
(e.g., an Alzheimer's therapeutic, anti-oxidant, etc.). Indeed, it is a
further aspect of this
invention to provide methods for enhancing prior art therapies and/or
pharmaceutical
compositions by co-administering a composition comprising selenium of this
invention. In
co-administration procedures, the agents may be administered concurrently or
sequentially.
In one embodiment, the compounds described herein are administered prior to
the other
active agent(s). The pharmaceutical formulations and modes of administration
may be any
of those described above. In addition, the two or more co-administered agents
may each be
administered using different modes or different formulations.
The agent or agents to be co-administered depends on the type of condition
being
treated. For example, when the condition being treated is a neurodegenerative
disease, the
additional agent can be an Alzheimer's therapeutic, an ALS therapeutic, a
Hunitington's
therapeutic, or the like. When the condition being treated is diabetes, the
additional agent
can be a diabetes therapeutic. When the condition being treated is cognitive
function, the
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CA 02524551 2012-08-31
additional agent can be an antioxidant. The additional agents to be co-
administered, such
as Alzheimer's therapeutics, diabetes therapeutics, or antioxidants, can be
any of the well-
known agents in the art, including, but not limited to, those that are
currently in clinical use.
Treatment of the various diseases and disorders described herein are often
generally
limited by the following two major factors: (1) the development of drug
resistance and (2)
the toxicity of known therapeutic agents. Some therapeutic agents have
deleterious side
effects, including non-specific lymphotoxicity and renal toxicity.
The methods described herein address both these problems. Drug resistance,
where
increasing dosages are required to achieve therapeutic benefit, is overcome by
co-
administering the compounds comprising selenium described herein with the
known agent.
In some embodiments, the compounds described herein sensitize target cells to
known
agents (and vice versa) and, accordingly, less of these agents are needed to
achieve a
therapeutic benefit.
The sensitizing function of the claimed compounds also addresses the problems
associated with toxic effects of known therapeutics. In instances where the
known agent is
toxic, it is desirable to limit the dosages administered in all cases, and
particularly in those
cases were drug resistance has increased the requisite dosage. Thus, in some
embodiments,
when the claimed compounds are co-administered with the known agent, they
reduce the
dosage required which, in turn, reduces the deleterious effects. Further,
because the
claimed compounds are themselves both effective and non-toxic in moderate
doses, co-
administration of proportionally more of these compounds than known toxic
therapeutics
will achieve the desired effects while minimizing toxic effects.
VI. Antioxidants
In some embodiments of the present invention, antioxidants are co-administered
with compositions or formulations of the present invention. The present
invention is not
limited by the type of antioxidant utilized. Indeed, a variety of antioxidants
are
contemplated to be useful in the present invention including, but not limited
to, alkylated
diphenylamines, N-alkylated phenylenediamines, phenyl-a-naphthylamine,
alkylated
phenyl-a-naphthylamine, dimethyl quinolines, trimethyldihydroquinolines and
oligomeric
compositions derived therefrom, hindered phenolics, alkylated hydroquinones,
hydroxylated thiodiphenyl ethers, alkylidenebisphenols, thiopropionates,
metallic
dithiocarbamates, 1,3,4-dimercaptothiadiazole and derivatives, oil soluble
copper
compounds, and the like, Naugalube 438, Naugalube 438L, Naugalube 640,
CA 02524551 2012-08-31
Naugalube 635, Naugalube 680, Naugalube AMS, Naugalube APAN, Naugard
PANA, Naugalube TMQ, Naugalube 531, Naugalube 431, Naugard BHT,
Naugalube 403, and Naugalube 420, ascorbic acid, tocopherols including alpha-
tocopherol, water-soluble antioxidants such as sulthydryl compounds and their
derivatives
(e.g., sodium metabisulfite and N-acetyl-cysteine), lipoic acid and
dihydrolipoic acid,
resveratrol, lactoferrin, ascorbic acid derivatives (e.g., ascorbyl palmitate
and ascorbyl
polypeptide), butylated hydroxytoluene, retinoids (e.g., retinol and retinyl
palmitate),
tocotrienols, ubiquinone, extracts containing flavonoids and isoflavonoids and
their
derivatives (e.g., genistein and diadzein), extracts containing resveratrol
and the like, grape
seed, green tea, pine bark, propolis, Irganox 1010, 1035, 1076, 1222
(manufactured by
Ciba Specialty Chemicals Co., Ltd.), Antigene P, 3C, FR, Sumilizer GA-80
(manufactured by Sumitomo Chemical Industries Co., Ltd.), beta-carotene,
lycopene,
vitamins C, E, and A, and other substances.
For example, in some embodiments, the present invention provides a method of
protecting against the by-products of oxidative stress in brain tissue
comprising
administering to a subject a composition comprising Sel-Plex . Although an
understanding of the mechanism is not necessary to practice the present
invention and the
present invention is not limited to any particular mechanism of action, in
some
embodiments, administering a composition comprising Sel-Plex to a subject
reduces the
expression of GST genes (e.g., Gstpl, Gstzl, and Gstm7) in the subject. In
some
embodiments, administering a composition comprising Sel-Plex to a subject
reduces the
level of DNA damage in brain tissue (e.g., neocortex) of a subject. Although
an
understanding of the mechanism is not necessary to practice the present
invention and the
present invention is not limited to any particular mechanism of action, in
some
embodiments, treatment with compositions and methods of the present invention
(e.g.,
dietary supplementation with Sel-Plex ) stabilizes cellular homeostasis (e.g.,
in the brain)
such that the expression of DNA-damage inducible genes (e.g., Gadd45b) is
reduced.
In some embodiments, the present invention provides a method of reducing
sensitivity of cells to H202 cytotoxicity comprising administering to the
cells a composition
comprising Sod-sel and/or Sel-Plex under conditions such that the expression
of SelW is
altered (e.g., increased) (See, e.g., Example 3). In some embodiments, the
present
invention provides a method of reducing the expression of SelW in a subject
comprising
administering a composition comprising selenium (e.g., Sel-Plex and/or Sod-
se!) and an
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CA 02524551 2012-08-31
antioxidant under conditions such that the expression of SelW is altered. In
some
embodiments, the present invention provides a method of promoting repair of
oxidatively
damaged proteins in a subject comprising administering to the subject a
composition
comprising Sod-se! and/or Sel-Plex under conditions such that the expression
of Se1R is
altered (e.g., increased) (See, e.g., Example 3).
The present invention further provides a method of reducing superoxide
radicals in
a subject (e.g., in a subject experiencing oxidative stress) comprising
administering a
composition (e.g., a nutritional supplement) comprising selenium (e.g., Sel-
Plexe) to the
subject. Furthermore, in some embodiments, the present invention provides that
subjects
receiving certain compositions comprising selenium (e.g., selenium supplements
comprising Sel-Plexe) have an enhanced ability to deal with oxidative stress.
Although an
understanding of the mechanism is not necessary to practice the present
invention and the
present invention is not limited to any particular mechanism of action, in
some
embodiments, subjects receiving a composition comprising selenium (e.g., a
dietary
supplement comprising Sel-Plexe) have an enhanced ability to cope with
oxidative stress
due to the ability of select forms of selenium (e.g., Sel-Plexe) to alter
(e.g., reduce) the
level of superoxide radicals in the subject. In some embodiments, reduction of
superoxide
radicals occurs in the brains (e.g., cerebral cortex) of subjects treated with
the compositions
and methods of the present invention (See e.g., Example 10, below).
It is contemplated that the compositions and methods of the present invention
will
find use in various settings, including research and clinical diagnostics. For
example,
compositions and methods of the present invention also find use in studies of
APP
metabolism (e.g., via analysis of proteins and pharmaceuticals capable of
altering levels
thereof) and in in vivo studies to observe Alzheimer's disease pathology. In
addition,
methods to quantitate oligomeric and/or fibrillar P-amyloid protein assemblies
in samples
find use in monitoring and/or determining the effectiveness of Alzheimer's
disease
treatment, as it is contemplated that decreasing levels of oligomeric P-
amyloid protein
assemblies in a subject's samples over time indicates the effectiveness of an
Alzheimer's
disease treatment.
Also provided herein is a method of identifying new treatments for
neurodegenerative disease (e.g., Alzheimer's disease) comprising treating a
subject having
neurodegenerative disease (e.g., Alzheimer's disease) with a composition
comprising
selenium (e.g., organic selenium (e.g., selenized yeast (e.g., Sel-Plex0)))
under conditions
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CA 02524551 2012-08-31
such that the expression level of a gene associated with neurodegenerative
disease (e.g.,
Alzheimer's disease) is altered (e.g., presenilin 1, presenilin 2), and then
co-administering
one or more test compounds, wherein the one or more test compounds are
examined for the
ability to alter the expression of a gene associated with neurodegenerative
disease (e.g.,
Alzheimer's disease) (e.g., presenilin 1, presenilin 2). Changes in the
expression levels of a
gene associated with neurodegenerative disease (e.g., Alzheimer's disease) is
indicative of a
compound that could be used for treating neurodegenerative disease (e.g.,
Alzheimer's
disease). These methods can be used to screen compounds for other diseases and
conditions (e.g., those described herein).
Uses of the compositions and methods provided by the present invention
encompass
human and non-human subjects and samples from those subjects, and also
encompass
research as well as diagnostic applications. Thus, it is not intended that the
present
invention be limited to any particular subject and/or application setting.
EXPERIMENTAL
The following examples are provided in order to demonstrate and further
illustrate
certain preferred embodiments and aspects of the present invention and are not
to be
construed as limiting the scope thereof.
Example 1
Materials and methods
Animal Care. Male C57BL/6J mice were housed singly and started on the
experimental diets described below immediately after weaning (21 days of age).
The mice
were maintained in the Shared Aging Rodent Facility at the William S.
Middleton
Memorial Veterans Administration Medical Center (Madison, WI). Temperature and
humidity were maintained at constant levels. Room light was controlled to
provide 12-hr
cycles of light and dark.
Experimental diets were created by Harlan Teklad (Madison, WI). Selenium
content
of the diets was determined by Covance Inc. (Madison, WI). Five (5) animals
were
included in each of the following treatment groups: a diet deficient in
selenium (SD); a diet
supplemented with selenomethionine (SM, obtained from Sigma,St. Louis, MO)
such that
the final selenium content of the diet was one (1) part per million; a diet
supplemented with
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CA 02524551 2012-08-31
sodium selenite (SS, Sigma) such that the final concentration of selenium in
this diet was
one (1) part per million; or a diet supplemented with the yeast selenium Sel-
Plex (SP,
Alltech, Lexington, Ky), such that the final concentration of selenium in this
diet was one
(1) part per million. Sel-Plex . Mice were provided with water and their
respective diet ad
libitum for 100 days. Diets were stored at the dark at 4 C and fresh diet was
added to
feeder twice weekly.
Tissue sample preparation and microarray analysis. Mice were killed at 100
days of
age by cervical dislocation. For intestinal expression studies, the intestine
was flushed
twice with saline solution and small intestine was measured and divided in
three equal
segments. A 3-cm region of the middle segment of the small intestine
corresponding to the
jejunum (-300 mg of tissue) was cut and rinsed again with physiological saline
to
completely remove contents, flash frozen in liquid nitrogen and stored at -80
C. For brain
(e.g., cerebral cortex) studies, the cerebral cortex was separated from the
surrounding brain
tissue and was flash frozen in liquid nitrogen and stored at -80 C.
Total RNA was isolated from using the guanidinium isothiocyanate method of
TRIZOL (Life Technologies, Grand Island, NY) and individual samples were used
for gene
expression profiles. Total RNA was cleaned up by RNeasy Mini kit (Qiagen,
Valencia,
CA). Target RNA was prepared by converting 5 mg total RNA into double-strand
cDNA
using GeneChip Expression 3'-Amplification Reagents One-Cycle cDNA Synthesis
Kit
(Affymetrix, Santa Clara, CA) with a T7- (dT)24 primer incorporating a T7 RNA
polymerase promoter. After cleaning up double-strand cDNA using Genechip
Sample
Cleanup Module (Affymetrix, Santa Clara, CA), biotin-labeled cRNA was
synthesized
from double-strand cDNA using GeneChip Expression 3'-Amplification Reagents
for IVT
Labeling (Affymetrix, Santa Clara, CA). The biotin-labeled cRNA was cleaned up
using
Genechip Sample Cleanup Module and was fragmented by heating (35 min at 94 C).
Fifteen (15)1..tg of cRNA fragments were hybridized (16 hat 45 C) to Mouse
Genome 430 2.0 Array (Affymetrix, Santa Clara, CA) using GeneChip
Hybridization Oven
640. After hybridization, the gene chips were automatically washed and stained
with
streptavidin-phycoerythrin biotinylated anti-streptavidin using Affymetrix
GeneChip
Fluidics Station 450. The DNA chips were scanned with Affymetrix GeneChip
Scanner
3000 (Affymetrix, Santa Clara, CA) to detect the cell signal intensities by
laser. All
calculations were performed with Affymetrix GeneChip Operating Software (GCOS)
version 1.3 after scanning.
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Data analysis.
1. The spreadsheet containing probe set identifiers and signal intensity
values was
opened in Microsoft Excel (version 11.1.1 for the Macintosh OS-X operating
system) and summary statistics were generated (mean signal intensity for each
treatment group, standard error of the mean). Two-tailed t-tests (equal
variance)
were performed for the following treatment groups: SM vs. SD, SS vs. SD, and
SP
vs. SD. Additionally, a "signal intensity score" was calculated for each probe
set as
the sum of the signal intensities for all chips (N=20).
2. The most recent annotation file was downloaded from the Affymetrix website.
The
data in this file was used to annotate the gene expression data in Step 1. The
resulting file was exported as a comma separated value (CSV) file.
3. The CSV file from Step 2 was imported into a database application (MySQL
version 4.1.12 for the Macintosh OS-X operating system).
4. Using MySQL, probe set identifiers ending with the letters "_x_at" and
"_s_at"
were removed from the data set. According to Affymetrix, probe sets with these
extensions do not map to unique genes (i.e., transcripts from more than one
gene
may hybridize to one probe set). After removing these probe sets, the data
were
exported as a CSV file.
5. The file from Step 3 was opened in Microsoft Excel to identify multiple
occurrences
of the same gene within the data set. When two (or more) probe sets were
determined to represent the same gene, the probe set with the largest "signal
intensity score" (see Step 2) was retained and the additional probe set(s)
were
deleted from the data set. At this stage, each probe set represent only a
single
transcript, and so hereafter the term "probe set" is interchangeable with the
term
"gene".
6. A new column in the data file was created to include information about how
the
expression of a particular gene was affected by the dietary treatment. Using
the p-
values from the t-tests described in Step 1, genes were sorted into one of the
following categories (and this information was noted in the new column); note
that
"statistically significant", as referred to below, means that the p-vaule(s)
of interest
were < (less than or equal to) 0.01:
a. "SelMeth specific": there was a statistically significant change in
expression
of this gene only in the SM vs. SD comparison (i.e., expression of the gene
was not statistically significantly different for either the SS vs. SD or the
SP
CA 02524551 2012-08-31
vs. SD comparisons.
b. "SodSel" specific": there was a statistically significant change in
expression
of this gene only in the SS vs. SD comparison
c. "SelPlex specific": there was a statistically significant change in
expression
of this gene only in the SP vs. SD comparison
d. "SelMeth-SodSel": there was a statistically significant change in
expression
of this gene in only for the SM vs. SD and the SS vs. SD comparisons
e. "SelMeth-SelPlex": there was a statistically significant change in
expression
of this gene in only for the SM vs. SD and the SP vs. SD comparisons
f. "SodSel-SelPlex": there was a statistically significant change in
expression
of this gene in only for the SS vs. SD and the SP vs. SD comparisons
g. "Unaffected": the expression of this gene was not significantly affected
by
the SM, SS or SP diets relative to the SD diets
h. "Affected by all": the expression of this gene was significantly
different for
the SM, SS, and SP groups relative to the SD group
7. The dataset was divided into two sub-sets, one containing "well-
characterized
genes" (essentially those transcripts that have a unique gene title and gene
symbol
according to Affymetrix's probeset annotation information) and
"Uncharacterized
transcripts" (all remaining probesets in the data set, including expressed
sequence
tags, cDNA sequences, etc.).
8. Each gene in the "well-characterized genes" subset of data was then
assigned a
"gene function" using the "GO Biological Process" column of the annotation
information provided by Affymetrix. in cases where multiple and diverse gene
ontology (GO) information is provided, information from the National Center
for
Biotechnology Information (NCBI) databases (Entrez-Gene, PubMed, etc.) were
used to generate a "consensus opinion" for the function of that gene.
Example 2
Dietary selenium alters gene expression in the mouse intestine
The ability of dietary selenium (e.g., derived from various sources such as
SeM, Sel-
sod, and Sel-Plex ) to alter the physiology (e.g., physiologic homeostasis)
and the
expression patterns (e.g., protein or gene expression patterns) of various
functional groups
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CA 02524551 2012-08-31
of proteins and various protein pathways in mouse intestine and brain (e.g.,
cerebral cortex)
was examined.
Thus, it was an object of the present invention to determine whether
compositions
and methods of the present invention could alter the expression levels (e.g.,
mRNA levels)
of various genes. One group of genes analyzed were genes classically
associated with
selenium. As described above, the expression levels of genes were analyzed
between mice
with and without dietary selenium, or, between mice fed different sources of
selenium)
(See, e.g., Table 1, below). No significant differences were observed in body
weights of
mice receiving a diet deficient in selenium, a diet comprising selomethionine
(Se-meth, or
SeM), a diet comprising sodium selenite (Sod-sel, or SS), or a diet comprising
Sel-Plex
(Sel-Plex , or SP) (See FIG. 1).
Selenium is known for its role in antioxidant systems, mainly because selenium
(as
selenocysteine) is a key component of glutathione peroxidases (GSH-Px).
Glutathione
peroxidases are a class of enzymes that metabolize or detoxify hydrogen
peroxide and lipid
hydroperoxides. Thus, they function to protect the cell against damage caused
by reactive
oxygen species (ROS) produced as by-products of aerobic cellular metabolism
(See, e.g.,
Arthur, Cell. Mol. Life Sci. 57, 1825, (2000)).
Accordingly, it was determined whether the expression level of GSH-Px would
change in subjects that received selenium supplementation (e.g., dietary
selenium
supplementation) versus those that did not (e.g., selenium deficient
subjects). Using the
compositions and methods of the present invention, it was demonstrated that
there was a
significant fold change (FC) in GSH-Px gene expression in subjects receiving
selenium
supplementation (e.g., receiving Se-meth, Sod-se!, and Sel-Plex ) compared to
selenium
deficient subjects. The fold change in expression levels of two GSH-Px genes
is described
in Table 1, below:
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CA 02524551 2012-08-31
Gene Se-meth Sod-sel Sel-Plex
Glutathione 4.9 4.1 4.7
Peroxidase 1 (all
p<0.01)
Glutathione 4.6 3.5 3.6
Peroxidase 3 (all
p<0.01)
Table 1.
Expression of other genes associated with selenium were also examined and
determined to be altered. For example, the upregulation of selenoenzymes
(e.g.,
Thioredoxin Reductase 1 (Trx-1), See, e.g., Rundlof and Amer, Anitoxidants and
Redox
Signaling, 6, 41(2004)) was observed. The thioredoxin system is a key defense
against
ROS and consists of Thioredoxin and Thioredoxin Reductase which reduces
Thioredoxin
using NADPH. In subjects receiving selenium supplementation, the fold increase
in
expression of the Thioredoxin Reductase 1 gene was as follows: SeM , 1.8;
SS,1.7; SP 1.8
(all with p values < 0.01). Thus, compositions and methods of the present
invention
functioned to alter the expression of genes previously known to be associated
with
selenium.
Another selenoenzyme, Type 1 iodothyronine deiodinase (See, e.g., Larsen and
Berry, Annu. Rev. Nutr., 15, 323 (1995)), also displayed increased expression
using the
compositions and methods of the present invention. This enzyme is responsible
for the
conversion of Thyroxin (T4) to bioactive thyroid hormone (T3). Selenium
supplementation
significantly increased the expression level (e.g., nucleic acid expression)
of Type 1
iodothyronine deiodinase as follows: SeM, 2.0 fold increase; SS, 2.8 fold
increase; SP, 2.1
fold increase.
Example 3
Dietary selenium alters the expression level of selenoprotein-encoding genes
in a
selenium source-dependent manner
Selenium (Se) is now known to be incorporated as selenocysteine in a number of
selenoproteins, glutathione peroxidase (GSH-Px, See Example 2) being the
prototypical
example. Selenocysteine is specifically encoded by the UGA codon, and inserted
in peptide
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CA 02524551 2012-08-31
chains by a cotranslational mechanism that is able to override the normal
function of UGA
as a termination codon. In eukaryotes, efficient selenocysteine incorporation
at UGA
codons requires a cellular protein factor and a cis-acting structural signal
usually located in
the inRNA 3'-untranslated region (3'-UTR), consisting of a selenocysteine
insertion
sequence (SECIS) in a characteristic stem-loop structure (See, e.g., Peterlin
et al., (1993),
In Human Retroviruses; Cullen, Ed.; Oxford University Press: New York; pp. 75-
100; Le
and Maize', Theor. Biol. 138:495 (1989)). The required protein factor is
presumed to be
present in certain cells types that express selenoproteins, such as liver
cells, lymphocytes,
macrophages, thrombocytes, and other blood cells. In such cell types, the
presence of a
SECIS element in an mRNA is necessary and sufficient for in-frame UGA codons
to be
translated as selenocysteine.
The expression levels of several selenoprotein-encoding genes were affected by
selenium supplementation. Importantly, the present invention demonstrates for
the first
time that there exists significant differences in the ability of various
sources of selenium to
alter the expression levels of the same genes (e.g., selenoprotein genes and
other genes
described herein). For example, the expression of Selenoprotein W (Se1W), was
not
significantly altered by SeM. However, Sod-sel and Sel-Plex upregulated SelW
5.1 fold.
SelW is expressed in many tissues, including brain, where its expression level
is maintained
in selenium deficiency. SelW is a glutathione-dependent antioxidant and it has
been shown
that overexpression of SelW in CHO cells and H1299 human lung cancer cells
markedly
reduces the sensitivity of both cell lines to H202 cytotoxicity (See, e.g.,
Jeong et al., FEBS
Letter, 517, 225 (2002)). Thus, in some embodiments, the present invention
provides a
method of reducing sensitivity of cells to H202 cytotoxicity comprising
providing to the
cells a composition comprising Sod-sel and/or Sel-Plex under conditions such
that the
expression of SelW is altered (e.g., increased).
Further illustrating the selenium source dependent nature of the ability to
alter gene
expression, the expression level of the gene for selenoprotein N1 (Sepnl), was
not
significantly affected by SeM or Sod-sel, but was increased 1.8-fold by Sel-
Plex
(p<0.02). It is thought that Sepnl plays an important role in muscle
integrity. For example,
in humans, multiminicore disease consists of a spectrum of congenital
neuromuscular
diseases with clinical conditions such as weakness and structural muscular
changes. It is
known that a third of all multiminicore disease cases are due to mutations in
the Sepnl gene
(See, e.g., Neuromuscul. Disord. 15 (4), 299-302 (2005); Am. J. Hum. Genet.
71(4), 739-
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CA 02524551 2012-08-31
749 (2002)). Thus, in some embodiments, the present invention provides a
method of
maintaining muscle integrity comprising providing to the cells a composition
comprising
Sel-Plex under conditions such that the expression of Sepnl is altered (e.g.,
increased).
Methionine sulfoxide reductases catalyze reduction of free and protein-bound
methionine sulfoxides to corresponding methionines (See, e.g., Brot et al.,
Proc. Natl.
Acad. Sci. USA 78, 2155 (1981); Weissbach et al., Arch. Biochem. Biophys. 397,
172
(2002)). The oxidation of methionine by reactive oxygen species (ROS)
generates a
diastereomeric mixture of methionine-S-sulfoxide (Met-S-SO) and methionine-R-
sulfoxide
(Met-R-SO). Two distinct enzyme families evolved for reduction of these
sulfoxides, with
methionine-S-sulfoxide reductase (MsrA) being stereospecific for Met-S-SO and
methionine-R-sulfoxide reductase (MsrB) for Met-R-SO. Previously described
functions of
these enzymes include repair of oxidatively damaged proteins, regulation of
protein
function and elimination of oxidants through reversible formation of
methionine sulfoxides
(See, e.g., Levine etal., IUBMB Life 50, 301 (2000)).
To date, two mammalian MsrB proteins have been identified: selenocysteine
(Sec)-
containing protein, designated selenoprotein R (Se1R; See, e.g., Kryukov et
al., J. Biol.
Chem. 274, 33888 (1999); , Proc. Natl. Acad. Sci. USA 99, 4245 (2002)) and its
homolog,
designated CBS-1, in which Cys is present in place of Sec (See, e.g., Jung
etal., FEBS
Lett. 527, 91(2002)). The Sec-containing MsrB has only been described in
mammals.
Members of the MsrB family have been characterized mechanistically (See, e.g.,
Kumar et
al., J. Biol. Chem. 277, 37527 (2002); Olry etal., J. Biol. Chem. 277, 12016
(2002)); and
structurally (Lowther et al., Nat. Struct. Biol. 9, 348 (2002)).
The gene for Se1R (also known as Selenoprotein X1) was not significantly
affected
by SeMet dietary supplementation but was upregulated 1.3-fold (p<0.01) and 1.2-
fold
(p<0.05) by Sod-sel and Sel-Plex , respectively. As described above, Se1R is a
methionine
sulfoxide reductase. Methionine residues in proteins are susceptible to damage
by ROS but
can be repaired via reduction of the resulting methionine sulfoxides by
enzymes such as
Se1R (See, e.g., Kim and Gladyshev, Mol Biol Cel 15, 1055, (2004)).
Accordingly, in some
embodiments, the present invention provides a method of promoting repair of
oxidatively
damaged proteins in a subject comprising providing to the subject a
composition
comprising Sod-sel and/or Sel-Plex under conditions such that the expression
of Se1R is
altered (e.g., increased).
CA 02524551 2012-08-31
Example 4
Selective forms of dietary selenium alter the expression of stress-inducible
proteins
The superoxide dismutase genes (e.g., SOD1 and SOD2) encode an
intramitochondrial free radical scavenging enzymes that are a first line of
defense against
superoxide (e.g., superoxide radicals) produced as a byproduct of oxidative
phosphorylation. (See, e.g., Li et al. Nature Genet 11: 376 (1995)).
Inactivation (e.g.,
homozygous mutants) of the Sod2 gene in transgenic mice by homologous
recombination
results in mice dying within the first 10 days of life with a dilated
cardiomyopathy,
accumulation of lipid in liver and skeletal muscle, and metabolic acidosis
(See, e.g., Li et
al. Nature Genet 11: 376 (1995)). Cytochemical analysis revealed a severe
reduction in
succinate dehydrogenase (complex II) and aconitase (a tricarboxylic acid cycle
enzyme)
activities in the heart and to a lesser extent in other organs. The findings
suggested that
MnSOD is required for normal biologic function of tissues by maintaining the
integrity of
mitochondrial enzymes susceptible to direct inactivation by superoxide.
Reactive oxygen species (ROS) have been implicated in a wide range of
degenerative processes including amyotrophic lateral sclerosis, ischemic heart
disease,
Alzheimer disease, Parkinson disease, and aging. ROS are generated by
mitochondria as
the toxic by-products of oxidative phosphorylation, their energy generating
pathway. As
noted above, genetic inactivation of the mitochondrial form of SOD in mice
results in
dilated cardiomyopathy, hepatic lipid accumulation, and early neonatal death
(See, e.g., Li
et al. Nature Genet 11: 376 (1995)). It has been reported that treatment with
a SOD
mimetic, MnTBAP, rescued Sod2 -/- mutant mice from this systemic pathology and
dramatically prolonged their survival (See, e.g., Melov et al., Nature Genet
18: 159
(1998)). Surviving animals developed a pronounced movement disorder
progressing to
total debilitation by 3 weeks of age. Neuropathologic evaluation showed a
striking
spongiform degeneration of the cortex and specific brainstem nuclei,
associated with gliosis
and intramyelinic vacuolization similar to that observed in cytotoxic edema
and disorders
associated with mitochondrial abnormalities such as Leigh disease and Canavan
disease. It
has been suggested that because of the failure of MnTBAP to cross the blood-
brain barrier
progressive neuropathology is caused by excessive mitochondrial production of
ROS (See,
e.g., Melov et al., Nature Genet 18: 159 (1998)).
Knockout mice for SOD1 exhibit typical progressive muscle atrophy and weakness
with selective damage to motor neurons that closely resembles human ALS. There
appears
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CA 02524551 2012-08-31
to be a causal relationship between mutant SOD1 secretion and neural toxicity
(e.g., the
mutant protein is not secreted). However, infustion of wild-type SOD in an ALS
rat model
significantly delays disease onset (See, e.g., J. Neurosci, 25, 108-117
(2005)).
Additionally, it has been shown that a copper (Cu) chaperone is required for
efficient
loading of Cu into SOD (See, e.g., Nat. Neurosci, 5, 301-307 (2002)). Thus,
the ability to
maintain normal levels of wild-type SOD or to enhance expression or function
of the same
may provide a beneficial therapeutic effect for ALS subjects.
Furthermore, it has been shown that regressive numbers of basal forebrain
cholinergic neurons appear in several areas of the brain of ALS subjects (See,
e.g.,
Neurochem Int. 46, 357-368, (2005)). Thus, the ability to upregulate genes
involved in
basal forebrain cholinergic neuron growth and/or maintenance may provide
beneficial
effects for a subject with ALS.
Thus, it was determined whether dietary selenium supplements could alter the
expression levels of SOD genes (e.g., SOD1 and SOD2). Subjects administered a
compositions comprising selenium (e.g., Sel-Plex or Sod-sel) exhibited and
enhanced
expression of SOD1 (e.g., 1.2 and 1.92 fold, respectively. Additionally, these
subjects also
exhibited an enhancement in the expression of the Cu chaperone for SOD, (CCS)
(1.19 fold
and 1.28 fold, respectively). Thus, the present invention provides a method of
treating a
subject with ALS comprising administering a composition comprising selenium
under
conditions such that the expression of SOD1 and/or CCS is enhanced.
In some embodiments, the present invention provides a method of reducing
superoxide radicals in a subject (e.g., in a subject experiencing oxidative
stress) comprising
providing a composition (e.g., a nutritional supplement) comprising selenium
(e.g., Sel-
PlexC) to the subject. Furthermore, in some embodiments, the present invention
provides
that subjects receiving certain compositions comprising selenium (e.g.,
selenium
supplements comprising Sel-PlexC) have an enhanced ability to deal with
oxidative stress.
Although an understanding of the mechanism is not necessary to practice the
present
invention and the present invention is not limited to any particular mechanism
of action, in
some embodiments, subjects receiving a composition comprising selenium (e.g.,
a dietary
supplement comprising Sel-PlexC) have an enhanced ability to cope with
oxidative stress
due to the ability of select forms of selenium (e.g., Sel-Plexe) to alter
(e.g., reduce) the
level of superoxide radicals in the subject. In some embodiments, reduction of
superoxide
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CA 02524551 2012-08-31
radicals occurs in the brains (e.g., cerebral cortex) of subjects treated with
the compositions
and methods of the present invention (See e.g., Example 10, below).
Another unique effect of Sel-Plex was its ability to significantly down-
regulate the
expression of the stress-inducible selenoprotein, type II iodothyronine
deiodinase, (Dio2).
Thyroid hormone has important regulatory effects in some mammalian tissues,
such
as the developing brain, the anterior pituitary gland, and brown adipose
tissue (See, e.g.,
Croteau et al. J. Clin. Invest 98: 405-417, (1996)). A relatively high
proportion of the
receptor-bound triiodothyronine is found within the tissue itself rather than
in plasma. The
expression in these tissues of type II iodothyronine deiodinase (Dio2), which
catalyzes
deiodination of thyroxine T4 exclusively on the outer ring (5-prime-position)
to yield T3,
suggests that Dio2 is responsible for this 'local' production of T3 and is
thus important in
influencing thyroid hormone action in these tissues. In addition, Dio2
activity is markedly
elevated in the hypothyroid state and appears to be responsible for catalyzing
the
production of a large proportion of the circulating T3 under such conditions.
It has been
noted that, from the cDNAs of iodothyronine deiodinase types I and III,
deiodinases contain
in-frame TGATGA codons that code for selenocysteine (See, e.g., Croteau et al.
J. Chin.
Invest 98: 405-417, (1996)). The catalytic properties and tissue patterns of
expression of
these selenoproteins differ from those of Dio2. Unlike Dio2, Dio 1 is
expressed in liver and
kidney and is capable of inner ring deiodination of sulfated thyroid hormone
conjugates.
Dio3 functions as an inner ring deiodinase to convert T4 and T3 to inactive
metabolites. Its
expression in placenta and several fetal tissues during early development
suggested that it
plays a role in preventing premature exposure of developing tissues to adult
levels of
thyroid hormones. Dio2 also is present in several fetal and neonatal tissues
and is essential
for providing the brain with appropriate levels of T3 during the critical
period of
development.
Dio2 is upregulated 10- to 50-fold in brown adipose tissue in response to cold
stress
(See, e.g., de Jesus et al., J. Clin. Invst., 108, 1379 (2001)).
It has been shown that selenium depletion reduced the basal endogenous Dio2
expression and activity in a mesothelioma cell line (See, e.g., J. Biol. Chem.
276: 30183
(2002)). This depletion could be reversed by selenium supplementation in a
dose- and
time-dependent fashion. Dio2 expression and activity also increased following
exposure to
a nonhydrolyzable cAMP analog. Exposure to the thyroxine substrate increased
the
degradation of DI02, resulting in decreased DI02 activity. The short half-life
of
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CA 02524551 2012-08-31
endogenous D102 (less than 1 hr) and the increased degradation of D102 in the
presence of
thyroxine were reduced or eliminated by exposure to proteasome inhibitors.
Experiments conducted using compositions and methods of the present invention
provided that SeMet and Sod-sel displayed no ability to alter the expression
levels of Dio2,
while Sel-Plex caused a significant, 2.3-fold down-regulation of this gene.
Thus, the
present invention provides a method of reducing stress (e.g., cellular stress)
in a subject
comprising providing to the subject a composition comprising selenium (e.g.,
Sel-Plex )
under conditions such that the expression of Dio2 is reduced. In some
preferred
embodiments, the present invention provides a method of stabilizing endocrine
function in
a subject comprising administering to the subject a composition comprising Sel-
Plex
under conditions such that the expression of Dio2 is reduced.
Although an understanding of the mechanism is not necessary to practice the
present invention and the present invention is not limited to any particular
mechanism of
action, in some embodiments, treating a subject with a composition comprising
selenium
(e.g., a dietary supplement comprising Sel-Plex ) reduces the expression of
Dio2, thereby
reducing cellular stress within the subject. Thus, the unique altering (e.g.,
reduction) of
expression of Dio2 demonstrates that subjects receiving certain forms of
selenium (e.g.,
Sel-Plex ) experience/are under less stress that those subject not receiving
treatment.
The expression of several other stress-associated genes was uniquely altered
(e.g.,
downregulated) by certain forms of selenium (e.g., expression altered by Sel-
Plex but not
altered by treatment with SeM or Sod-sel). One example was the gene for
Glyoxalase 1
(Glo1). Glyoxalase is the main detoxification pathway for methyl-glyoxal, a
cytotoxic by-
product of aerobic glycolysis (See, e.g., Amicarelli et al., Carcinogenesis,
19, 519 (1998)).
It has been shown that the Glol gene was upregulated approximately 1.6-fold in
brain tissue of a transgenic mouse model of Alzheimer disease (AD) and
frontotemporal
dementia (See, e.g., Chen et al., Proc. Nat. Acad. Sci. 101: 7687 (2004)).
GLO1 was also
elevated in human Alzheimer disease brains compared to nondemented controls,
and GLO1
immunohistochemistry detected intensely stained flame-shaped neurons in AD
brains. Data
demonstrated the potential of transcriptomics applied to animal models of
human diseases
and suggested a previously unidentified role for glyoxalase I in
neurodegenerative disease
(See, e.g., Chen et al., Proc. Nat. Acad. Sci. 101: 7687 (2004).
Experiments conducted using compositions and methods of the present invention
provide that the expression levels of Glo I were not significantly affected by
SeMet or Sod-
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CA 02524551 2012-08-31
Sel. However, treatment (e.g., dietary supplementation) with Sel-Plex
resulted in a 1.3
fold reduction of expression (p<0.01). Accordingly, the present invention
provides a
method of treating a subject (e.g., an Alzheimer disease subject) comprising
providing to
the subject a composition comprising selenium (e.g., Sel-Plex or derivatives
thereof)
under conditions such the expression of Glol in the subject is reduced.
The expression of growth arrest and DNA damage-inducible genes was also
altered
(e.g., reduced) by selenium supplementation (See, e.g., Table 2, below). Thus,
in some
embodiments, the present invention provides compositions (e.g., comprising Sel-
Plex )
and methods that reduce DNA damage in a subject, as evidenced by the down-
regulation of
genes associated with DNA damage and growth arrest in subjects that received
certain
forms of selenium supplementation (e.g., Sel-Plex ). Thus, in some
embodiments, the
present invention provides a method of reducing DNA damage in a subject
comprising
providing to the subject a composition comprising selenium (e.g., Sel-Plex )
under
conditions such that DNA damage is reduced.
Gene Title/Symbol FC SM FC SS FC SP Functional
Class
Growth arrest and NS NS -1.3 (P<0.05) Signal
DNA-damage- Transduction
inducible 45 beta.
(Gadd45b)
Growth arrest and -1.5 (p<0.05) -2.0 (p<0.01) -2.2 (p<0.05) Stress
DNA-damage- response
inducible 45
gamma.(Gadd45g)
P53 and DNA NS NS -1.3 (P<0.05) Stress
damage-regulated response
1.(Pdrg1).
Table 2
Another class of proteins whose expression was altered with selenium treatment
is
prohibitins. Prohibitins are proteins that have been ascribed various
functions within the
cell, including cell cycle regulation, involvement in apoptosis and assembly
of
mitochondrial respiratory chain enzymes. They are present in the inner
mitochondrial
membrane and their expression is known to be induced by metabolic stress
caused by an
imbalance in the synthesis of mitochondrial and nuclear-encoded mitochondrial
proteins.
Prohibitins act in cooperation with each other to modulate mitochondrial
activity,
particularly in situations of mitochondrial stress (See, e.g., Coates et al.,
Exp. Cell.
Research, 265, 262 (2001)). Generally, with an increase in age, the is a
concomitant
increase in mitochaondrial stress.
CA 02524551 2012-08-31
Using compositions and methods of the present invention, it was observed that
subjects treated with certain forms of selenium (e.g., Sel-Plex )
significantly
downregulated the expression of Prohibitin (Phb) 1.3-fold (p<0.05), whereas
Sod-sel did
not significantly alter Phb expression and where SeM significantly upregulated
Phb
expression 1.6-fold (p<0.05). Thus, in some embodiments, the present invention
provides a
method of altering age associated expression of a prohibitin gene in a subject
comprising
administering to said subject a composition comprising Sel-Plex under
conditions such
that age associated expression of a prohibitin gene is reduced. Although an
understanding
of the mechanism is not necessary to practice the present invention and the
present
invention is not limited to any particular mechanism of action, providing
certain forms of
selenium (e.g., Sel-Plex ) reduces mitochondrial stress associated with aging
whereas
other forms of selenium (e.g., selenomethionine) are incapable of reducing
mitochondrial
stress and may even increase it. Thus, this provides further support for the
use of certain
compositions comprising certain forms of selenium (e.g., Sel-Plex ) and not
other types of
selenium (e.g., SeM or Sod-sel) in order to reduce stress (oxidative or other
forms) in a
subject. Thus, in general, the present invention provides compositions
comprising certain
forms of selenium (e.g., Sel-Plex ) that, when administered (e.g., via a
dietary supplement)
to a subject, do not induce the expression of stress inducible genes that are
induced by the
administration of other forms of selenium (e.g., SeM and/or Sod-sel).
Accordingly, in
some embodiments, the present invention provides a method of reducing cellular
stress
(e.g., metabolic stress) in a subject comprising providing to the subject a
composition
comprising selenium (e.g., Sel-Plex ) under conditions such that the
expression of Phb is
reduced.
Example 5
Selective forms of dietary selenium alter neuronal gene expression
Basal forebrain cholinergic neurons (BFCNs) are involved in cognitive
functions
such as learning and memory and are affected in several neurodegenerative
diseases, such
as Alzheimer's disease (AD). The LIM homeobox protein 8 gene (Lhx8), is
important for
the proper development and maintenance of BFCNs (See, e.g., Mori et al., Eur.
J.
Neurosci., 19, 3129 (2004)).
It has been reported that mice with a null mutation in the Lhx8 gene are
deficient in
the development of forebrain cholinergic neurons (Zhao et al.õ Proc. Nat.
Acad. Sci. 100:
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CA 02524551 2012-08-31
9005 (2003)). The Lhx8 mutants lacked the nucleus basalis, a major source of
the
cholinergic input to the cerebral cortex. In addition, the number of
cholinergic neurons was
reduced in several other areas of the subcortical forebrain in these mutants.
Although
cholinergic neurons were not formed, initial steps in their specification
appeared to be
preserved, as indicated by a presence of cells expressing a truncated Lhx8
mRNA and
inRNA of the homeobox gene Gbxl. These results provide genetic evidence
supporting an
important role for Lhx8 in development of cholinergic neurons in the
forebrain.
Using compositions and methods of the present invention, the observed
expression
level of Lhx8 was not significantly different between Se-deficient subjects
and that of
subjects receiving certain forms of selenium (e.g., SeM or Sod-sel). However,
when
subjects were treated (e.g., received a dietary supplement) with a composition
comprising
Sel-Plex , the expression of Lhx8 was upregulated 12.9-fold (p<0.01). Thus, in
some
embodiments, the present invention provides methods of maintaining and/or
stabilizing
neurologic function (e.g., cholinergic neuron growth and function) in a
subject comprising
providing to the subject a composition comprising Sel-Plex under conditions
such that the
expression of Lhx8 is enhanced.
In addition, the product of another gene, transforming growth factor beta 2
(TGF-
132), is known to increase neuronal proliferation in the developing cerebellum
(See, e.g.,
Elvers et al., Mechanisms of Development, 122, 587 (2004)). Furthermore, it
has been
shown that TGF-132 is a growth and survival factor for granule cell precursors
in the
cerebellum and that antibody-mediated neutralization of endogenous TGF-132
represses
proliferation of cerebellar granule cell precursors and induces
neurodegeneration. It has
also been demonstrated that knocking out (e.g., deleting) TGF-02 is a lethal
phenotype with
TGF-132 deficient mice developing a range of defects and dying before
development of the
cerebellum occurs (See, e.g., Sanford et al., Development, 124, 2659 (1997)).
Using compositions and methods of the present invention, the expression level
of
TGF-132 was not altered, compared to controls, in subjects receiving certain
forms of
selenium (e.g., SeM or Sod-Sel). However, when subjects were treated (e.g.,
received a
dietary supplement) with a composition comprising Sel-Plex , the expression
TGF-13 was
upregulated 2.4-fold. Thus, in some embodiments, the present invention
provides a method
of increasing cerebellum function in a subject comprising providing to the
subject a
composition comprising Sel-Plex . Although an understanding of the mechanism
is not
necessary to practice the present invention and the present invention is not
limited to any
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CA 02524551 2012-08-31
particular mechanism of action, in some embodiments, providing a subject a
composition
comprising selenium (e.g., a daily dietary supplement comprising Sel-P1ex411)
increases
neuronal activity (e.g., increases neuronal proliferation) and/or inhibits
neurodegeneration
(See, e.g., Example 10 below).
Example 6
Selective forms of dietary selenium alter the expression of diabetes related
genes
Neurogenin 3 (Neurog3) is a key transcription factor in the differentiation of
the
endocrine pancreas. Neurog3 is an important part of the activation pathway for
insulin
gene expression and helps to ameliorate glucose tolerance (See, e.g., Watada,
Endocrine
Journal, 51, 255 (2004)). It is thought that lower than normal levels (e.g.,
under-
expression) of Neurog 3 plays a role in certain types of Diabetes (See, e.g.,
Lee et al.,
Genes Dev. 16: 1488 (2002)). Using compositions and methods of the present
invention, it
was determined that Neurog3 expression was significantly upregulated I.7-fold
in subjects
receiving a composition comprising Sel-Plex , whereas subjects that received
SeM or Sod-
sel treatments displayed no significant alteration of Neurog3 expression.
Thus, in some
embodiments, the present invention provides a method of treating a subject
(e.g., a subject
with diabetes) comprising administering to the subject a composition
comprising Sel-Plex
under conditions such that the expression of Neurog3 is altered (e.g.,
enhanced) in the
subject. Although an understanding of the mechanism is not necessary to
practice the
present invention and the present invention is not limited to any particular
mechanism of
action, in some embodiments, providing a subject with diabetes a composition
comprising
Sel-Plex ameliorates glucose tolerance in the subject via up-regulating the
expression of
Neurog3 expression.
Example 7
Selective forms of dietary selenium up-regulate the expression of genes
associated with
enhanced respiratory system function
The Drosophila respiratory system and the mammalian lung are both formed by a
process of branching morphogenesis, which depends on epithelial and
mesenchymal
interactions mediated by signaling between members of the fibroblast growth
factor (FGF)
family and their cognate receptors. Branchless, a Drosophila FGF homologue, is
expressed
in the tips of tracheal branches (See, e.g., Sutherland et al., Cell 87, 1091
(1996)).
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CA 02524551 2012-08-31
Branchless activates an FGF receptor homologue termed Breathless (See, e.g.,
Glazer and
Shilo, Genes Dev 5, 697 (1991)), which directs tracheal cell migration as well
as inducing
secondary and terminal branches.
The sprouty gene (Spry2) product functions as an FGF antagonist in Drosophila:
overexpression of sprouty blocks activation of downstream effectors in the
Branchless
pathway, whereas sprouty null mutation enhances the function of Branchless
downstream
genes, resulting in enhanced tracheal branching (See, e.g., Hacohen, et al.,
Cell 92, 253
(1998)). In Drosophila and mice, the product of the Spry2 gene has been
demonstrated to
negatively modulate respiratory organogenesis (See, e.g., Teftt et al.,
Current Biology, 9,
219 (1999)).
Using compositions and methods of the present invention, it was demonstrated
that
Sel-Plex possessed a unique ability to downregulate the sprouty homolog 2
gene (Spry2).
Specifically, subjects receiving a composition comprising Sel-Plex displayed
a significant
reduction in Spry2 gene expression (1.7-fold reduction), while subjects
receiving
compositions comprising other forms of selenium (e.g., SeM or Sod-sel)
displayed no
alteration in expression levels of Spry2 relative to Se-deficient controls.
Thus, the present
invention provides a method of enhancing respiratory system function in a
subject
comprising providing to the subject a composition comprising selenium (e.g.,
Sel-Plex ).
Although an understanding of the mechanism is not necessary to practice the
present
invention and the present invention is not limited to any particular mechanism
of action, in
some embodiments, treating a subject with a composition comprising selenium
(e.g., Sel-
Plex ) enhances respiratory system function via reducing Spry2 gene
expression.
Example 8
Selective forms of dietary selenium alter the expression of genes associated
with aging
and cognitive function
Aging is well known to be associated with increased oxidant generation (See,
e.g.,
Peinado et al., Anat Rec, 247, 420 (1997)). For example, highly reactive
oxygen species
(ROS) promote a wide spectrum of cell damage, including DNA damage, lipid
peroxidation, alteration of intracellular redox balance and inactivation of
enzymes. A key
host mechanism in the defense against ROS is performed by the family of
Glutathione-S-
Transferases (GSTs) that protect against the by-products of oxidative stress
through a
variety of reactions (See, e.g., Hayes et al., Annu. Rev. Phramacol. Toxicol.,
45, 51,
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CA 02524551 2012-08-31
(2004)). In the area of neurodegeneration, oxidation of cathecholamines yields
aminochrome, dopachrome, noradrenochrome and adrenochrome that are harmful
because
they can produce 02 by redox cycling. These quinone-containing compounds can
be
conjugated with GSH through the actions of GSTs, a reaction that prevents
redox cycling
- 5 (See, e.g., Dagnino-Subiabre et al., Biochem. Biophys. Res. Commun.,
274, 32 (2000)). 0-
quinones formed from dopamine can also be conjugated with GSH by GSTs, and
this
reaction is thought to combat degenerative processes in the dopaminergic
system in the
human brain (e.g., loss of the ability to combat this process may play a role
in disease such
as Parkinson's disease).
In microbes, plants, flies, fish and mammals, expression of GSTs is
upregulated by
exposure to pro-oxidants and, indeed, the promoter regions of cytosolic GSTs
contain anti-
oxidant response elements through which they are transcriptionally activated
during
exposure to Michael reaction acceptors and oxidative stress (See, e.g., Hayes
et al., Annu.
Rev. Phramacol. Toxicol., 45, 51, (2004)).
Thus, compositions and methods of the present invention were analyzed to
determine if they were capable of altering the expression levels of GST genes.
Compositions comprising various forms of selenium (e.g., SeM, Sod-se!, and Sel-
Plex8)
were administered to subjects and the expression levels of GST genes
monitored. The
expression level of several GST genes were altered by selenium supplementation
compared
with control subjects receiving Se-deficient diets (See Table 3, below).
Gene name Symbol FC SeM FC Sod-Se! FC Se!-Plex
Glutathione S- Gsta3 NS -2.3 -2.5
transferase,
alpha 3
Glutathione S- Gsta4 NS NS -1.7
transferase,
alpha 4
Glutathione S- Gstml NS -2.4 NS
transferase,
mul
Glutathione S- Gstm2 NS -2.1 -2.1
transferase, mu
2
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Glutathione S- Gstm3 NS -2.7 -2.3
transferase, mu
3
Glutathione S- Gsttl NS NS -1.4
transferase,
theta 1
Glutathione S- Gstt2 NS -1.3 NS
transferase,
theta 2
Surprisingly, subjects receiving free dietary selenomethionine (SeM)
demonstrated
no alteration in the expression pattern of these genes (e.g., the GST genes
were not down-
regulated). However, subjects receiving Sod-sel and Sel-Plex displayed an
altered (e.g.,
reduced) expression of GST genes. Thus, the present invention provides that
distinct
differences exist in the ability of different selenium sources to elicit
responses in the
expression profiles of genes (e.g., GST genes and those described elsewhere
herein).
Although an understanding of the mechanism is not necessary to practice the
present invention and the present invention is not limited to any particular
mechanism of
action, in some embodiments, treating a subject with a composition comprising
Sel-Plex
brings about less stress in the subject (e.g., provides lower oxidative stress
levels), thereby
permitting a general down-regulation of the expression of GST genes in
subjects receiving
Sel-Plex . Accordingly, the present invention provides a method of reducing
oxidative
stress in a subject comprising providing to the subject a composition
comprising selenium
(e.g., Sel-Plex or sod-sel) under conditions such that the expression of GST
genes (e.g.,
Gstt2, Gsttl, Gsta3, Gsta4, Gstml, Gstm2, or Gstm3) are reduced. In some
embodiments,
two or more different forms of selenium (e.g., Sel-Plex and Sod-se) are
administered to a
subject. In some embodiments, administering two of more forms of selenium
provides an
additive effect (e.g., provides an additive reduction of GST expression). In
some
embodiments, administering two of more forms of selenium provides a more than
additive
(e.g., synergistic) effect of reducing GST gene expression. In some
embodiments,
administering two of more forms of selenium to a subject does not negate the
effect of
either selenium source to reduce the expression of GST genes. In some
embodiments, the
present invention provides a method of treating a subject with Parkinson's
disease
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CA 02524551 2012-08-31
comprising providing to the subject a composition comprising selenium (e.g., a
dietary
supplement comprising Sel-PlexS) under conditions such that the expression of
GST genes
ubiquitin genes are down-regulated.
In some embodiments, the present invention provides a method of retarding age-
related progression (e.g., increase in oxidative stress levels) in a subject
comprising
providing to the subject a composition comprising selenium (e.g., a dietary
supplement
comprising Sel-Plex8). In other embodiments, the present invention provides a
method of
inhibiting neuronal degeneration (e.g., reducing oxidative stress that leads
to or is causative
of neuronal degeneration) in a subject comprising providing to the subject a
composition
comprising selenium (e.g., a dietary supplement comprising Sel-Plex8).
Although an
understanding of the mechanism is not necessary to practice the present
invention and the
present invention is not limited to any particular mechanism of action, in
some
embodiments, age-retardation and prevention of neurodegeneration is attained
by treating a
subject with a composition comprising selenium (e.g., a dietary supplement
comprising Sel-
Plexe) that leads to the down-regulation of stress induced genes (e.g., GST
genes).
Furthermore, the present invention demonstrates that certain forms of selenium
(e.g., Sel-
Plexe) are capable of altering various gene expression profiles in a subject
that other forms
of selenium (e.g., SeM and/or Sod-sel) are not. Thus, the present invention
provides that, in
some embodiments, Sel-Plex is superior to other forms of selenium (e.g., SeM
or Sod-sel)
for use in nutritional interventions (e.g., for maintaining and prolonging
optimal cognitive
function and retarding agedness).
As described below, data demonstrating the selenium source dependent nature of
gene expression alteration generated in intestinal tissue (e.g., See Examples
2-8 above) is
also observed in other tissue (e.g., brain tissues). Additionally, as
described in Example 9,
below, certain forms of selenium (e.g., Sel-Plexe) demonstrates superior
biological
availability in terms of the amount of selenium deposited (e.g., in brain
tissue) relative to a
variety of other selenium sources.
Example 9
Effect of Various Selenium Sources on Brain Selenium Concentrations in White-
Egg-
Laying Hens and their Offspring
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Compositions and methods of the present invention were utilized to evaluate
the
effects of various Se sources on the accumulation of brain Se concentrations
in hens and
their offspring.
The study was conducted at the Coldstream Research Facility from June 28, 2004
until November 16, 2004. Six dietary treatments were fed to a total of 48 hens
(r=8) that
were bred on three consecutive days starting on September 16, 2004.
The dietary treatments were as follows:
Basal (no added Se)
Selenite (.3 ppm)
Se! Plex (.3 ppm)
Tepsel (.3 ppm)
Se 2000 (.3 ppm)
Selenosource (.3 ppm)
Chicks that hatched were divided into two groups. Half of the chicks were
killed
for brain collection and the remaining chicks were allowed to grow for 14 days
on a Se
deficient diet, at which time they were terminated for brain collection. Hen
brains were
analyzed for Se content individually, whereas the chick brains were
homogenized and
pooled because of small sample size.
Brain Se concentrations for the hens and chicks are shown in Tables 4 and 5,
below,
respectively. The Selenosource value represents only one hen brain analyzed
and therefore
was not included in the statistical analysis.
Hens fed Sel-Plex had the highest concentration of Se compared with all other
treatments included in the model. No increase in brain Se content was observed
due to any
of the remaining treatments when compared with the basal treatment. The brain
Se
concentration in the chick brains represents the numerically highest among all
treatments.
Table 4. Hen Brain Se Concentration Table 5. Chick Brain Se
Concentration at day 14
Treatment ppb S.E. Treatment ppb
1. Basal 870 46 Basal 786
2. Selenite 850 65 Selenite 784
3. Sel-Plex 1125 46 Sel-Plex 995
4. Tepsel 825 55 TepSel 862
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5. Se2000 909 73 Se 2000 884
6. Selenosource 1102* NE Selenosource 872
contrast P =
1 vs. 3 0.0006
3 vs. 5 0.0184
2 vs. 3 0.0018
3 vs. 4 0.0002
*NE = Not estimated due to one sample
SE ¨ standard error
Thus, in addition to being preferred for use in methods of the present
invention
(e.g., for altering gene expression profiles), compositions comprising Sel-
Plex also
provide (e.g., when provided to a subject as a dietary supplement or through
other means),
when compared to equal consumption of other forms of selenium, the highest
levels of
bioavailable selenium (e.g., in brain tissue).
Example 10
Influences of selective forms of dietary selenium on brain (e.g., cerebral
cortex) gene
expression:
Compositions and methods of the present invention were tested to determine
whether they could play a role in altering the aging process (e.g., altering
the level of gene
expression known to be associated with aging). In general, subjects treated
with
compositions and methods of the present invention display gene expression
profiles
consistent with reversal or retardation of the aging process. For example, by
comparing the
gene expression profiles obtained with compositions and methods of the present
invention,
to the gene expression profiles obtained from the brain tissue of very old
(e.g., 30-month
old) mice (See, e.g., Lee, et al., Nature Genetics 25:294 (2000)), nearly
opposite gene
expression patterns were observed between the two cohorts.
For example, in aged animals, a concerted induction of the complement cascade
genes C4, Clqa, Clqb and Clqc was observed (See, e.g., Lee, et al., Nature
Genetics
25:294 (2000)).
The complement system is a complex cascade involving proteolytic cleavage of
serum glycoproteins often activated by cell receptors. This cascade ultimately
results in
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induction of the inflammatory response, phagocyte chemotaxis and opsonization,
and cell
lysis (See, e.g., Villiers et al., Crit Rev Imrnunol.;24:465 (2004); Morgan et
al., Immunol
Lett. 97:171 (2005)).
Complement factors C3a, C5a and C4 can induce vasodilatation, increased
capillary, permeability, and expression of leukocyte adhesion molecules.
Complements C3a
and C4b are opsonins that bridge phagocytes to microorganisms. Comlements C3a
and C4a
promote phagocyte chemotaxis. Complement C3b may be an opsonin for antgen-
antibody
complexes which helps prevent damage from the formation of large, insoluble
immune
aggregates. Complement C5a, like C3a is an anaphylatoxin, and is a chemotactic
attractant
for induction of neutrophilic release of antimicrobial proteases and oxygen
radicals. A
complex of complements C5b,C6, C7, and C8 mediates the polymerization of up to
eighteen C9 molecules into a tube-like membrane attack complex that is
inserted into the
plasma membrane of an unwanted organisms such as of gram-negative bacteria and
viral
infected cells. This channel through the lipid bilayer results in lysis of the
cell. Ischaemic
infarction may also cause initiation of the complement cascade. Excessive
deposits of
membrane attack complexes in tissues may occur following ischaemic injury.
Other
deleterious effects of complement activation include, degranulation of
neutrophils,
basophils and mast cells, unwanted release of the neutrophil products elastase
and oxygen
radicals, and extracorporeal blood circulation. Complement inhibitors have
been suggested
as potential therapeuics for immune diseases and Alzheimer's disease.
The Complement Pathways. Three pathways have been elucidated through which
the complement cascade can be initiated; Classical, Alternate and Lectin
Pathways. All
three pathways merge through at common intersection, complement C3 (See, e.g.,
FIG. 2).
The Classical Pathway: The classical pathway mediates specific antibody
responses. The classical pathway is initiated by the binding of antibodies to
cell surface
antigens. Subsequent binding of the antibody to complement Clq subunits of Cl
result in
catalytically active Cis subunits. The two activated Cls subunits are then
able to catalyze
the assembly of the C3 convertase (complement C4b2a) from complements C2 and
C4.
The Alternate Pathway: The alternate pathway does not require the action of
antibodies to inititate the cascade, but is initiated by foreign cell surface
components. In the
alternate pathway complement C3 undergoes spontaneous cleavage resulting in
complement B binding to C3b. Diffusion of the Ba subunit results in an active
alternate
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pathway C3 convertase (C3bBb). C3bBb is stabilized by binding to properdin
prior to
merging into the common pathway and conversion of C3.
The Lectin Pathway: The lectin pathway is similar to the classical pathway.
Clq is
not involved in the lectin pathway. Instead an opsonin, mannan binding protein
(MBP), is
involved in the initiation process.
Production of complement proteins in the brain leads to the generation of pro-
inflammatory peptides and contributes to neuronal damage associated with
stroke.
Importantly, it has been documented that activated components of the
complement pathway
are associated with Alzheimer's disease (AD) lesions and other
neurodegenerative
disorders such as Multiple Sclerosis (See, e.g., Yasojima et al., Am. J.
Pathology, 154, 927
(1999); Schwab and McGeer, Exp. Neurology, 174, 81 (2002)). Studies in AD
brain have
shown vigorous up-regulation of complement genes (e.g., rnRNAs) and the
appearance of
strong bands in Western blots for complement activation products. The fold
change in
complement components in the brains of old mice versus young mice were as
follows:
Complement C4, up-regulated 4.9 fold; Clqa, up-regulated 1.7 fold; Clqb, up-
regulated 1.8
fold; Clqc, up-regulated 1.8 fold (See, e.g., Lee, et al., Nature Genetics
25:294 (2000)).
Accordingly, it was determined whether compositions and methods of the present
could alter the expression of complement genes in the cerebral cortex. The
effects of
selenium supplementation, using various sources of selenium, on the expression
levels of
genes encoding components of the complement system were as follows:
Gene FC SeM FC Sod-Sel FC Sel-Plex
Complement component 1.12 1.11 -1.28*
1, q subcomponent
binding protein,
Cl qbp
Complement component _1.11 -1.18 -1.58*
1, q subcomponent,
alpha polypeptide,
Cl qa
Complement component _1.15 -1.35 -1.51*
1, q subcomponent, beta
polypeptide,
C 1 qbp
Complement component 1.0 -1.07 -1.49*
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1, q subcomponent,
gamma polypeptide,
Cl qg
Complement component 1.04 -1.29 -1.58*
1, r subcomponent,
Clr
Table 6.
As illustrated in Table 6, above, compositions and methods of the present
invention
were able to alter the expression of various complement genes (e.g., that have
been
demonstrated to be aberrantly expressed in neurodegenerative diseases such as
Alzheimer's
disease). Specifically, a statistically significant down-regulation of
complement component
genes was brought about by selenium (e.g., Sel-Plex , p<0.01, whereas
providing subjects
with compositions comprising SeM or Sod-sel did not provide a statistically
significant
alteration of complement component genes). The ability of selenium (e.g., Sel-
Plexe) to
reduce the expression of genes associated with the complement cascade produces
gene
expression profiles (e.g., reduced expression level) that are highly similar
to that seen in
multiple tissues of calorie-restricted (aging-retarded) mice (See, e.g., Sohal
and Weindrich,
Science, 273, 59 (1996)).
Accordingly, in some embodiments, the present invention provides a method of
retarding age related expression of complement associated genes (e.g., Clq,
Clq alpha, Clq
beta, Clq gamma, and Clqr) in a subject comprising providing to the subject a
composition
comprising selenium (a dietary supplement comprising Sel-Plexe) under
conditions such
that complement associated gene expression is reduced. In some embodiments,
the present
invention provides a method of treating an Alzheimer's disease patient
comprising
providing to the Alzheimer's disease patient a composition comprising selenium
(e.g., Sel-
Plex(11)) under conditions such that symptoms of Alzheimer's disease in the
patient are
reduced. Although an understanding of the mechanism is not necessary to
practice the
present invention and the present invention is not limited to any particular
mechanism of
action, in some embodiments, providing a composition comprising selenium
(e.g., Sel-
Plex3D) to an Alzheimer's subject reduces symptoms associated with Alzheimer's
through
reducing the expression of complement associated genes (e.g., Clq, Clq alpha,
Clq beta,
Clq gamma, and Clqr). In some embodiments, compositions and methods of the
present
invention are used as a prophylactic treatment in order to prevent the onset
of Alzheimer's
disease. In some embodiments, compositions and methods of the present
invention are
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used in combination with other known therapeutic treatments for the treatment
of
neurologic disease (e.g., Alzheimer's disease). In other embodiments,
compositions and
methods of the present invention are used to prevent neurodegeneration (e.g.,
by inhibiting
expression of complement associated genes, or inhibiting the expression of
other genes
described herein as having detrimental effects to cellular homeostasis, such
as GST genes).
Compositions and methods of the present invention also altered the expression
of a
novel member of the TNF/Clq/adiponectin superfamily, CORS-26. CORS-26 displays
structural homolgies to adiponectin, which exerts proinflammatory and
destructive
properties in arthritic synovium (See, e.g., Tamer et al., Arthritis Res.&
Therapy, 7, 23
(2005)). Subjects treated with certain forms of selenium (e.g., SeM and Sod-
Sel) displayed
no alteration in expression levels of CORS-26, whereas subjects that received
a dietary
supplement comprising other forms of selenium (e.g., Sel-Plex ) displayed a
reduction in
expression of 4.61 fold. Thus, in some embodiments, the present invention
provides a
method of treating arthritis in a subject comprising providing to the subject
a composition
comprising selenium (e.g., Sel-Plex ) under conditions such that symptoms
associated
with arthritis are reduced. Although an understanding of the mechanism is not
necessary to
practice the present invention and the present invention is not limited to any
particular
mechanism of action, in some embodiments, providing a composition comprising
Sel-
Plex to a subject with arthritis reduces symptoms associated with arthritis
by reducing
CORS-26 gene expression.
Another class of genes that display a significant level of expression in aged
mice
compared to young mice are the cathepsins (e.g., cathepsins D, S and Z, See,
e.g., Lee et
al., Lee, et al., Nature Genetics 25:294 (2000)). Cathepsins are major
components of the
lysosomal proteolytic system and have been implicated in the processing of
amyloid
precursor protein (APP) to amyloid 13-peptides. Importantly, they are induced
in the brain
of AD patients (See, e.g., Lemere et al., Am. J. Pathology, 146, 848 (1995)).
Using
compositions and methods of the present invention, the expression of genes
encoding a
number of cathepsins was downregulated in response to selenium
supplementation, most
notably by sodium selenite and Sel-Plex (See Table 7, below).
Gene FC SM FC SS FC SP
Cathepsin B -1.03 -1.13* -1.16*
Cathepsin D 1.02 -1.24* -1.29*
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Cathepsin Z -1.13 -1.30* -1.48*
Cathepsin 0 -1.16 -1.18 -1.25*
Table 7. * downregulation significant relative to Se-deficient mice
It was further demonstrated that other genes involved in processing amyloid
precursor protein (APP) were downregulated in response to selenium
supplementation.
One example is 7-secretase. The enzyme complex, 7 -secretase cleaves APP
resulting in the
release of amyloid-11 peptide, a principal component of AD plaques. Nicastrin
is a
transmembrane glycoprotein that interacts with presenilin, Aph-1 and Pen-2 to
form the
high molecular weight complex with 7-secretase activity (Confaloni et al.,
Molecular Brain
Research, 136, 12 (2005)). The expression levels of the genes encoding
nicastrin and
presenilin were downregulated in response to treatment with certain
compositions
comprising selenium of the present invention (e.g., most notably, and
significantly, by Sel-
Plex8).
Gene FC SM FC SS FC SP
Nicastrin 1.04 -1.67*
Presenilin 1 1.02 -1.11 -1.22*
Table 8. *Significant relative to Se-deficient animals. P<0.01.
Furthermore, a number of genes involved in the generation of beta amyloid
peptide
were downregulated in response to treatment with compositions and methods of
the present
invention (e.g., selenium supplementation, See Table 9 below). For example,
the amyloid
beta (A4) precursor protein binding, family B, member 1 gene, (Apbb1/Fe65).
Apbbl/Fe65
is an adaptor protein expressed mainly in the nervous system. APP is cleaved
in the
transmembrane region by 7-secretase. Gamma-cleavage of APP produces the
extracellular
amyloid beta peptide of Alzheimer disease and releases an intracellular tail
fragment. It has
been demonstrated that the cytoplasmic tail of APP forms a multimeric complex
with the
nuclear adaptor protein Fe65 and the histone acetyltransferase TIP60 (See,
e.g., Cao and
Sudhof, Science 293: 115 (2001)). Apbbl/Fe65 binds to APP and the interaction
is
mediated via a phosphotyrosine binding domain in Apbbl/Fe65 and the carboxy-
terminal
cytoplasmic domain of APP. Fe65 modulates trafficking and processing of APP,
including
production of the beta-amyloid peptide that is central to the pathogenesis of
AD (See, e.g.,
Kesavapany et al., Neuroscience, 115, 951, (2002)).
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Gene FC SM FC SS FC SP
Amyloid beta (A4) 1.02 -1.32* -1.21*
precursor protein-
binding, family B,
member 1 (Apbb 1
or Fe65)
Amyloid beta (A4) 1.02 -1.55* -1.45*
precursor-like
protein (Aplp 1)
Amyloid beta (A4) 1.08 -1.41 -1.61*
precursor protein-
binding, family A,
member 1(Apba1)
Table 9. *Significant relative to Se-deficient animals. P<0.01
Thus, in some embodiments, the present invention provides a method of treating
an
Alzheimer's disease patient comprising providing to the Alzheimer's disease
patient a
composition comprising selenium (e.g., Sel-Plexe) under conditions such that
signs and
symptoms of Alzheimer's disease in the patient are reduced. Although an
understanding of
the mechanism is not necessary to practice the present invention and the
present invention
is not limited to any particular mechanism of action, in some embodiments,
providing a
composition comprising selenium (e.g., Sel-Plexe) to an Alzheimer's subject
reduces
symptoms associated with Alzheimer's through reducing the expression of genes
that
encode proteins involved in processing amyloid precursor protein (APP) (e.g.,
Nicastrin,
Presenilin 1, Cathepsin B, Cathepsin D, Cathepsin Z, or Cathepsin 0) or genes
involved in
the generation of beta amyloid peptide (e.g., Apbb 1, Aplp 1, and Apbal). In
some
embodiments, compositions and methods of the present invention are used as a
prophylactic
treatment in order to prevent the onset of Alzheimer's disease. In some
embodiments,
compositions and methods of the present invention are used in combination with
other
known therapeutic treatments for the treatment of neurodegenerative disease
(e.g.,
Alzheimer's disease, Parkinson's disease, Huntington's disease, ALS, etc.). In
other
embodiments, compositions and methods of the present invention are used to
prevent
neurodegeneration (e.g., by inhibiting expression of genes that encode
proteins involved in
processing amyloid precursor protein or genes involved in the generation of
beta amyloid
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peptide), conversely, enhancing expression of genes that provide a beneficial
effect on
cognitive function (e.g., Lhx8).
Studies in the aging mouse brain also identified the induced expression of
early
response genes, Junb and Fos , that are co-induced in response to neocortical
injury or
hypoxic stress (See, e.g., Lee, et al., Nature Genetics 25:294 (2000); Hermann
et al.,
Neuroscience, 88, 599 (1999)). In neocortex, Junb was upregulated 1.8-fold.
The present
invention demonstrates that it is possible to down-regulate the expression of
Junb using
compositions and methods of the present invention. Specifically, the present
invention
provides that it is possible to down-regulate the expression of early response
genes (e.g.,
Junb) in brain tissue (e.g., the neocortex) using compositions and methods
(e.g., dietary
supplementation with Sel-P1ex0) of the present invention.
Gene FC SM FC SS FC SP
Junb -1.38 -1.59 -2.01*
Table 10. *Significant relative to Se-deficient animals.
Similar to data generated in intestinal tissue, a downregulation in DNA-damage
inducible genes was noted in response to treatment with compositions and
methods of the
present invention. For example, in intestinal tissue, a decreased expression
was
demonstrated for Gadd45b with Sel-Plex treatments (p<0.05) and a decreased
expression
of Gadd45glp for all selenium treatments (e.g., SeM, Sod-se! and Sel-Plexe)
(p<0.05) was
demonstrated. In brain, gene expression was altered using compositions and
methods of the
present invention as follows:
Gene FC SM FC SS FC SP
Gadd45b -1.26 -1.39* -1.48
Gadd45glp (Growth 1.02 -1.12 -1.37*
arrest and DNA-
damage inducible
45 gamma
interacting protein
Table 12. *Significant relative to Se-deficient animals.
Other similarities between intestinal and brain data were noted in the area of
Glutathione-S-Transferase (GST) expression. For example. in intestine, a
decreased
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expression of the GST genes, Gsta3, Gsta4 and Gstm3 was demonstrated in the
Sod-se! and
Sel-plex groups (p<0.05). In brain, gene expression of a number of other GST
genes was
altered using compositions and methods of the present invention as follows:
Gene FC SM FC SS FC SP
Gst pi 1 (Gstpl) -1.04 1.02 -1.14*
Gst zeta 1 (Gstzl) -1.08 -1.3 -1.41*
Gst mu 7 (Gstan7) -1.05 -1.25* -1.24*
Table 12. *Significant relative to Se-deficient animals.
Thus, the present invention provides a method of protecting against the by-
products
of oxidative stress in brain tissue comprising providing to a subject a
composition
comprising selenium (e.g., Sel-Plex8). Although an understanding of the
mechanism is not
necessary to practice the present invention and the present invention is not
limited to any
particular mechanism of action, in some embodiments, providing a composition
comprising
selenium (e.g., Sel-Plexe) to a subject reduces the expression of GST genes
(e.g., Gstpl,
Gstz 1, and Gstm7) in the subject In some embodiments, providing a composition
comprising selenium (e.g., Sel-Plex8) to a subject reduces the level of DNA
damage in
brain tissue (e.g., neocortex) of a subject Although an understanding of the
mechanism is
not necessary to practice the present invention and the present invention is
not limited to
any particular mechanism of action, in some embodiments, treatment with
compositions
and methods of the present invention (e.g., dietary supplementation with Sel-
Plexe)
stabilizes cellular homeostasis (e.g., in the brain) such that the expression
of DNA-damage
inducible genes (e.g., Gadd45g1p) is reduced.
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