U.S. patent application number 14/456718 was filed with the patent office on 2014-11-27 for compositions and methods for establishing and/or maintaining pregnancy.
The applicant listed for this patent is Alltech, Inc.. Invention is credited to Thomas P. Lyons, Ronan Power.
Application Number | 20140348872 14/456718 |
Document ID | / |
Family ID | 40027754 |
Filed Date | 2014-11-27 |
United States Patent
Application |
20140348872 |
Kind Code |
A1 |
Lyons; Thomas P. ; et
al. |
November 27, 2014 |
COMPOSITIONS AND METHODS FOR ESTABLISHING AND/OR MAINTAINING
PREGNANCY
Abstract
The present invention relates to compositions comprising
selenium (e.g., organic selenium (e.g., selenized yeast (e.g.,
SEL-PLEX))) and methods of using the same (e.g., as a therapeutic
and/or prophylactic treatment). For example, the present invention
provides compositions comprising selenium (e.g., organic selenium
(e.g., selenized yeast (e.g., SEL-PLEX))) and methods of using the
same for treating and/or preventing one or more conditions (e.g.,
problems) disorders, and/or diseases related to establishing and/or
maintaining a pregnancy. Compositions and methods of the present
invention find use in, among other things, research and clinical
(e.g., preventative and therapeutic) applications.
Inventors: |
Lyons; Thomas P.;
(Nicholasville, KY) ; Power; Ronan; (Lexington,
KY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Alltech, Inc. |
Nicholasville |
KY |
US |
|
|
Family ID: |
40027754 |
Appl. No.: |
14/456718 |
Filed: |
August 11, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12122331 |
May 16, 2008 |
8846111 |
|
|
14456718 |
|
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60930752 |
May 18, 2007 |
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Current U.S.
Class: |
424/195.16 |
Current CPC
Class: |
A61K 48/00 20130101;
A61K 33/04 20130101; A61P 15/00 20180101; A61K 36/064 20130101;
A61K 36/06 20130101 |
Class at
Publication: |
424/195.16 |
International
Class: |
A61K 36/064 20060101
A61K036/064 |
Claims
1. A method of altering expression of one or more genes associated
with establishment of and/or maintenance of pregnancy in a subject
comprising: administering an effective amount of a composition
comprising a dried, nonviable selenium-enriched yeast to the
subject wherein the effective amount enhances the expression of one
or more genes associated with establishment of and/or maintenance
of pregnancy in the subject compared to a control subject.
2. The method of claim 1, wherein the control subject is not
administered selenium.
3. The method of claim 1, wherein the control subject is
administered a composition comprising selenium, wherein the
selenium is not present in a dried, nonviable selenium enriched
yeast.
4. The method of claim 1, wherein the one or more genes associated
with establishment of and/or maintenance of pregnancy is serum and
glucocorticoid inducible-protein kinase (sgk).
5. The method of claim 1, wherein enhancing expression of one or
more genes associated with establishment of and/or maintenance of
pregnancy reduces and/or eliminates the risk of perinatal morbidity
and/or mortality in the subject.
6. The method of claim 1, wherein enhancing expression of one or
more genes associated with establishment of and/or maintenance of
pregnancy increases fetal growth in the subject.
7. The method of claim 7, wherein increasing fetal growth in said
subject comprises increasing fetal growth rate in said subject.
8. The method of claim 1, wherein enhancing expression of one or
more genes associated with establishment of and/or maintenance of
pregnancy increases the duration of gestation in said subject.
9. The method of claim 1, wherein enhancing expression of one or
more genes associated with establishment of and/or maintenance of
pregnancy promotes the health of the uterus.
10. The method of claim 10, wherein promoting the health of the
uterus comprises promoting connective tissue formation and/or
rearrangement.
11. The method of claim 1, wherein enhancing expression of one or
more genes associated with establishment of and/or maintenance of
pregnancy promotes proper formation of the pubis.
12. The method of claim 1, wherein enhancing expression of one or
more genes associated with establishment of and/or maintenance of
pregnancy promotes the health of the embryo and/or fetus in said
subject.
13. The method of claim 13, wherein promoting the health of the
embryo and/or fetus in said subject comprises promoting limb and
nervous system development.
14. The method of claim 13, wherein promoting the health of the
embryo and/or fetus in said subject comprises promoting muscle
growth and differentiation.
15. The method of claim 13, wherein promoting the health of the
embryo and/or fetus in said subject comprises promoting neural tube
development.
16. The method of claim 13, wherein enhancing expression of one or
more genes associated with establishment of and/or maintenance of
pregnancy reduces the risk of and/or incidence of an event selected
from the group consisting of spontaneous abortion, miscarriage, and
birthing complications.
17. The method of claim 1, wherein the subject is a human.
18. The method of claim 1, wherein the subject is a non-human
mammal.
19. The method of claim 1, wherein the subject is selected from the
group consisting of a subject from an avian species and a subject
from an aquatic species.
20. The method of claim 1, wherein said expression is enhanced two
fold or greater in said subject compared to the control
subject.
21. The method of claim 1, wherein said composition comprising
selenium is administered in such a way so as to provide between 100
and 400 .mu.g of selenium to said subject each day.
22. The method of claim 1, wherein said subject is administered
said composition for a period of no less than 6 months prior to
establishing and/or attempting to establishing a pregnancy.
23. The method of claim 1, wherein said subject is administered
said composition during pregnancy.
24. The method of claim 1, wherein said one or more genes
associated with establishment of and/or maintenance of pregnancy in
a subject are selected from the group consisting fibroblast growth
factor 2 (FGF2), SNF1-like kinase, platelet-derived growth factor
alpha (PDFGA), cyclin D1 (CCND1), activin A receptor type 1
(ACVR1), Sp3 transcription factor (SP3), homeobox A7, homeobox D12,
retinoic acid receptor beta (RARB), insulin-like growth factor
binding protein 7, and Protein Kinase A.
Description
[0001] This application claims the benefit of and priority to U.S.
Provisional Patent Application Ser. No. 60/930,752 filed May 18,
2007, hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions comprising
selenium (e.g., organic selenium (e.g., selenized yeast (e.g.,
SEL-PLEX))) and methods of using the same (e.g., as a therapeutic
and/or prophylactic treatment). For example, the present invention
provides compositions comprising selenium (e.g., organic selenium
(e.g., selenized yeast (e.g., SEL-PLEX))) and methods of using the
same for treating and/or preventing one or more conditions (e.g.,
problems) disorders, and/or diseases related to establishing and/or
maintaining a pregnancy. Compositions and methods of the present
invention find use in, among other things, research, agriculture,
and clinical (e.g., preventative and therapeutic) applications.
BACKGROUND OF THE INVENTION
[0003] Infertility and other complications related to pregnancy are
a major problem in the United States and abroad in human and
non-human populations. It is estimated that around 10 million
individuals are affected by infertility and/or other conditions
related to the inability to conceive and/or have children.
[0004] A wide variety of factors are thought to reduce and/or
obstruct fertility including, among other things, genetic factors,
physical factors, health, age, diet, stress, and drug and alcohol
consumption.
[0005] For example, when a woman ages, her reproductive system
(e.g., comprising the reproductive tract, uterus, and ova) also
ages. As a woman ages, she may become less likely to become
pregnant and if she does become pregnant, she runs the risk of
having complications related to the pregnancy.
[0006] What is needed are compositions and methods for increasing
fertility (e.g., increasing conditions favorable for establishing
and/or maintaining pregnancy (e.g., in human and non-human species
(e.g., mammals, birds, etc.))).
SUMMARY OF THE INVENTION
[0007] The present invention relates to compositions comprising
selenium (e.g., organic selenium (e.g., selenized yeast (e.g.,
SEL-PLEX))) and methods of using the same (e.g., as a therapeutic
and/or prophylactic treatment). For example, the present invention
provides compositions comprising selenium (e.g., organic selenium
(e.g., selenized yeast (e.g., SEL-PLEX))) and methods of using the
same for treating and/or preventing one or more conditions (e.g.,
problems) disorders, and/or diseases related to establishing and/or
maintaining a pregnancy. Compositions and methods of the present
invention find use in, among other things, research, agriculture,
and clinical (e.g., preventative and therapeutic) applications.
[0008] Accordingly, in some embodiments, the present invention
provides a method of treating and/or preventing a condition (e.g.,
a problem) a disorder, and/or a disease related to establishing
and/or maintaining a pregnancy comprising administrating to a
subject a composition comprising selenium (e.g., organic selenium
(e.g., selenized yeast (e.g., SEL-PLEX))), for example, under
conditions such that the expression of one or more genes involved
in establishing and/or maintaining pregnancy are altered (e.g.,
enhanced) in the subject.
[0009] In some embodiments, the present invention provides a method
of altering expression of one or more genes associated with
establishment of and/or maintenance of pregnancy in a subject
comprising: providing a subject; and a composition comprising
selenium, wherein the selenium is present within a dried, nonviable
selenium-enriched yeast; and administering the composition to the
subject under conditions such that the expression of one or more
genes associated with establishment of and/or maintenance of
pregnancy is enhanced in the subject compared to a control subject.
In some embodiments, the control subject is not administered
selenium. In some embodiments, the control subject is administered
a composition comprising selenium, wherein the selenium is not
present in a dried, nonviable selenium-enriched yeast. In some
embodiments, the one or more genes associated with establishment of
and/or maintenance of pregnancy is relaxin. In some embodiments,
the one or more genes associated with establishment of and/or
maintenance of pregnancy is serum and glucocorticoid
inducible-protein kinase (sgk). In some embodiments, enhancing
expression of one or more genes associated with establishment of
and/or maintenance of pregnancy reduces and/or eliminates the risk
of perinatal morbidity and/or mortality in the subject. In some
embodiments, enhancing expression of one or more genes associated
with establishment of and/or maintenance of pregnancy increases
fetal growth in the subject. In some embodiments, increasing fetal
growth in the subject comprises increasing fetal growth rate in the
subject. In some embodiments, enhancing expression of one or more
genes associated with establishment of and/or maintenance of
pregnancy increases the duration of gestation in the subject. In
some embodiments, enhancing expression of one or more genes
associated with establishment of and/or maintenance of pregnancy
promotes the health of the uterus. In some embodiments, promoting
the health of the uterus comprises promoting connective tissue
formation and/or rearrangement. In some embodiments, enhancing
expression of one or more genes associated with establishment of
and/or maintenance of pregnancy promotes proper formation of the
pubis. In some embodiments, enhancing expression of one or more
genes associated with establishment of and/or maintenance of
pregnancy promotes the health of the embryo and/or fetus in the
subject. In some embodiments, promoting the health of the embryo
and/or fetus in the subject comprises promoting limb and nervous
system development. In some embodiments, promoting the health of
the embryo and/or fetus in the subject comprises promoting muscle
growth and differentiation. In some embodiments, promoting the
health of the embryo and/or fetus in the subject comprises
promoting neural tube development. In some embodiments, enhancing
expression of one or more genes associated with establishment of
and/or maintenance of pregnancy reduces the risk of and/or
incidence of an event selected from the group comprising
spontaneous abortion, miscarriage, and birthing complications. In
some embodiments, the subject is a human. In some embodiments, the
subject is a non-human mammal. In some embodiments, the subject is
a subject from an avian species. In some embodiments, the subject
is a subject from an aquatic species. In some embodiments, the
expression is enhanced two fold or greater in the subject compared
to the control subject. In some embodiments, expression is enhanced
three fold or greater in the subject compared to the control
subject. In some embodiments, the expression is enhanced greater
than two fold (e.g., three fold, four fold, five fold, six fold or
more) in the subject compared to the control subject. In some
embodiments, expression is enhanced three fold or greater in the
subject compared to the control subject. In some embodiments, the
composition comprising selenium is administered in such a way so as
to provide between 100 and 400 .mu.g of selenium to the subject
each day, although embodiments in which greater than 400 .mu.g and
less than 100 .mu.g are administered are also contemplated. In some
embodiments, the composition comprising selenium is administered in
such a way so as to provide 200 .mu.g of selenium to the subject
each day. In some embodiments, the subject is administered the
composition for a period of no less than 6 months prior to
establishing and/or attempting to establishing a pregnancy. In some
embodiments, a subject is administered the composition for more
than 9 months, more than 12 months, more than 18 months, or more
than two years prior to establishing and/or attempting to establish
a pregnancy. In some embodiments, a subject is administered a
composition of the present invention for less than six months prior
to establishing and/or attempting to establish a pregnancy. In some
embodiments, the subject is administered the composition during
pregnancy. In some embodiments, the one or more genes associated
with establishment of and/or maintenance of pregnancy in a subject
are selected from the group comprising fibroblast growth factor 2
(FGF2), SNF1-like kinase, platelet-derived growth factor alpha
(PDFGA), cyclin D1 (CCND1), activin A receptor type 1 (ACVR1), Sp3
transcription factor (SP3), homeobox A7, homeobox D12, retinoic
acid receptor beta (RARB), insulin-like growth factor binding
protein 7, and Protein Kinase A.
[0010] The present invention also provides a method of treating a
subject comprising: identifying a subject that will benefit from
the treatment; providing: the subject; and a composition comprising
selenium, wherein the selenium is present within a dried, nonviable
selenium-enriched yeast; and administering the composition to the
subject under conditions such that the expression of one or more
genes associated with establishment of and/or maintenance of
pregnancy is enhanced in the subject compared to a control subject.
In some embodiments, identifying the subject comprises detecting
the level of expression of one or more genes associated with
establishment of and/or maintenance of pregnancy. In some
embodiments, the one or more genes associated with establishment of
and/or maintenance of pregnancy is relaxin. In some embodiments,
the one or more genes associated with establishment of and/or
maintenance of pregnancy is serum and glucocorticoid
inducible-protein kinase (sgk). In some embodiments, the one or
more genes associated with establishment of and/or maintenance of
pregnancy is selected from the group comprising fibroblast growth
factor 2 (FGF2), SNF1-like kinase, platelet-derived growth factor
alpha (PDFGA), cyclin D1 (CCND1), activin A receptor type 1
(ACVR1), Sp3 transcription factor (SP3), homeobox A7, homeobox D12,
retinoic acid receptor beta (RARB), insulin-like growth factor
binding protein 7, and Protein Kinase A. In some embodiments,
detecting the level of expression of one or more genes associated
with establishment of and/or maintenance of pregnancy comprises
detecting nucleic acid expression and/or protein expression. In
some embodiments, the method further comprises: d) detecting the
level of one or more genes associated with establishment of and/or
maintenance of pregnancy after administration of the composition.
The present invention contemplates the identification of different
types of subjects that may benefit from the compositions and
methods disclosed herein including, but not limited to, a subject
desiring reduction and/or elimination of the risk of perinatal
morbidity and/or mortality; a subject desiring an increase in fetal
growth in the subject; a subject desiring an increase in fetal
growth rate in the subject; a subject desiring an increase in the
duration of gestation in the subject; a subject desiring the
promotion of connective tissue formation and/or rearrangement in
the subject; a subject desiring promotion of proper formation of
the pubis; a subject desiring the promotion of the health of the
embryo and/or fetus in the subject; a subject desiring promotion of
limb and nervous system development in an embryo and/or fetus in
the subject; a subject desiring promotion of muscle growth or
differentiation in an embryo and/or fetus in the subject; a subject
desiring promotion of neural tube development in an embryo and/or
fetus in the subject; a subject desiring a reduction in the risk
and/or incidence of spontaneous abortion; a subject desiring a
reduction in the risk and/or incidence of miscarriage; a subject
desiring a reduction in the risk and/or incidence of stillbirth;
and a subject desiring a reduction in the risk and/or incidence of
birthing complications.
[0011] 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. 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-PLEX))) and one or more different forms
of selenium provides an additive benefit (e.g., enhancement of gene
expression) to the subject. In some embodiments, providing selenium
(e.g., organic selenium (e.g., selenized yeast (e.g., SEL-PLEX)))
and one or more different forms of selenium provides a synergistic
(e.g., more than additive) benefit (e.g., enhancement of gene
expression) to the subject. In some embodiments, providing selenium
(e.g., organic selenium (e.g., selenized yeast (e.g., SEL-PLEX)))
and one or more different forms of selenium provides altered (e.g.,
enhanced) expression of more genes than are altered (e.g.,
enhanced) with either form of selenium alone.
[0012] 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 25-800
.mu.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))) is administered to the subject
so as to provide between 200-400 .mu.g 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 25 and 75
.mu.g 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 .mu.g of
selenium each day.
DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows the fold change (FC) in the expression of
GSH-PX genes in oviduct, relative to selenium deficient hens
induced by sodium selenite (SS) and SEL-PLEX (SP).
[0014] FIG. 2 shows the changes in expression profiles of multiple
genes involved in reproduction and embryogenesis in response to
sodium selenite or SEL-Plex. The numbers indicate Fold Change (FC)
relative to selenium deficiency, grey background indicates a
statistically significant up-regulation (p<0.05) and the black
background no significant change relative to selenium
deficiency.
[0015] FIG. 3 shows the changes in expression profiles of multiple
genes involved in reproduction and embryogenesis in response to
sodium selenite or SEL-PLEX administration. The numbers indicate
Fold Change (FC) relative to selenium deficiency, grey background
indicates a statistically significant up-regulation (p<0.05) and
the black background no significant change relative to selenium
deficiency.
[0016] FIG. 4 shows the changes in expression profiles of Protein
Kinase A, transcription factor SP3 and
Serum/glucocorticoid-regulated kinase (Sgk), and an interrelation
among the same, in response to sodium selenite or SEL-PLEX
administration. The numbers indicate Fold Change (FC) relative to
selenium deficiency.
[0017] FIG. 5 shows the effect on relaxin gene expression in
response to sodium selenite or SEL-PLEX administration.
DEFINITIONS
[0018] As used herein, the terms "pregnant subject," "pregnancy"
and "pregnant" (e.g., when used in reference to a subject) refers
to a subject (e.g., human or non-human mammal, avian species,
aquatic species (e.g., fish)) carrying one or more developing
offspring within its body. The pregnant subject may have conceived
naturally or may have become pregnant through an artificial
technique (e.g., artificial reproductive technology (e.g., in vitro
fertilization or embryo transfer)). The developing offspring may be
at any stage of gestation (e.g., embryonic or fetal stage (e.g.,
depending upon how much time has passed since fertilization (e.g.,
in humans, this transition occurs at about eight weeks following
implantation of the embryo))).
[0019] As used herein, the term "mammal" refers to any of the
various warm-blooded vertebrate animals of the class Mammalia.
[0020] As used herein, the term "gestation" refers to the period of
development in the uterus from the time of conception until
parturition/birth, and "gestational age" refers to the length of
this time period.
[0021] Conception refers to the formation of one or more viable
zygotes through the union of sperm and egg.
[0022] Birth refers to the emergence and separation of offspring
from the mother, whether by natural or assisted means.
[0023] As used herein, the terms "peptide," "polypeptide" and
"protein" 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).
[0024] 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.
[0025] 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. 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.
[0026] As used herein, the term "SEL-PLEX" 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.
[0027] 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.
[0028] 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). 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.
[0029] As used herein, the term "oxidative stress" refers to the
cytotoxic effects of oxygen radicals (e.g., superoxide anion
(O.sub.2.sup.-), hydroxy radical (OH), and hydrogen peroxide
(H.sub.2O.sub.2)), generated, for example, as byproducts of
metabolic processes that utilize molecular oxygen (See e.g., Coyle
et al., Science 262:689-695 (1993)).
[0030] 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, goats,
swine, avian species (e.g., domesticated fowl (e.g., chickens,
turkeys, ducks, geese, and guinea fowl, etc.) and other game birds
(e.g., quail, pheasants, grouse, partridges, emu, ostriches,
pigeons, etc.), as well as aquatic species (e.g., fresh water and
marine (salt water) species (e.g., including, but not limited to,
fish (e.g., bass, catfish, carp, cod, crappie, eel, flounder,
grouper, haddock, halibut, herring, kingfish, mackerel, mahi mahi,
orange roughy, perch, pike, Pollock, salmon, sardine, shark,
snapper, sole, tilapia, trout, tuna, and walleye), crustaceans
(e.g., crab, crayfish, lobster, prawn, and shrimp), mollusks, etc.)
that is studied, analyzed, tested, diagnosed or treated (e.g.,
using the compositions and methods of the present invention). As
used herein, the terms "host," "subject" and "patient" are used
interchangeably, unless indicated otherwise.
[0031] 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 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.).
[0032] As used herein, the terms "auto-antibody" or
"auto-antibodies" refer to any immunoglobulin 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."
[0033] As used herein, the tem). "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 immunogens, 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.
[0034] 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.
[0035] 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 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.
[0036] 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).
[0037] 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.
[0038] The terms "Western blot," "Western immunoblot" "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).
[0039] 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 Protocols
in Molecular Biology, Ch. 11, John Wiley & Sons, Inc., New York
(1994)). In addition, there are numerous commercially available
ELISA test systems.
[0040] 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 definition 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.
[0041] 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.
[0042] As used herein, the teem "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.
[0043] 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.
[0044] 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, SelW, Sepn1,
SelR, Sod2, Dio2, Glo1, Phb, Lhx8, TGF-J31, glutathione
peroxidases, fibroblast growth factor 1 (FGF1), fibroblast growth
factor 2 (FGF2), SNF1-like kinase, platelet-derived growth factor
alpha (PDFGA), cyclin D1 (CCND1), activin A receptor type 1
(ACVR1), Sp3 transcription factor (SP3), homeobox A7, homeobox D12,
retinoic acid receptor beta (RARB), insulin-like growth factor
binding protein 7, Protein Kinase A, Serum and Glucocorticoid
inducible-protein kinase (Sgk), and relaxin). Examples of suitable
reagents include, but are not limited to, nucleic acid probes
capable of specifically hybridizing to mRNA or cDNA, and antibodies
(e.g., monoclonal or polyclonal antibodies).
[0045] As used herein, the term "effective amount" refers to the
amount of a composition (e.g., comprising selenium (e.g.,
SEL-PLEX)) sufficient to effect beneficial or desired results. For
example, in the case of pregnancy, an "effective amount" is an
amount necessary to improve the likelihood of establishing and/or
maintaining pregnancy (e.g., from embryonic to fetal state, or from
first to second trimester, or from second to third trimester,
etc.). In some embodiments, an effective amount is sufficient to
achieve a successful result in at least 55%, at least 65%, at least
75%, at least 85%, or at least 95% of the occasions administered
(e.g., in the absence of other factors). 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.
[0046] 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 mouth (oral),
through the eyes (ophthalmic), 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.
[0047] 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., embryonic and/or fetal nutrient and/or
medicament, 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.
[0048] As used herein, the term "treatment" or grammatical
equivalents encompasses the improvement and/or reversal of symptoms
of disease and/or disorder related to, and/or a problem with
establishing and/or maintaining a pregnancy (e.g., intrauterine
growth retardation, placental and/or corpus luteum insufficiency).
A compound that causes an improvement in any parameter associated
with a disease, disorder, or a problem (e.g., with establishing
and/or maintaining a pregnancy) when 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 identified as having a
disease, disorder, or a problem (e.g., with establishing and/or
maintaining a pregnancy) as well as those in which a disease,
disorder, or a problem (e.g., with establishing and/or maintaining
a pregnancy) is to be prevented (e.g., using a prophylactic
treatment of the present invention).
[0049] As used herein, the term "at risk for having a problem with
establishing and/or maintaining a pregnancy" refers to a subject
(e.g., a human) that is predisposed to experiencing a problem with
establishing and/or maintaining a pregnancy. This predisposition
may be genetic (e.g., a particular genetic tendency to experience a
problem with establishing and/or maintaining a pregnancy, such as
heritable disorders), or due to other factors (e.g., age, prior
experience of a problem with establishing and/or maintaining a
pregnancy, drug or alcohol use, 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 problem with establishing and/or
maintaining a pregnancy.
[0050] As used herein, the term "experiencing a problem with
establishing and/or maintaining a pregnancy" refers to a subject
(e.g., a human) that is experiencing a particular problem with
establishing and/or maintaining a pregnancy. It is not intended
that the present invention be limited to any particular signs or
symptoms, nor problem. Thus, it is intended that the present
invention encompass subjects that are experiencing any type of
problem related to establishing and/or maintaining a pregnancy
wherein the subject exhibits at least some indicia (e.g., sign
and/or symptom) associated with the problem with establishing
and/or maintaining a pregnancy.
[0051] 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.
[0052] 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, problem with establishing
and/or maintaining a pregnancy, 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 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).
[0053] 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.
[0054] 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.
[0055] As used herein, the term "pharmaceutical composition" refers
to the combination of an active agent (e.g., composition comprising
SEL-PLEX) with a carrier, inert or active, making the composition
especially suitable for diagnostic or therapeutic use in vitro, in
vivo or ex vivo.
[0056] 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.
[0057] 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).
[0058] 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), incorporated herein by reference).
[0059] 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 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.
[0060] 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
NW.sub.4.sup.+, wherein W is C.sub.1-4 alkyl, and the like.
[0061] 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
Na.sup.+, NH.sub.4.sup.+, and NW.sub.4.sup.+ (wherein W is a
C.sub.1-4 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.
[0062] 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.
[0063] 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-carboxymethylaminomethyl-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-methoxy-aminomethyl-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.
[0064] 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.
[0065] As used herein, the teens "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 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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 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.
[0070] 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.
[0071] 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.
[0072] 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 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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 T.sub.m 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."
[0077] As used herein, the term "T.sub.m" 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 T.sub.m of nucleic acids is well known
in the art. As indicated by standard references, a simple estimate
of the T.sub.m value may be calculated by the equation:
T.sub.m=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 T.sub.m.
[0078] 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.
[0079] "High stringency conditions" when used in reference to
nucleic acid hybridization comprise conditions equivalent to
binding or hybridization at 42.degree. C. in a solution consisting
of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4.H.sub.2O
and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,
5.times.Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm
DNA followed by washing in a solution comprising 0.1.times.SSPE,
1.0% SDS at 42.degree. C. when a probe of about 500 nucleotides in
length is employed.
[0080] "Medium stringency conditions" when used in reference to
nucleic acid hybridization comprise conditions equivalent to
binding or hybridization at 42.degree. C. in a solution consisting
of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4.H.sub.2O
and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,
5.times.Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm
DNA followed by washing in a solution comprising 1.0.times.SSPE,
1.0% SDS at 42.degree. C. when a probe of about 500 nucleotides in
length is employed.
[0081] "Low stringency conditions" comprise conditions equivalent
to binding or hybridization at 42.degree. C. in a solution
consisting of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l
NaH.sub.2PO.sub.4.H.sub.2O and 1.85 g/l EDTA, pH adjusted to 7.4
with NaOH), 0.1% SDS, 5.times.Denhardt's reagent
(50.times.Denhardt's contains per 500 ml: 5 g Ficoll (Type 400,
Pharamcia), 5 g BSA (Fraction V; Sigma)) and 100 .mu.g/ml denatured
salmon sperm DNA followed by washing in a solution comprising
5.times.SSPE, 0.1% SDS at 42.degree. C. when a probe of about 500
nucleotides in length is employed.
[0082] 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").
[0083] 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.
[0084] 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.
[0085] 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).
[0086] 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-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 immunoglobulins 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.
[0087] 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.
[0088] 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.
[0089] 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, DEAF-dextran-mediated
transfection, polybrene-mediated transfection, electroporation,
microinjection, liposome fusion, lipofection, protoplast fusion,
retroviral infection, and biolistics.
[0090] 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.
[0091] 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.
[0092] 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 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.sup.- 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.sup.- 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.
[0093] 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.
[0094] 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).
[0095] 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 the natural environment (e.g., an animal or a cell) and
to processes or reaction that occur within a natural
environment.
[0096] 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 problem with establishing
and/or maintaining a pregnancy, a disease, illness, sickness, or
disorder of bodily function. 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.
[0097] 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.
[0098] 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.
[0099] 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 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 posttranscriptional gene silencing in plants.
[0100] 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
[0101] 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, Annu.
Rev. Biochem. 52, 711-760 (1983); Deleve and Kaplowitz, Pharm.
Ther. 52, 287-305 (1991); Palmer and Paulson, Nutr. Rev. 55,
353-361 (1997)).
[0102] 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)).
[0103] Selenium is important for optimum fertility in males, in
both humans and animals. For example, it has been shown that
selenium supplementation of rooster diets significantly reduces the
incidence of sperm abnormalities (See, e.g., Edens et al.,
Practical applications for selenomethionine: broiler breeder
reproduction. In: Nutritional Biotechnology in the Feed and Food
Industries: Proceedings of Alltech's 18.sup.th Annual Symposium (T.
P. Lyons and K. A. Jacques, eds), Nottingham University Press,
Nottingham, UK, pp. 29-42, 2002). Considerably less information is
available on the effects of selenium in female fertility.
[0104] 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.
[0105] 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.
[0106] 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., Furnsinn 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.
[0107] 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 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 et al., 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 Prev, 24: 305-13
(2000)).
[0108] 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
[0109] 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 might 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, metabolic systems, etc.) of a subject (e.g., a
human or non-human mammal, avain species, bovine or other animal).
Furthermore, understanding how various forms of selenium differ in
their ability to exert effects on a subject may provide 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 may also be identified and avoided.
[0110] Accordingly, the present invention relates to compositions
comprising selenium (e.g., organic selenium (e.g., selenized yeast
(e.g., SEL-PLEX))) and methods of using the same (e.g., as a
therapeutic and/or prophylactic treatment). For example, the
present invention provides compositions comprising selenium (e.g.,
organic selenium (e.g., selenized yeast (e.g., SEL-PLEX))) and
methods of using the same for treating and/or preventing one or
more conditions (e.g., problems) disorders, and/or diseases related
to establishing and/or maintaining a pregnancy. Compositions and
methods of the present invention find use in, among other things,
research and clinical (e.g., preventative and therapeutic)
applications.
[0111] In some embodiments, the present invention provides a method
for treating and/or preventing one or more events (e.g., problems
(e.g., a condition, a disorder, and/or a disease)) related with
establishing and/or maintaining a pregnancy in a female subject,
comprising administrating to the subject a composition comprising
selenium (e.g., organic selenium (e.g., selenized yeast (e.g.,
SEL-PLEX))) under conditions such that one or more genes associated
with the establishment of and/or maintenance of pregnancy are
altered (e.g., enhanced). The present invention identified a number
of genes (e.g., the expression of which has been documented to be
instrumental in the establishment and maintenance of pregnancy
and/or healthy embryogenesis) that were upregulated in subjects
after administration of selenium (e.g., organic selenium (e.g.,
selenized yeast (e.g., SEL-PLEX))) to the subjects compared to
subjects that were not administered selenium (See Example 1). For
example, these genes include, but are not limited to, TGF-.beta.1,
glutathione peroxidases, fibroblast growth factor 1 (FGF1),
fibroblast growth factor 2 (FGF2), SNF 1-like kinase,
platelet-derived growth factor alpha (PDFGA), cyclin D1 (CCND1),
activin A receptor type 1 (ACVR1), Sp3 transcription factor (SP3),
homeobox A7, homeobox D12, retinoic acid receptor beta (RARB),
insulin-like growth factor binding protein 7, Protein Kinase A,
Serum and Glucocorticoid inducible-protein kinase (Sgk), and
relaxin.
[0112] Relaxin is a 6 kDa protein hormone member of the
insulin-like growth factor family (See, e.g., Shirota et al., Ann N
Y Acad Sci. 2005 May; 1041:144-6; Dschietzig and Stangl, 2003 Cell
Mol Life Sci. 2003 April; 60(4):688-700). Relaxin exerts pronounced
effects on the female reproductive tract (e.g., that are involved
in the maintenance of pregnancy and successful parturition).
Relaxin is important for normal delivery in several mammalian
species because of its marked rearrangement of reproductive tract
connective tissue. In rats, there is a major pre-labor surge in
circulating relaxin levels and relaxin is critical for cervical
dilation. In guinea pigs, relaxin significantly increases the
intrapubic ligament to enlarge the diameter of the pubis. It is
also essential for normal delivery in pigs. In humans, relaxin is
involved in the establishment and maintenance of pregnancy and
successful parturition. For example, relaxin stimulates production
of several endometrial products including prolactin, glycodelin,
insulin-like growth factor binding factor 1 (IGFBP-1) and vascular
endothelial growth factor in progesterone-primed human endothelial
cells in vitro. In rhesus monkeys, relaxin promotes uterine weight
as well as endometrial lymphocyte and arteriole number. Overall,
relaxin is viewed as a very significant factor in the establishment
and/or maintenance of pregnancy (See, e.g., Goldsmith et al., 2004.
Proceedings of the National Academy of Sciences,
101:4685-4689).
[0113] According to Brackett et al., (See, e.g., Bracket et al.,
1997. General and Comparative Endocrinology, 105:155-163; Bracket
et al., 1985. Biology of Reproduction, 32(Suppl. 1):43), at the
time of onset of puberty or following molting in hens, a sevenfold
increase in pubic bone width occurs. The widening of pubic bone
width is analogous to the effect of relaxin on lengthening and
widening of the pelvic girdle in other species (e.g., the guinea
pig) during pregnancy (See, e.g., Zarrow, 1947. Proceedings of the
Society for Experimental Biology and Medicine, 66:489-491). Thus,
in hens, relaxin plays a role in oviposition by influencing
biochemical as well as morphological alterations in the uterus,
oviduct and pelvic ligaments. Furthermore, teachings regarding
relaxin effects in hens are not limited to birds and/or animals.
Indeed, the hen oviduct is viewed as one of the most valuable model
systems for studying the general effects of sex hormones (e.g.,
estrogen and progesterone) as applied to all species (See, e.g.,
Dougherty and Sanders, 2005. Estrogen action: revitalization of the
chick oviduct model. Trends in Endocrinology and Metabolism,
16:414-419).
[0114] Fibroblast growth factor 1 (FGF1) is involved in
angiogenesis, cell proliferation and differentiation, embryonic
development, organogenesis, and tissue repair, (See, e.g., Krauss
et al., J Cell Sci. 2005 Jun. 1; 118(Pt 11):2355-62). Fibroblast
growth factor 2 (FGF2) is involved in limb and nervous system
development, cell cycle regulation, as well as muscle
growth/differentiation (See, e.g., Poole et al., Dev Dyn. 2001
January; 220(1):1-17; Mehler, Results Probl Cell Differ. 2002;
39:27-52). SNF1-like kinase is involved in regulation of insulin
receptor signaling, cell cycle regulation and muscle growth and
differentiation (See, e.g., Stephenson et al., Genomics. 2004 June;
83(6):1105-1). Platelet-derived growth factor alpha (PDFGA) is
involved in growth factor activity, regulation of progression
through the cell cycle, and cell proliferation (See, e.g.,
Betsholtz, Cytokine Growth Factor Rev. 2004 August; 15(4):215-28).
Cyclin D1 is involved in cell cycle and cell division regulation
(See, e.g., Nemer and Horb, Cell Cycle. 2007 January; 6(2):117-21).
Activin A receptor type 1 (ACVR1) is involved in activin signaling,
FSH production, and follicular development in the ovary (See, e.g.,
Florio et al., Steroids. 2003 November; 68(10-13):801-7). Sp3
transcription factor (SP3) and homeobox (HOX) proteins (e.g., HOX
A7 and HOX D12) are transcription factors involved in developmental
processes (See, e.g., Koos et al., Ann N Y Acad Sci. 2005 May;
1041:233-47; Li et al., Biochem Cell Biol. 2004 August;
82(4):460-71; Bondos, Sci STKE. 2006 Oct. 3; 2006(355):pe38;
Hombria, Differentiation. 2003 October; 71(8):461-76; and DeLise,
Osteoarthritis Cartilage. 2000 September; 8(5):309-34). Retinoic
acid receptor beta (RARB) is involved in vertebrate development,
steroid hormone receptor/retinoic acid receptor activity, as well
as neural tube development in the embryo (See, e.g., Mark et al.,
Annu Rev Pharmacol Toxicol. 2006; 46:451-80; Underhill et al.,
Novartis Found Symp. 2001; 232:171-85; discussion 185-8; and Jettan
et at, Prog Nucleic Acid Res Mol Biol. 2001; 69:205-47).
Insulin-like growth factor binding protein 7 is involved with
regulation of cell growth and insulin-like growth factor binding
(See, e.g., Kleinberg et al., Endocrinology. 2007 March;
148(3):1080-8; Blum et al., J Clin Endocrinol Metab. 2007 January;
92(1):219-28).
[0115] Serum and Glucocorticoid inducible-protein kinase (Sgk) is
an immediate-early gene known to be important in the proliferation
and differentiation of granulosa cells surrounding the developing
oocyte (See, e.g., Alliston et al., 1997 Molecular Endocrinology,
11:1934-1949; Chudgar et al., Mol Vis. 2006 Sep. 30; 12:1117-26).
Granulosa cells produce steroids and a range of other growth
factors known to be essential for oocyte development (See, e.g.,
Wang et al., Fertil Steril. 2006 November; 86(5):1392-401; Machelon
et al., J Gynecol Obstet Biol Reprod (Paris). 2006 September; 35(5
Pt 2):2S14-2S18).
[0116] Accordingly, in some embodiments, the present invention
provides a method of enhancing the expression relaxin in a subject
comprising providing a subject and a composition comprising
selenium (e.g., organic selenium (e.g., selenized yeast (e.g.,
SEL-PLEX))) and administrating the composition to the subject under
conditions such that the expression of relaxin is enhanced in the
subject (e.g., compared to a subject not administered the
composition) (See, e.g., Example 1 and FIG. 5).
[0117] In some embodiments, administration of selenium (e.g.,
organic selenium (e.g., selenized yeast (e.g., SEL-PLEX))) to a
female subject (e.g., a subject that is, or that is attempting to
become, pregnant) promotes fertilization and/or healthy
embryogenesis and/or gestation in the subject (e.g., normal embryo
development (e.g., limb and nervous system development, muscle
growth and differentiation and neural tube development (e.g., due
to the expression of one or more genes (e.g., fibroblast growth
factor 2 (FGF2), SNF1-like kinase, platelet-derived growth factor
alpha (PDFGA), cyclin D1 (CCND1), activin A receptor type 1
(ACVR1), Sp3 transcription factor (SP3), homeobox A7, homeobox D12,
retinoic acid receptor beta (RARB), insulin-like growth factor
binding protein 7, Protein Kinase A, Serum and Glucocorticoid
inducible-protein kinase (Sgk), and/or relaxin (See FIG. 2) being
altered (e.g., upregulated) upon administration of selenium))).
[0118] In some embodiments, fertilization and/or healthy
embryogenesis and/or gestation in the subject (e.g., subsequent to
administration of selenium (e.g., organic selenium (e.g., selenized
yeast (e.g., SEL-PLEX))) results from proper rearrangement of
connective tissues (e.g., within the uterus (e.g., of a human)
and/or oviduct (e.g., in a hen)) during pregnancy. In some
embodiments, fertilization and/or healthy embryogenesis and/or
gestation in the subject (e.g., subsequent to administration of
selenium (e.g., organic selenium (e.g., selenized yeast (e.g.,
SEL-PLEX))) results from correction of corpus luteum and/or
placental insufficiency (e.g., due to the expression of genes
induced subsequent to administration of selenium). In some
embodiments, fertilization and/or healthy embryogenesis and/or
gestation in the subject (e.g., subsequent to administration of
selenium (e.g., organic selenium (e.g., selenized yeast (e.g.,
SEL-PLEX))) results from proper formation (e.g., widening) of the
pubis. In some embodiments, fertilization and/or healthy
embryogenesis and/or gestation in the subject (e.g., subsequent to
administration of selenium (e.g., organic selenium (e.g., selenized
yeast (e.g., SEL-PLEX))) results from increasing intrauterine fetal
growth (e.g., due to induction of genes expression (e.g., within
the female subject (e.g., within granulosa cells (e.g., that
promote oocyte development)))). In some embodiments, genes induced
by the granulosa cells are steroids and/or growth factors.
[0119] In some embodiments, fertilization and/or healthy
embryogenesis and/or gestation in the subject (e.g., subsequent to
administration of selenium (e.g., organic selenium (e.g., selenized
yeast (e.g., SEL-PLEX))) results from proper generation and/or
maintenance of endometrial products subsequent to administration of
selenium. In some embodiments, fertilization and/or healthy
embryogenesis and/or gestation in the subject (e.g., subsequent to
administration of selenium (e.g., organic selenium (e.g., selenized
yeast (e.g., SEL-PLEX))) results from proper formation and/or
health of the oviduct, uterus and/or pelvic ligaments (e.g., in a
hen administered selenium).
[0120] Accordingly, in some embodiments, because the present
invention provides that the administration of selenium (e.g.,
organic selenium (e.g., selenized yeast (e.g., SEL-PLEX))) results
in a significant elevation of expression of genes involved in the
establishment of and/or maintenance of a pregnancy, the present
invention identifies one or more classes of subjects that may
benefit from administration of selenium (e.g., organic selenium
(e.g., selenized yeast (e.g., SEL-PLEX))). For example,
compositions (e.g., that enhance the expression of genes related to
establishing and/or maintaining pregnancy when administered to a
subject) and methods (e.g., of administering a composition
comprising selenium to a subject) of the present invention may be
utilized by any subject that is attempting to become or that is
pregnant.
[0121] In some embodiments, compositions and methods of the present
invention may be utilized (e.g., prescribed to and/or administered
to) by a subject in which one or more of the following are desired:
proper rearrangement of connective tissues (e.g., within the uterus
(e.g., of a human) and/or oviduct (e.g., in a hen)) during
pregnancy (e.g., related to increase expression of relaxin and/or
other genes disclosed herein (e.g., the expression of which is
altered by administration of selenium); correction of corpus luteum
and/or placental insufficiency (e.g., due to the expression of
genes induced subsequent to administration of selenium (e.g.,
relaxin and/or gsk); proper formation (e.g., widening) of the pubis
(e.g., during pregnancy (e.g., related to the expression of
relaxin); increased intrauterine fetal growth (e.g., due to induced
expression of genes (e.g., within the female subject (e.g., within
granulosa cells (e.g., that promote oocyte development (e.g.,
steroids and/or growth factors (e.g., those identified
herein))))))))); proper generation and/or maintenance of
endometrial products; healthy embryogenesis and/or gestation (e.g.,
due to expression of one or more genes described herein (e.g.,
relaxin and/or gsk); and/or proper formation and/or health of the
oviduct, uterus and/or pelvic ligaments (e.g., in a hen
administered selenium (e.g., due to expression of relaxin and/or
one or more other genes whose expression is upregulated subsequent
to administration of selenium as described herein))).
[0122] In some embodiments, compositions and methods of the present
invention may be utilized by (e.g., prescribed to and/or
administered to) a subject identified (e.g., via a test and/or
procedure (e.g., diagnostic test and/or procedure)) as one who will
benefit from the compositions and methods of the present invention
(e.g., identified as a subject at risk for having a problem with
establishing and/or maintaining a pregnancy). For example, in some
embodiments, prior to administrating compositions and methods of
the present invention to a subject, a subject is first identified
as one who has one or more of the following: placental
insufficiency (See, e.g., Lepercq and Mahieu-Caputo, 1998, Horm.
Res. 49(suppl 2):14-19); a specific maternal weight and/or height
prior to pregnancy (e.g., that is identified as being at risk for
non full-term pregnancy); low weight gain during pregnancy;
maternal history of non-full term pregnancies (e.g., spontaneous
abortion, stillbirth, neonatal death, and/or premature
parturition); previous offspring with low birth weight; specific
maternal activities placing the pregnancy at risk (e.g., smoking,
alcohol and/or drug use, and/or poor nutrition); early intrauterine
infections; maternal medical diseases; multiparous pregnancies; a
history of or newly experienced complications arising during
pregnancy; and/or a general desire for establishing pregnancy
and/or maintenance of a healthy pregnancy.
[0123] In some embodiments, a subject is identified (e.g., as a
candidate for compositions and methods of the present invention) by
measuring expression levels of certain genes (e.g., nucleic acid
levels (e.g., cDNA, RNA, etc.) and/or proteins. For example, in
some embodiments, the expression level of one or more genes
associated with (e.g., important for) establishing pregnancy and/or
maintaining pregnancy are measured in a subject, and a subject is
identified as a candidate for receiving compositions and methods of
the present invention. For example, in some embodiments, the
expression level of one or more genes from the group fibroblast
growth factor 2 (FGF2), SNF1-like kinase, platelet-derived growth
factor alpha (PDFGA), cyclin D1 (CCND1), activin A receptor type 1
(ACVR1), Sp3 transcription factor (SP3), homeobox A7, homeobox D12,
retinoic acid receptor beta (RARB), insulin-like growth factor
binding protein 7, Protein Kinase A, Serum and Glucocorticoid
inducible-protein kinase (Sgk), and relaxin are measured and
subjects identified.
[0124] In some embodiments, a subject with low to non-detectable
levels of one or more genes associated with (e.g., important for)
establishing pregnancy and/or maintaining pregnancy (e.g., relaxin
and/or sgk) compared to a normal, healthy control, and/or a to a
level identified (e.g., through standardization) to be that of a
subject likely to establish and/or maintain a healthy pregnancy,
identified using the compositions and methods of the present
invention, can be administered a composition comprising selenium
(e.g., organic selenium (e.g., selenized yeast (e.g., SEL-PLEX)))
in order to elevate expression of one or more of the genes with low
to non-detectable expression levels. In some embodiments, at some
point after administration (e.g., a point during administration
(e.g., during a course of administration as described herein) or
subsequent to the last administration), the expression level (e.g.,
nucleic acid and/or protein expression levels) of one or more of
the genes can be monitored (e.g., detected (e.g., in order to
characterize the effectiveness of the treatment)). In some
embodiments, depending upon the expression level of the one or more
genes, the administration of selenium may be altered (e.g.,
increased or decreased), terminated, or left unchanged. As
described herein, the present invention is not limited by the
method of detecting the expression of any particular protein and/or
nucleic acid.
[0125] In some embodiments, a subject may begin utilizing
compositions (e.g., comprising selenium (e.g., organic selenium
(e.g., selenized yeast (e.g., SEL-PLEX)))) and methods of the
present invention after any type of pregnancy related test,
including, but not limited to, a test indicating that a subject is
or is not pregnant (e.g., a blood or urine test (e.g., for human
chorionic gonadotropin (hCG))), after any type of fetal screening
test (e.g., tests used to identify and/or characterize certain
traits or characteristics (e.g., tests performed to identify and/or
characterize birth defects (e.g., amniocentesis, sonograms, nuchal
translucency testing, and genetic screening (e.g., to identify
and/or characterize down syndrome, spina bifida, cleft palate, Tay
Sachs disease, sickle cell anemia, thalassemia, cystic fibrosis, or
fragile x syndrome)))), a test indicating the size of a fetus
(e.g., after use of morphometric and/or Doppler ultrasonic
measurements to measure fetus size (e.g., size of head, limbs,
etc.), a test indicating the size and/or formation and/or location
of the uterus (e.g., use of morphometric and/or Doppler ultrasonic
measurements to measure uterine size, location, formation, etc.), a
test indicating multiple gestation, etc.). In some embodiments, a
subject begins utilizing (e.g., administration of) compositions and
methods of the present invention within 24 hours (e.g., within 18
hours, 9 hours, 6 hours, 3 hours, 1 hour or less) of such a
pregnancy related test or after receiving the results of the same.
In some embodiments, a subject begins administration of
compositions and methods of the present invention within 1 week,
within 3 days, within 2 days, or within 1 day of a pregnancy
related test or after receiving the results of the same.
[0126] In some embodiments, a subject is identified as a candidate
for the compositions and methods of the present invention because
the subject displays low, non-normal gene and/or protein expression
(e.g., of one or more genes and/or proteins) that correlates with
poor fertility and/or poor fetal development and/or low birth
weight. The present invention is not limited by the type of subject
that may benefit from the compositions and methods described
herein. Indeed, any subject with any problem related to
establishing and/or maintaining pregnancy is contemplated to
benefit from the compositions and methods disclosed herein. For
example, in some embodiments, compositions and methods of the
present invention are utilized by (e.g., prescribed to and/or
administered to) a subject because the subject has been identified
as one who has tried to become pregnant for more than six weeks,
for more than two months, for more than 4 months, for more than 6
months, for more than a year, for more than two years, or for more
than three years (e.g., without establishing a pregnancy).
[0127] In some embodiments, after a subject is identified as one
that may benefit from the compositions and/or methods of the
present invention, and is administered a composition of the present
invention, and subsequently characterized for one or more
beneficial results (e.g., alteration of gene expression involved
with establishment of and/or maintenance of pregnancy) due to
administration of a composition of the present invention, the
subject may also be further classified and/or characterized post
such characterization. For example, a subject may be characterized
as one with high, medium or low probability of successful
parturition and/or mature pregnancy (e.g., without additional help
and/or medical intervention); or as a subject with a likelihood of
complication resulting in non-full term gestation, miscarriage,
etc. (e.g., without additional help and/or medical
intervention).
[0128] In some embodiments, a subject administered and/or utilizing
compositions and methods of the present invention may keep a record
of one or more pieces of information throughout the treatment
process. This information may include, but is not limited to, sleep
habits, emotional status, temperature, eating habits, level of
stress, and working habits.
[0129] The present invention provides that certain forms of
selenium (e.g., organic selenium (e.g., selenized yeast (e.g.,
SEL-PLEX))) compared to other types of selenium (e.g., sodium
selenite) are able to produce a significant elevation of expression
of genes involved in the establishment of and/or maintenance of a
pregnancy (e.g., relaxin). Thus, the present invention
distinguishes between different forms of selenium that are useful
and that are not useful in methods of the present invention (e.g.,
for enhancing expression of genes and/or proteins involved with
establishing and/or maintenance of pregnancy).
[0130] For example, in some embodiments, the present invention
provides a method of altering expression of one or more genes
associated with establishment of and/or maintenance of pregnancy
(e.g., relaxin, Sgk, etc.) in a subject comprising providing a
subject; and a composition comprising 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 (e.g.,
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 with residual inorganic
selenium being eliminated (e.g., using a rigorous washing process)
and not exceeding 2% of the total selenium content)); and
administering the composition to the subject under conditions such
that the expression of one or more genes associated with
establishment of and/or maintenance of pregnancy (e.g., relaxin,
Sgk, etc.) is enhanced the subject (e.g., compared to a subject not
administered the composition, or that is administered a different
type of composition comprising selenium (e.g., sodium selenite)).
In some embodiments, the one or more genes associated with
establishment of and/or maintenance of pregnancy is relaxin. In
some embodiments, enhancing expression of one or more genes
associated with establishment of and/or maintenance of pregnancy
(e.g., relaxin, Sgk, etc.) reduces and/or eliminates the risk of
perinatal morbidity and/or mortality in the subject. In some
embodiments, enhancing expression of one or more genes associated
with establishment of and/or maintenance of pregnancy (e.g.,
relaxin, Sgk, etc.) increases fetal growth (e.g., fetal growth rate
(e.g., due to nutrition, blood and/or oxygen supply, etc.) in the
subject. In some embodiments, enhancing expression of one or more
genes associated with establishment of and/or maintenance of
pregnancy (e.g., relaxin, Sgk, etc.) increases the duration of
gestation (e.g., into the second trimester, into the third
trimester, and/or to full term). In some embodiments, enhancing
expression of one or more genes associated with establishment of
and/or maintenance of pregnancy (e.g., relaxin, Sgk, etc.) promotes
the health of the uterus (e.g., promotes connective tissue
formation and/or rearrangement). In some embodiments, enhancing
expression of one or more genes associated with establishment of
and/or maintenance of pregnancy (e.g., relaxin, Sgk, etc.) promotes
the health of the embryo and/or fetus (e.g., promotes limb and
nervous system development, muscle growth and differentiation,
and/or neural tube development). In some embodiments, enhancing
expression of one or more genes associated with establishment of
and/or maintenance of pregnancy (e.g., relaxin, Sgk, etc.) reduces
the risk and/or incidence of spontaneous abortion, miscarriage,
and/or birthing complications (e.g., of a human and/or non-human
mammal, or of an avian species. In some embodiments, enhancing
expression of one or more genes associated with establishment of
and/or maintenance of pregnancy (e.g., relaxin, Sgk, etc.) promotes
the health and development of hens and/or the hen's offspring.
[0131] In some embodiments, compositions and methods of the present
invention are utilized in in vitro fertilization and/or embryo
transfer settings. For example, in some embodiments, enhancing
expression (e.g., in a subject administered a composition
comprising selenium) of one or more genes associated with
establishment of and/or maintenance of pregnancy (relaxin, Sgk,
etc.) increases the likelihood of success for a successful
pregnancy resulting from in vitro fertilization (IVF) and/or embryo
transfer (ET) procedure. In some embodiments, a subject that is
planning on undergoing an IVF or ET procedure (e.g., to benefit the
health of the uterus and/or other supporting tissues important for
pregnancy (e.g., in an aged (e.g., older than 20, older than 25,
older than 30, older than 35, older than 40 years of age) female
wishing to become pregnant) begins administration of a composition
comprising selenium (e.g., organic selenium (e.g., selenized yeast
(e.g., SEL-PLEX))) in advance of the procedure (e.g., 1 month
ahead, 3 months ahead, six months ahead, 9 months ahead, 1 year
ahead, or two years ahead). However, a subject that is planning on
undergoing an IVF or ET procedure may also begin administration of
a composition comprising selenium (e.g., organic selenium (e.g.,
selenized yeast (e.g., SEL-ALEX))) greater than two years or less
than 1 month ahead of the procedure. In some embodiments, a subject
who has an IVF or ET procedure performed is administered a
composition of the present invention during the procedure and/or
after the procedure.
[0132] In some embodiments, the present invention provides
compositions and methods for the treatment, diagnosis and/or
prevention of conditions (e.g., problems), disorders, and/or
diseases involving fetal growth (e.g., including, but not limited
to, intrauterine growth retardation and placental insufficiency).
In some embodiments, the invention provides methods for modulating
signaling pathways related to the polypeptide hormone, relaxin. In
some embodiments, the present invention provides methods relating
to the modulation of relaxin expression (e.g., nucleic acid and/or
protein expression) and/or synthesis, relaxin receptor synthesis,
relaxin binding to its receptor, and/or relaxin signaling (See,
e.g., U.S. Patent Application Publication Nos. 20060247172 and
20010053553, each of which is hereby incorporated by reference in
their entireties for all purposes).
[0133] In some embodiments, the present invention provides a method
of increasing intrauterine fetal growth rate, comprising the step
of administering to a pregnant subject a therapeutically effective
amount of a composition comprising selenium (e.g., organic selenium
(e.g., selenized yeast (e.g., SEL-PLEX))) for a time sufficient to
increase the expression of one or more genes associate with fetal
growth (e.g., relaxin (e.g., thereby increasing fetal growth
rate)). In some embodiments, the composition comprising selenium
(e.g., organic selenium (e.g., selenized yeast (e.g., SEL-PLEX)))
is administered during the first, second, and/or third trimester of
pregnancy. In some embodiments, the composition comprising selenium
(e.g., organic selenium (e.g., selenized yeast (e.g., SEL-PLEX)))
is administered for at least 2 weeks starting at ovulation. In yet
another embodiment, the composition comprising selenium (e.g.,
organic selenium (e.g., selenized yeast (e.g., SEL-PLEX))) is
administered before and after ovulation. In yet another embodiment,
the composition comprising selenium (e.g., organic selenium (e.g.,
selenized yeast (e.g., SEL-PLEX))) is administered for about a week
before ovulation and about four weeks after ovulation. The present
invention is not limited to these particular time frames for
administration of a composition comprising selenium (e.g., organic
selenium (e.g., selenized yeast (e.g., SEL-PLEX))). Indeed, a
composition comprising selenium may be administered months to years
prior to any particular ovulation as well as one to 9 months or
more after any particular ovulation.
[0134] In some embodiments, the present invention provides a method
of increasing intrauterine fetal growth rate wherein a composition
comprising selenium (e.g., organic selenium (e.g., selenized yeast
(e.g., SEL-PLEX))) is administered in an amount sufficient to
result in the birth of a baby of at least around normal birth
weight. In some embodiments, the composition comprising selenium
(e.g., organic selenium (e.g., selenized yeast (e.g., SEL-PLEX)))
is administered in an amount sufficient to maintain a desired serum
concentration of one or more gene products associated with
establishing and/or maintaining a pregnancy described herein (e.g.,
relaxin) in the pregnant subject (e.g., of at least around 0.5-3.0
ng/mL (e.g., 0.1 ng/mL) although concentrations above 3.0 and below
0.5 ng/mL are also contemplated).
[0135] In some embodiments, the present invention provides a method
of increasing intrauterine fetal growth rate comprising the step of
administering to a pregnant subject a therapeutically effective
amount of a composition comprising selenium (e.g., organic selenium
(e.g., selenized yeast (e.g., SEL-PLEX))) for a time sufficient to
increase fetal growth rate, wherein the increase in fetal growth
rate is assessed by an imaging technique. The present invention is
not limited by the type of imaging technique utilized. In some
embodiments, the imaging technique is selected from a group
comprising ultrasonic imaging and magnetic resonance imaging. In
some embodiments, the subject (e.g., the imaged subject) is
diagnosed and/or identified as hosting a fetus with intrauterine
growth retardation (e.g., due to one or more of the conditions,
diseases and/or disorders described herein). In some embodiments,
diagnosing and/or identification of intrauterine growth retardation
is obtained via use of an imaging technique (e.g., ultrasonic
imaging or magnetic resonance imaging).
[0136] In some embodiments, the present invention provides a method
of increasing intrauterine fetal growth rate comprising the step of
administering to a pregnant subject a therapeutically effective
amount of a composition comprising selenium (e.g., organic selenium
(e.g., selenized yeast (e.g., SEL-PLEX))) wherein the subject has a
condition, disease and/or disorder that increases the risk of fetal
intrauterine growth retardation and/or low birth weight. The
present invention is not limited by the type of condition, disease
and/or disorder that may result in low birth weight and/or
intrauterine growth retardation. Indeed, a variety of conditions,
disorders and/or diseases are contemplated including, but not
limited to, autoimmune disease (e.g., lupus), hyperthyroidism,
hypertension, preeclampsia, infection, serum antiphospholipid
antibodies, a history of spontaneous abortion or other non-full
term pregnancy (e.g., miscarriage, stillbirth, premature
parturition), a history of intrauterine growth retardation, a
history of having children with low birth weight, a
multiple-gestation pregnancy, and a pregnancy resulting from in
vitro fertilization or embryo transfer.
[0137] The present invention is not limited by the subject treated
with a composition comprising selenium (e.g., organic selenium
(e.g., selenized yeast (e.g., SEL-PLEX))) of the present invention
(e.g., for increasing the likelihood of successfully establishing
and/or maintaining a healthy pregnancy in the subject (e.g., but
enhancing expression of one or more genes involved in establishment
and/or maintenance of pregnancy)). In some embodiments, the subject
treated is a human. In some embodiments, the compositions and
methods of the present invention may be used to treat pregnant
human subjects or human subjects that desire to become pregnant. In
some embodiments, the compositions and methods may be used to treat
non-human mammals (e.g., a horse, cow, sheep, goat, swine, deer,
dog, cat, rat, and/or a mouse (e.g., that are pregnant or that are
desired to become pregnant)). In some embodiments, the subject
treated is from an avian species (e.g., domesticated fowl (e.g.,
chickens, turkeys, ducks, geese, and guinea fowl, etc.) and other
game birds (e.g., quail, pheasants, grouse, partridges, emu,
ostriches, pigeons, etc.). In some embodiments, the compositions
and methods of the present invention may be used to treat (e.g.,
administered to) aquatic species (e.g., fresh water and marine
(salt water) species (e.g., including, but not limited to, fish
(e.g., bass, catfish, carp, cod, crappie, eel, flounder, grouper,
haddock, halibut, herring, kingfish, mackerel, mahi mahi, orange
roughy, perch, pike, Pollock, salmon, sardine, shark, snapper,
sole, tilapia, trout, tuna, and walleye), crustaceans (e.g., crab,
crayfish, lobster, prawn, and shrimp), mollusks, etc.).
[0138] In some embodiments, the present invention provides a method
of reducing the risk or incidence of spontaneous abortion in a
pregnant subject, the method comprising administering to the
pregnant subject an amount of a composition comprising selenium
(e.g., organic selenium (e.g., selenized yeast (e.g., SEL-PLEX)))
effective to reduce the risk or incidence of spontaneous abortion.
In some embodiments, the composition comprising selenium (e.g.,
organic selenium (e.g., selenized yeast (e.g., SEL-PLEX))) is
administered during and/or prior to the first trimester of
pregnancy. In some embodiments, the composition comprising selenium
(e.g., organic selenium (e.g., selenized yeast (e.g., SEL-PLEX)))
is administered during the first, second, and/or third trimester of
pregnancy.
[0139] In some embodiments, compositions and/or methods of the
present invention are utilized in conjunction with monitoring of
patients undergoing clinical evaluation for treatment of fetal
growth abnormalities, infertility, and/or one or more complications
related to pregnancy. In some embodiments, such methods also
include monitoring of the size of a fetus with respect to its
gestational age.
[0140] Thus, in some embodiments, compositions and methods
described herein may be performed in conjunction with, prior to, or
subsequent to one or more techniques for measuring fetal size
and/or growth. For example, upon identifying a subject (e.g.,
human) exhibiting higher or lower levels of one or more genes
involved in the establishment and/or maintenance of pregnancy
(e.g., relaxin (e.g., in serum)) relative to that of a
corresponding control subject, the size and/or growth rate of one
or a plurality of fetuses carried by the subject can be measured to
further clarify whether the one or plurality of fetuses exhibit
abnormal growth. In some embodiments, compositions and methods
described herein may be used in combination (e.g., concurrently)
with diagnostic techniques to identify abnormal fetal growth. In
some embodiments, a diagnosis involves an assessment the size
and/or gestational age of the fetus. For example, an early
ultrasonographic examination can be used to determine the
gestational age (See, e.g., Benson and Doubilet, 1991, AJR Am. J.
Roentgenol. 157:1275-1277). Other imaging techniques, such as
magnetic resonance imaging, can also be used to estimate
gestational age and/or to monitor intrauterine growth of the fetus
as well as growth of the placenta. In the absence of an accurate
estimate of gestational age, serial measurements of fetal size may
be used to assess whether or not fetal size is appropriate. In some
embodiments, an analysis utilizing morphometric and/or Doppler
ultrasonic measurements may be used to identify abdominal
circumference and estimated fetal weight (e.g., based on
measurements of head size, abdominal size and femur length (e.g.,
to identify fetuses likely to be large or small for their
gestational age (See, e.g., Chang et al., 1992, Obstet. Gynecol.
80:1030-8)).
[0141] The present invention is not limited by any particular
method of detecting the expression (e.g., nucleic acid and/or
protein expression) of one or more genes related to the
establishment and/or maintenance of pregnancy in a subject (e.g.,
prior to, during and/or after administration of a composition of
the present invention). In some embodiments, the detection of one
or more genes (e.g., wild type and/or variant forms thereof (e.g.,
polymorphic and/or mutant foams) related to the establishment
and/or maintenance of pregnancy in a subject involves, for example,
their amplification (e.g., by PCR (e.g., that may be followed by
the detection of the amplified molecules using techniques well
known to those of skill in the art)). Methods of detecting wild
type and variant forms of genes, as well as detecting gene products
(e.g. using antibodies), are well known to those of skill in the
art (e.g., for exemplary methods of detecting wild type and mutant
forms of relaxin, and relaxin gene products, See, U.S. Patent
Publication No. 20060247172, hereby incorporated by reference in
its entirety).
[0142] In some embodiments, the present invention demonstrates how
specific forms of selenium (e.g., organic selenium, (e.g.,
selenized-yeast (e.g., SEL-PLEX))) may be used to benefit a
subject. In particular, the present invention provides that
compositions and methods of the present invention can be used to
stabilize or increase the general health of a developing embryo,
fetus, and/or uterus within a female subject. For example, as
described in detail herein, the present invention provides
compositions and methods that can enhance expression of genes
and/or proteins that are important in the establishment and/or
maintenance of pregnancy (e.g., a full term and/or healthy
pregnancy).
[0143] In some embodiments, the present invention also provides
that a composition comprising selenium (e.g., organic selenium,
(e.g., selenized-yeast (e.g., SEL-PLEX))) may be used in
combination (e.g., co-administered with) other agents that promote
the establishment and/or maintenance of pregnancy (e.g., a full
term and/or healthy pregnancy). One or ordinary skill in the art
knows well that there a number of such agents currently available.
Examples of such agents include, but are not limited to, agents
utilized in IVF and/or embryo transfer procedures (e.g.,
urofollitropin, follitropin alpha and follitropin beta, chorionic
gonadotropins, Clomiphene citrate, gonadorelin, leuprolide,
menotropins, estrogen, prolactin, testosterone, progesterone),
agents utilized to promote the health of the embryo, fetus, and/or
mother (e.g., vitamins, minerals, hormones, etc.). Thus, in some
embodiments, the present invention provides one or more forms of
selenium (e.g., SEL-PLEX) that is biologically available and is
administered alone or co-administered with an agent used for
promoting the establishment and/or maintenance of pregnancy (e.g.,
a full term and/or healthy pregnancy).
[0144] In some embodiments, 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 1). 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 promoting
ovulation) to form a selenium-agent derivative.
[0145] Once the desired form of selenium is chosen, it can be
administered alone or in combination with one or more agents used
for promoting the establishment and/or maintenance of pregnancy
(e.g., a full term and/or healthy pregnancy). 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)).
[0146] The present invention is not limited by the composition
and/or formulation 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- or di- or trimethylated
derivatives, of which the monomethylated derivatives are most
toxic. (See, e.g., Bedwal, R. S., et al., 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. Clin. 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)).
[0147] 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.
[0148] As described above, the present invention demonstrates that
certain forms of selenium (e.g., SEL-ALEX) 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 promoting the establishment and/or
maintenance of pregnancy (e.g., a full term and/or healthy
pregnancy)), 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.
[0149] 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.
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
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 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).
[0150] Other forms of selenium that find use in various embodiments
of the present invention are described in U.S. Pat. Nos. 6,911,550,
6,197,295, 5,221,545, and 6,576,233, and U.S. Pat. App. Nos.
20010043925, 20050069594, and 20050089530, herein incorporated by
reference in their entireties.
[0151] 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, and may be administered in any sterile,
biocompatible pharmaceutical carrier, including, but not limited
to, saline, buffered saline, dextrose, and water.
[0152] Selenium (e.g., SEL-PLEX) can be administered to a subject
(e.g., a patient) by a variety of means including, but not limited
to, via pill or tablet form and/or 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. Formulations also contemplated
in the present invention include those useful for parenteral
administration, such as intravenous, subcutaneous, intramuscular,
and intraperitoneal.
[0153] 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.
[0154] 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 subjects (e.g., that is pregnant or that desires to become
pregnant). 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.
[0155] Depending on the target sought to be altered by treatment
(e.g., gene expression associated with establishing and/or
maintaining pregnancy), 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.
[0156] 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.
[0157] 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.
[0158] 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.,
relaxin and/or gsk). Determination of effective amounts is well
within the capability of those skilled in the art, especially in
light of the disclosure provided herein.
[0159] 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.
[0160] 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).
[0161] 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.
[0162] 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,
mannitol, 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.
[0163] 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).
[0164] 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.
[0165] 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.
[0166] 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
mM-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.
[0167] 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.
[0168] A therapeutically effective dose, in the context of
establishing and/or maintaining pregnancy, is an amount necessary
to improve the likelihood of establishing and/or maintaining
pregnancy (e.g., from embryonic to fetal state, or from first to
second trimester, or from second to third trimester, etc.). In some
embodiments, an effective amount is sufficient to achieve a
successful result in at least 55%, at least 65%, at least 75%, at
least 85%, or at least 95% of the occasions administered (e.g., in
the absence of other factors). 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.
[0169] Toxicity and therapeutic efficacy of compositions of the
present invention can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals (e.g., for
determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (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 LD.sub.50/ED.sub.50. Compounds that 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 ED.sub.50 with 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.
[0170] 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 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.
[0171] 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 .mu.g per day (e.g., SEL-PLEX is administered
to a subject in such a way so as to provide between 25 and 800
.mu.g 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 .mu.g per day. In other preferred embodiments, selenium
is administered at a daily dose of between 200 and 400 .mu.g per
day. Doses outside of 25 and 800 .mu.g 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 administered 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 .mu.g of selenium. In other preferred embodiments,
the daily dose is 200 .mu.g of selenium (e.g., organic selenium
(e.g., selenized yeast (e.g., SEL-PLEX))).
[0172] 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.
[0173] Pharmaceutical compositions and formulations for topical
administration may include transdermal patches, ointments, lotions,
creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional pharmaceutical carriers, aqueous, powder or oily
bases, thickeners and the like may be necessary or desirable.
[0174] 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.
[0175] 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.
[0176] 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.
[0177] 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.
[0178] 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.
[0179] 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.
[0180] 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.
[0181] 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., a
hormone utilized for IVF procedure). In some embodiments, two or
more combined agents may be used together or sequentially.
[0182] The present invention also includes methods involving
co-administration of compounds comprising selenium described herein
with one or more additional active agents (e.g., a hormone utilized
for IVF procedure). 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 additional
agent(s) to be co-administered (e.g., a hormone utilized for IVF
procedure) can be any of the well-known agents in the art,
including, but not limited to, those that are currently in clinical
use.
[0183] Treatment of the various conditions, diseases and/or
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.
[0184] 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.
[0185] 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.
[0186] It is contemplated that the compositions and methods of the
present invention will find use in various settings, including
research and clinical diagnostics and therapeutics. For example,
compositions and methods of the present invention also find use in
studies of embryogenesis, fetal development, and birthing (e.g., of
human and non-human mammal subjects, as well as avian species).
Thus, 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
[0187] 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
Administration of Selenium Enhances Reproduction Potential
[0188] In an effort to examine possible benefits of administration
of selenium on female reproductive performance, hens were randomly
selected from a broiler-breeder flock housed at Coldstream poultry
facility, University of Kentucky. The hens were maintained on a
selenium-deficient diet from 6 weeks of age until 22 weeks of age
and were then placed on the following dietary treatments for over
forty weeks until the time of sampling: 1) Control,
selenium-deficient, Torula yeast-based diet (0.02 ppm selenium); 2)
The control diet supplemented with 0.3 ppm selenium from sodium
selenite; and 3) The control diet supplemented with 0.3 ppm
selenium from SEL-ALEX. Hens were taken from each of the three
experimental groups, their oviducts removed, flash-frozen and
stored at -80.degree. C. prior to RNA isolation.
[0189] Labeled RNA preparations from oviduct were hybridized with
AFFYMETRIX chicken genome arrays containing 38,000 probe sets using
standard methodology. Overall, it was found that 5,105 transcripts
were significantly affected by at least one selenium treatment in
hen oviduct.
[0190] Dietary selenium supplementation increased the expression
levels of classical selenium-associated genes. For example, as
described herein, selenium is known for its role in antioxidant
systems, mainly because selenium (as selenocysteine) is a key
component of glutathione peroxidases (GSH-Px). These are a class of
enzymes that detoxify hydrogen peroxides and lipid hydroperoxides.
Thus, glutathione peroxidases are believed to function to protect a
cell against reactive oxygen species (ROS) produced as byproducts
of aerobic cellular metabolism.
[0191] For example, the fold change (FC) in the expression of two
Glutathione Peroxidases (GSH-PX) genes in oviduct (relative to
selenium deficient hens) induced by sodium selenite (SS) and
Sel-Plex (SP) are shown in FIG. 1. Significant induction of both
Glutathione Peroxidases analyzed was noted for hens administered
selenium in the form of SEL-PLEX, whereas significant induction of
only Glutathione Peroxidase 3 was noted for administration of
selenium in the form of sodium selenite. Administration of selenium
to the hens also resulted in the up-regulation of genes encoding a
variety of selenoproteins, including Selenoprotein P, Selenoprotein
T, Selenoprotein 15 kDa and Selenoprotein K.
[0192] Administration of selenium to hens also resulted in a
heretofore undocumented alteration of expression of genes involved
with the establishment of and/or maintenance of pregnancy (e.g.,
genes encoding transcription and growth factors in hen oviduct).
For example, overall changes in the expression profiles of genes
associated with establishing and/or maintaining pregnancy in
response to sodium selenite or Sel-Plex are shown in FIGS. 2 and 3.
The numbers indicate Fold Change (FC) relative to selenium
deficiency, the grey background indicates a statistically
significant up-regulation (p<0.05) and the black background no
significant change relative to selenium deficiency. The biological
processes in which these growth and transcription factors are
involved in are briefly described and generally relate to factors
that are involved in key areas of reproduction and embryogenesis
(e.g., including, but not limited to follicular development in
ovary, and embryonic limb and nervous system development, muscle
growth and differentiation and neural tube development).
[0193] Additionally, a number of linked effects were also observed
that have not previously been associated with administration of
selenium that also have the ability to alter (e.g., enhance)
fertility and oocyte development. For example, follicle stimulating
hormone (FSH) activates protein kinase A, which in turn activates
the transcription factors SP1 or SP3. These transcription factors
can activate serum and glucocorticoid inducible-protein kinase,
sgk. Sgk is an immediate-early gene known to be important in the
proliferation and differentiation of granulosa cells surrounding
the developing oocyte (See, e.g., Alliston et al., 1997. Molecular
Endocrinology, 11:1934-1949). Granulosa cells produce steroids and
a range of other growth factors known to be essential for oocyte
development. In the case of hens administered selenium relative to
selenium-deficient hens, up-regulation of the genes encoding
protein kinase A, SP3 and Sgk, the interrelation among them, and
their potential biological effects are shown in FIG. 4.
Administration of SEL-PLEX to the hens led to significant elevation
of expression of Protein Kinase A, SP3 and Sgk, whereas
administration of sodium selenite did not induce significant
expression of SP3 or Sgk.
[0194] Another striking and unexpected effect of administration of
selenium was observed with the gene encoding the hormone relaxin.
Relaxin is a 6 kDa protein hormone member of the insulin-like
growth factor family (See, e.g., Shirota et al., Ann N Y Acad Sci.
2005 May; 1041:144-6; Dschietzig and Stangl, 2003 Cell Mol Life
Sci. 2003 April; 60(4):688-700). In many mammalian species, relaxin
exerts pronounced effects on the female reproductive tract that are
involved in the maintenance of pregnancy and successful
parturition. Relaxin is important for normal delivery in several
mammalian species because of its marked rearrangement of
reproductive tract connective tissue. In rats, there is a major
pre-labor surge in circulating relaxin levels and relaxin is
critical for cervical dilation. In guinea pigs, relaxin
significantly increases the intrapubic ligament to enlarge the
diameter of the pubis. It is also essential for normal delivery in
pigs. In humans, relaxin stimulates production of several
endometrial products including prolactin, glycodelin, insulin-like
growth factor binding factor 1 (IGFBP-1) and vascular endothelial
growth factor in progesterone-primed human endothelial cells in
vitro. In rhesus monkeys, relaxin stimulated uterine weight as well
as endometrial lymphocyte and arteriole number. Overall, relaxin is
viewed as a very significant factor in the establishment and/or
maintenance of pregnancy (See, e.g., Goldsmith et al., 2004.
Proceedings of the National Academy of Sciences,
101:4685-4689).
[0195] According to Brackett et al., (See, e.g., Bracket et al.,
1997. General and Comparative Endocrinology, 105:155-163; Bracket
et al., 1985. Biology of Reproduction, 32(Suppl. 1):43), at the
time of onset of puberty or following molting, a sevenfold increase
in pubic bone width occurs. The widening of pubic bone width is
analogous to the effect of relaxin on lengthening and widening of
the pelvic girdle in the guinea pig during pregnancy (See, e.g.,
Zarrow, 1947. Proceedings of the Society for Experimental Biology
and Medicine, 66:489-491). Thus, in hens, relaxin may assisting in
oviposition by influencing biochemical as well as morphological
alterations in the uterus, oviduct and pelvic ligaments.
[0196] Thus, the ability to alter (e.g., increase) relaxin levels
in various subjects therefore may exist as one means of improving
reproductive function in the host subject (e.g., human or non-human
animal, avian species, etc.) This is particularly true when it is
considered that hen oviduct is viewed as one of the most valuable
model systems for studying the general effects of sex hormones,
such as estrogen and progesterone as applied to all species (See,
e.g., Dougherty and Sanders, 2005. Estrogen action: revitalization
of the chick oviduct model. Trends in Endocrinology and Metabolism,
16:414-419).
[0197] When subjects were administered selenium, the gene encoding
relaxin was significantly upregulated (e.g., 2.52-fold) by SEL-PLEX
(p=0.0033) relative to selenium deficient animals, whereas sodium
selenite had no significant effect on relaxin gene expression
levels (See FIG. 5). Accordingly, the present invention provides a
method of enhancing the expression relaxin in a subject comprising
providing a subject and a composition comprising selenium (e.g.,
organic selenium (e.g., selenized yeast (e.g., SEL-PLEX))) and
administrating the composition to the subject under conditions such
that the expression of relaxin is enhanced in the subject (e.g.,
compared to a subject not administered the composition).
[0198] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described compositions and
methods of the invention will be apparent to those skilled in the
art without departing from the scope and spirit of the invention.
Although the invention has been described in connection with
specific preferred embodiments, it should be understood that the
invention as claimed should not be unduly limited to such specific
embodiments. Indeed, various modifications of the described modes
for carrying out the invention that are obvious to those skilled in
the relevant fields are intended to be within the scope of the
present invention.
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