Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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USE OF EQUOL FOR AMELIORATING OR PREVENTING
NEUROPSYCHIATRIC AND NEURODEGENERATIVE
DISEASES OR DISORDERS
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0001] This invention was made with Government support under Grant No.
NRI 2002-00798 and Grant No. NRI 2004-01811, awarded by the U.S. Dept. of
Agriculture (USDA).
[0002] The Government has certain rights in this invention.
BACKGROUND
1. Technical Field
[0003] This invention relates to equol and its use as a therapeutic compound
for treating and preventing physiological and pathophysiological conditions
medi-
ated by androgens. Specifically, the present invention relates to the use of
equol
for preventing or treating neuropsychiatric or neurodegenerative diseases or
disor-
ders.
2. Background Information
[0004] In recent years phytoestrogens have received increased investigative
attention due to their potential protective effects against age-related
diseases (e.g.
cardiovascular disease and osteoporosis) and hormone-dependent cancers (i.e.,
breast and prostate cancer). There are three main classifications of
phytoestro-
gens: 1) isoflavones (derived principally from soybeans), 2) lignans (found in
flax-
seed in large quantities) and 3) coumestans (derived from sprouting plants
like al-
falfa). Of these three main classifications, human consumption of isoflavones
has
the largest impact due to its availability and variety in food products
containing
soy. Of the isoflavones, genistein and daidzein are thought to exert the most
po-
tent estrogenic hormone activity and thus most attention has been directed
toward
these molecules (Knight D. C. et al., Obstet Gyneco, 187:897-904, (1996);
Setchell, K. D. R. Am J Clin Nutt , 129:1333S-1346S (1998); Kurzer, M. S. et
al.,
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Annu Rev Nutr, 17:353-381(1997)). However, these isoflavone molecules do not
exist at high levels in their biologically active form in soy foods, but
rather are at
high abundance in a precursor form. For example, genistin, the precursor of
gen-
istein, is the glycosidic form that contains a carbohydrate portion of the
molecule.
Additionally, malonylglucoside and acetylglucoside forms also are found. These
conjugates are metabolized in the GI tract by intestinal bacteria, which
hydrolyze
the carbohydrate moiety to the biologically active phytoestrogen, genistein.
The
same metabolic step occurs for the aglycone daidzein, which is converted from
the
glycosidic form daidzein. Daidzein is then further metabolized to equol in an
"equol-producing" mammal. Thereafter, equol circulates in the blood stream at
very high concentrations. Equol is not normally present in the urine of most
healthy adults unless soy is consumed. The formation of equol in vivo is exclu-
sively dependent on intestinal microflora as evidenced from the finding that
germ-
Phyto-Free animals do not excrete equol, and that equol is not found in the
plasma
and urine of newborn or 4-month old infants fed exclusively soy foods from
birth
due to the fat that the intestinal flora has not yet developed in neonates.
See
Setchell K. D. R. et al., The Lancet 1997; 350:23-27.
[0005] The phenolic ring structures of isoflavones enable these compounds to
bind estrogen receptors (ER) and mimic estrogen. Although genistein and
daidzein bind to ER, it is with a lower affinity when compared to estradiol,
and
with a greater affinity for ER(3 than to ERa. Additionally, phytoestrogens
have
been reported to act like natural selective estrogen receptor modulators
(SERMs)
at various tissue sites throughout the body. In some tissues, there is
evidence that
phytoestrogens act as estrogen agonists, whereas in others, they display
antagonis-
tic characteristics comparable to that of tamoxifen or raloxifene where SERM
ac-
tivity appears to be sex-hormone and gender dependent.
[0006] While the bulk of the scientific literature has focused on the natural
isoflavones in soy or clover, little has been reported on the actions or
effects of
their intestinally derived metabolites.
[0007] Equol (7-hydroxy-3(4'hydroxyphenyl)-chroman) represents the major
metabolite of the phytoestrogen daidzein, one of the main isoflavones found
abun-
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dantly in soybeans and soy-foods. Equol, however, is not a phytoestrogen, be-
cause it is not a natural constituent of plants. Equol does not occur
naturally in
any plant-based products. Rather, it is a non-steroidal isoflavone that is
exclu-
sively a product of intestinal bacterial metabolism (relatively few
individuals, -30-
400%, have the micro flora necessary to convert soy isoflavones to equol).
Previ-
ous research with equol has identified that equol possess some weak estrogenic
properties, binds sex hormone binding globulin, binds a-fetoprotein, and has
anti-
oxidant activity. However, equol is unique among the plant-derived isoflavones
in
that it possesses a chiral center and as such exists as two distinct
enantiomeric
forms, the R- and S-enantiomers. We have shown that the S-enantiomer of equol
is the exclusive equol forin found in the urine and plasma of "equol-
producing"
mammals consuming soy, and is the only equol enantiomer made by human intes-
tinal bacteria. All previous studies on equol appear to have been conducted
with
the racemic forin of equol. There has in general been a lack of appreciation
that
two forms of equol exist and to our knowledge no previous study has reported
on
the specific actions or activity of the individual enantiomers. The R- and S-
enantiomers conformationally differ, which subsequently influences their
biologi-
cal activity. For example, only the S-enantiomer of equol binds estrogen
receptor
(ER) with sufficient affinity to make it relevant to bind circulating equol
levels
reported in humans. Compared to 17(3-estradiol the relative binding affinities
of
the R- and S-equol enantiomer for ERa are 210.6 and 49.2 fold less
respectively.
However, the S-equol enantiomer seems to be largely ER(3-selective with a rela-
tively high affinity for ERP. Enantiomer S-equol binds ER(3 at approximately
20% that of 170-estradiol [equol, Kd=0.7 nM vs. 17(3-estradiol, Kd=0.15 nM],
while the R-equol enantiomer binds at approximately 100 fold less. R-Equol, al-
though not naturally occurring, is of considerable importance because of its
ability
to modulate androgen-mediated processes in the body.
[0008] The prostate gland depends on androgen hormone action for its devel-
opment and growth, and the development of human benign prostatic hyperplasia
(BPH) clearly requires a combination of testicular androgens during the aging
process. However, testosterone is not the major androgen responsible for
growth
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of the prostate. The principal prostatic androgen is 5a-dihydrotestosterone
(5a-
DHT), as evidenced by current treatments of prostatic cancer are directed
toward
reducing 5a-DHT with 5a-reductase inhibitors. Although not elevated in human
BPH, 5a-DHT levels in the prostate remain at a normal level with aging,
despite a
decrease in the plasma testosterone. Testosterone is converted to 5a-DHT by 5a-
reductase in prostatic stromal and basal cells. 5a-DHT is primarily
responsible for
prostate development and the pathogenesis of BPH. Inhibitors of 5a-reductase
re-
duce prostate size by 20% to 30%. This reduction in glandular tissue is
achieved
by the induction of apoptosis, which is histologically manifested by ductal
atro-
phy. 5a-reductase occurs as 2 isoforms, type 1 and type 2, with the prostate
ex-
pressing predominantly the type-2 isoform, and the liver and skin expressing
pri-
marily the type-1 isoform. Patients have been identified with deficiencies in
the
type-2 5a-reductase, but not type 1. Knockout mice with the type-2 5a-
reductase
null-mutation demonstrate a phenotype similar to that seen in men with 5a-
reductase deficiency. Type-1 5a-reductase knockout male mice are
phenotypically
normal with respect to reproductive function. Enzymatic activity for 5a-
reductase
or immunohistochemical detection has been noted in other genitourinary
tissues,
such as the epididymis, testes, gubemaculum, and corporal cavemosal tissue.
[0009] Quantitatively, women secrete greater amounts of androgen than of es-
trogen. The major circulating steroids generally classified as androgens
include
dehydroepiandrosterone sulphate (DHEAS), dehydroepiandrosterone (DHEA),
androstenedione (A), testosterone (T), and 5a-DHT in descending order of serum
concentration, though only the latter two bind the androgen receptor to a
signifi-
cant degree. The other three steroids are better considered as pro-androgens.
5a-
DHT is primarily a peripheral product of testosterone metabolism. Testosterone
circulates both in its free form, and bound to protein including albumin and
sex
steroid hormone-binding globulin (SHBG), the levels of which are an important
determinant of free testosterone concentration. The postmenopausal ovary is an
androgen-secreting organ and the levels of testosterone are not directly
influenced
by the menopausal transition or the occurrence of menopause.
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[0010] The work of some research has focused on the development of ster-
oidal compounds for the treatment of androgen dependent diseases such as: hir-
sutism, androgenic alopecia, benign prostatic hyperplasia (BPH) and prostate
can-
cer. 5a-DHT has been implicated as a causative factor in the progression of
these
diseases, largely through the clinical evaluation of males who are genetically
defi-
cient of steroid 5a-reductase enzyme. As a result of such studies, the
inhibition of
this enzyme has become a pharmacological strategy for the design and synthesis
of
new antiandrogenic drugs. However, it is unclear whether inhibition of 5a-
reductase will have a deleterious impact on the system, as evidenced by
contrain-
dications arising from reported side effects of conventional treatments using
5a-
reducatse inhibitors. The development of different strategies that target the
inhibi-
tion of 5a-DHT effects would be a major advance in the therapy of androgen-
mediated conditions.
SUMMARY
[0011] The present invention is a method of preventing or ameliorating a neu-
ropsychiatric or neurodegenerative disease or disorder in a subject. The
method
includes administering a composition comprising equol in an amount sufficient
to
prevent or ameliorate the neuropsychiatric or neurodegenerative disease or
disor-
der.
[0012] The equol may be a racemic mixture of R-equol and S-equol. Prefera-
bly, the equol is enantiomerically enriched with R-equol. Alternatively, the
equol
may be enantiomerically enriched with S-equol.
[0013] The neuropsychiatric or neurodegenerative disease or disorder may be
depression, anxiety, bipolar disorder, obsessive-compulsive disorder,
hyperactive
disorder, weight gain or obesity. The neuropsychiatric or neurodegenerative
dis-
ease or disorder may be Alzheimer's disease or Parkinson's disease. The neuro-
psychiatric or neurodegenerative diseases or disorder may be a peri- or
postmeno-
pausal symptom.
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[0014] The composition may further comprise an excipient. Preferably, the
composition is in an oral formulation selected from the group consisting of a
tab-
let, capsule, powder, trouche, buccal tablet, and sublingual tablet. The
composi-
tion may be an oral formulation comprising at least about 0.25 mg of equol.
The
composition may be a food product and a beverage. The composition may be a
delayed or a sustained release formulation.
[0015] Preferably, the equol is in an amount sufficient to bind free 5a-
dihydrotestosterone and inhibit its binding with androgen receptors.
Preferably,
the equol is in an amount sufficient to bind free 5a-dihydrotestosterone and
inhibit
its binding with androgen receptors, and sufficient to bind estrogen receptor
sub-
types.
[0016] In another embodiment, the present invention is a method of reducing
obesity in a subject. The method includes administering a composition
comprising
a therapeutically effective amount of equol.
[0017] In another embodiment, the present invention is a method of reducing
depression in a subject. The method includes administering a composition com-
prising a therapeutically effective amount of equol.
[00181 In yet another embodiment, the present invention is a method of reduc-
ing anxiety in a subject. The method includes administering a composition com-
prising a therapeutically effective amount of equol.
[00191 In further embodiment, the present invention is a method of providing a
personalized treatment of a neuropsychiatric or neurodegenerative disease or
dis-
order in a subject. The method includes assessing a condition of the
emotional,
mental or endocrine health of the patient; assessing an equol-producer status
of a
patient; and determining an optimally beneficial course of treatment, selected
from
the group consisting of a) mode of administration, b) a dose amount, and c) a
dose
interval.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 shows the chemical structures of S-equol and R-equol enanti-
omers.
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[0021] FIG. 2A shows an appearance/disappearance plot of R-equol in plasma
after oral administration of R-equol to a healthy adult.
[0022] FIG. 2B shows an appearance/disappearance plot of S-equol and R-
Equol in plasma (mean levels) after oral administration of S-equol or R-equol
in
healthy human adults (from: K.D. Setchell et al., Am J. Clin. Nutr., 81: 1072-
1091,
2005).
[0023] FIG. 3 shows a mass chromatogram of the elution of the equol enanti-
omers from a sample of urine from an adult consuming soy food, compared
against pure enantiomeric standards that had been characterized by optical
dichro-
ism.
[0024] FIG. 4 shows the GC-MS analysis of the trimethylsilyl ether derivative
of synthesized product.
[0025] FIG. 5 shows a mass chromatogram of a chiral separation of S-equol
and R-equol from a racemic mixture.
[0026] FIG. 6A shows prostate weight for in intact male rats subcutaneously
injected with DMSO or equol.
[0027] FIG. 6B shows lutenizing hormone (LH) for in intact male rats subcu-
taneously injected with DMSO or equol.
[0028] FIG. 7 shows a distinct peak in [3H] DHT+equol but not [3H] DHT
alone.
[0029] FIG. 8A shows two distinct peaks in [3H]-DHT+equol incubated with
prostate (A), while,
[0030] FIG. 8B shows only a single peak is present in [3H]-DHT incubated
with prostate (B).
[0031] FIG. 9 shows the specific binding of equol to [3H]-DHT.
[0032] FIG. 10A shows prostate weight in gonadectomized (GDX) male rats sc
injected with DMSO, 5a-DHT, equol, or both 5a-DHT and equol.
[0033] FIG. I OB shows plasma LH in gonadectomized (GDX) male rats sc in-
jected with DMSO, 5a-DHT, equol, or both 5a-DHT and equol.
[0034] FIG. 11 shows plasma 5a-DHT levels in rats treated with DMSO,
DHTP, equol, or both DHTP and equol.
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[0035] FIG. 12 shows the histological effects of equol in the prostate gland
of
GDX (A-D) and intact (E & F) rats treated with either Trent: DMSO (A & E),
equol (B & F), DHT (C), or DHT plus equol (D).
[0036] FIG. 13 shows the histological effects of equol on the epididymis of in-
tact rats treated with DMSO (A) or equol (B).
[0037] FIG. 14 shows body weight in male rats fed either an isoflavone-rich
(Phyto-600) or a phytoestrogen-free (Phyto-Free) diet.
[0038] FIG. 15 shows the white adipose tissue mass in male rats fed either a
Phyto-600 or Phyto-Free diet.
[0039] FIGS. 16A and 16B show food and water intake in male rats fed a
Phyto-600 or a Phyto-Free diet, respectively.
[0040] FIGS. 17A and 17B show plasma leptin and insulin levels from male
rats fed a Phyto-600 or a Phyto-Free diet, respectively.
[0041] FIG. 18 shows serum glucose levels from male rats (non-fasting) fed
either a Phyto-600 or Phyto-Free diet.
[0042] FIG. 19 shows thyroid (T3) serum levels in male rats fed either a Phyto-
600 or Phyto-Free diet.
[0043] FIG. 20 shows body weights of female rats fed either a Phyto-600 or
Phyto-Free diet.
[0044] FIG. 21 shows the white adipose tissue mass from female rats fed either
a Phyto-600 or a Phyto-Free diet.
[0045] FIG. 22 shows serum glucose levels from female rats fed either a
Phyto-600 or Phyto-Free diet.
[0046] FIG. 23 shows serum T3 levels from female rats fed either a Phyto-600
or Pllyto-Free diet.
[0047] _ FIG. 24 shows the body weights of female rats fed either a Phyto-600
or a Phyto-Free diet after 50 days of age.
[0048] FIG. 25 shows the white adipose tissue mass of female rats fed either a
Phyto-600 or al Phyto-Free diet after 50 days of age.
[0049] FIG. 26 shows the serum leptin levels of female rats fed either a Phyto-
600 or a Phyto-Free diet after 50 days of age.
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[0050] FIG. 27 shows the serum insulin levels of female rats fed either a
Phyto-600 or a Phyto-tree diet after 50 days of age.
[0051] FIG. 28 shows body weights of OVX rats fed either a Phyto-600 (black
bars) or a Phyto-Free (white bars) diet after and placed on a behavioral
estrus in-
duction regimem.
[0052] FIG. 29 shows the white adipose tissue mass of OVX rats fed either a
Phyto-600 or 4 Phyto-Free diet.
[0053] FIG. 30 shows the serum leptin levels of OVX rats fed either a Phyto-
600 or a Phyto-Free diet.
[0054] FIG. 31 shows the body weights of 112-day-old male rats fed AIN-76,
Phyto-Free, Phyto-200, or Phyto-600 diets.
[0055] FIG. 32 shows the body weights of 279-day-old male rats fed AIN-76,
Phyto-Free, Phyto-200, or Phyto-600 diets.
[0056] FIG. 33 shows the body weights of 350-day-old male rats fed AIN-76,
Phyto-Free, Phyto-200, or Phyto-600 diets.
[0057] FIG. 34 shows the adipose tissue mass from 350-day-old male rats fed
AIN-76, Phyto-Free, Phyto-200, or Phyto-600 diets.
[0058] FIG. 35 shows serum insulin levels in 350-day-old male rats fed AIN-
76, Phyto-Free, Phyto-200, or Phyto-600 diets.
[0059] FIG. 36 shows serum leptin levels in 350-day-old male rats fed AIN-76,
Phyto-Free, Phyto-200, or Phyto-600 diets.
[0060] FIG. 37 shows body weights of 112-day-old female rats fed AIN-76,
Phyto-Free, Phyto-200, or Phyto-600 diets.
[0061] FIG. 38 shows body weights of 279-day-old female rats fed AIN-76,
Phyto-Free, Phyto-200, or Phyto-600 diets.
[0062] FIG. 39 shows body weights of 145-day-old male Noble rats fed Phyto-
Free or Phyto-600 diets.
[0063] FIG. 40 shows white adipose tissue mass from 145-day-old male Noble
rats fed Phyto-Free or Phyto-600 diets.
[0064] FIG. 41 shows body weights of 145-day-old female Noble rats fed
Phyto-Free or Phyto-600 diets.
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[0065] FIG. 42 shows white adipose tissue mass from 145-day-old female No-
ble rats fed Phyto-Free or Phyto-600 diets.
[0066] FIG. 43 shows baseline body weights of three groups of rats on a
Phyto-Free diet prior to receiving equol injections.
[0067] FIG. 44 shows body weights of three groups of rats after 21 days on a
Phyto-Free diet prior to receiving equol or vehicle injections.
[0068] FIG. 45 shows body weights of three groups of rats on a Phyto-Free
diet 7 days after receiving equol or vehicle injections.
[0069] FIG. 46 shows body weights of three groups of rats on a Phyto-Free
diet 15 days after receiving equol or vehicle injections.
[0070] FIG. 47 shows body weights of three groups of rats on a Phyto-Free
diet 22 days after receiving equol or vehicle injections.
[0071] FIG. 48 shows body weights of three groups of rats on a Phyto-Free
diet 28 days after receiving equol or vehicle injections.
[0072] FIG. 49 shows adipose tissue mass from three groups of rats on a
Phyto-Free diet 28 days after receiving equol or vehicle injections.
[0073] FIG. 50 shows testes weight from three groups of rats on a Phyto-Free
diet 28 days after receiving equol or vehicle injections.
[0074] FIG. 51 shows number elevated-plus maze anxiety-related behavior
(entries into open arms) of 300-day-old male rats fed 4 different diets.
[0075] FIG. 52 shows elevated-plus maze anxiety-related behavior (time in
open arms) of 300-day-old male rats fed 4 different diets.
[0076] FIG. 53 shows elevated-plus maze anxiety-related behavior (entries into
open arms) of 330-day-old female rats fed 4 different diets.
[0077] FIG. 54 shows elevated-plus maze anxiety-related behavior (time in
open arms) of 330-old female rats fed 4 different diets.
[0078] FIG. 55 shows serum isoflavone levels in 300-day-old male rats fed 4
different diets.
[0079] FIG. 56 shows serum isoflavone levels in 330-day-old female rats fed 4
different diets.
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[0080] FIG. 57 shows the observed change in BMI for individuals after 5
weeks of strict adherence to a diet containing isoflavones.
[0081] FIG 58 is a graph of the total distance traveled in meters before the
equol injections (A) and after the equol injections (B) in Phyto-free fed mid-
aged
female rats compared to animals fed a Phyto-600 diet in the Porsolt swim test.
[0082] FIG. 59 is a graph of the overall average speed (in meters per second)
before the equol injections (A) and after the equol injections (B) in Phyto-
free fed
mid-aged female rats compared to animals fed a Phyto-600 diet in the Porsolt
swim test.
[0083] FIG. 60 is a graph of the total time mobile in seconds before the equol
injections (A) and after the equol injections (B) in Phyto-free fed mid-aged
female
rats compared to animals fed a Phyto-600 diet in the Porsolt swim test.
[0084] FIG. 61 is a graph of the total number of dives before the equol injec-
tions (A) and after the equol injections (B) in Phyto-free fed mid-aged female
rats
compared to animals fed a Phyto-600 diet in the Porsolt swim test.
[0085] FIG. 62 is a graph of the total time immobile in seconds before the
equol injections (A) and after the equol injections (B) in Phyto-free fed mid-
aged
female rats compared to animals fed a Phyto-600 diet in the Porsolt swim test.
[0086] FIG. 63 is a graph of the total number of boli excreted before the
equol
injections (A) and after the equol injections (B) in Phyto-free fed mid-aged
female
-rats compared to animals fed a Phyto-600 diet in the Porsolt swim test.
[0087] FIG. 64 is a graph of the body weight loss in equol-treated adult male
rats compared to Phyto-(Free) fed control animals, n = 8 per treatment group.
[0088] FIG. 65 is a graph of the number of entries into the open arms of the
elevated plus maze (EPM).
[0089] FIG. 66 is a graph of the time spent in the open arms of the EPM.
[0090] FIGA7 is an illustration of the hormone sensitive hypothalamic regions
of the brain in adult male rats after being treated with equol for 25
consecutive
days.
[0091] FIG. 68 is a graph of the number of entries into the open arms of the
EPM.
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[0092) FIG. 69 is another graph of the time spent in the open arms of the EPM
of male rats treated during prenatal and early postnatal development with
equol
coinpared to genistein or control animals.
DETAILED DESCRIPTION OF THE DRAWINGS AND THE PRES-
ENTLY PREFERRED EMBODIMENTS
[0093] Despite the recent gains in understanding the pharmacology of equol as
it pertains to estrogen actions, our research showing potent antiandrogen
effects of
equol is unique and novel and opens new approaches to preventing or treating
an-
drogen-related conditions. Binding or sequestering 5a-DHT would provide a
means for inhibiting its effect on 5 a-DHT- sensitive tissues. There is no
known
ligand that is specific for 5a-DHT, but such an agent would have distinct
advan-
tages over non-discriminatory compounds that target the androgen receptor di-
rectly or the enzymes involved in androgen synthesis.
[0094] The invention is based on the unexpected discovery by the inventor
that equol may be used to prevent and/or treat neuropsychiatric disorders,
such as
depression, anxiety; and neurodegenerative disorders, such as Alzheimer's dis-
ease, Parkinson's disease or minor dementia. The inventor further noted that
equol may influence motivation for and regulation of feeding and weight
control.
[0095] These findings have important ramifications in health and disease and
indicate potential broad and important usage for equol in the treatment of
andro-
gen mediated pathologies. Without intending to be bound by a particular mecha-
nism it is believed that equol can act as an anti-androgen. The anti-
androgenic
properties of equol are unique in that equol does not bind the androgen
receptor
(AR) but specifically binds 5a-dihydrotestosterone (5a-DHT) with high affinity
and thereby prevents 5a-DHT from binding the AR. Furtherinore, both the R- and
S-enantiomers of equol may specifically bind 5a-DHT, sequester 5a-DHT from
the AR and block 5a-DHT's actions in physiological processes in vivo. Racemic
equol, which constitutes R-equol and S-equol and R-equol or S-equol alone,
selec-
tively bind 5a-DHT.
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[0096] In mammals, there are two principal androgens, testosterone and its 5a-
reduced metabolite, 5a-DHT. 5a-DHT is recognized as the most potent androgen
in the mammalian body. The AR, which is encoded by a single-copy gene located
on the human X-chromosome specifically mediates the actions of androgens. Al-
though both testosterone and 5a-DHT bind the AR, certain tissues (i.e.
prostate
gland, hair follicles, etc.) that are only slightly influenced by testosterone
are
greatly influenced by 5a-DHT. Furthermore, 5a-DHT has been implicated in a
number of diseases and disorders. Because, as mentioned above, equol specifi-
cally binds and prevents the actions of 5a-DHT, there is an indication for a
broad
and important usage for equol in the treatment of androgen-mediated
pathologies.
Specifically, the inventors discovered that equol may be used to prevent or
treat
neuropsychiatric and neurodegenerative diseases or disorders.
[0097] Equol has a structure similar to the steroidal estrogen estradiol.
Equol
is unique among the isoflavones in that it possesses a chiral center and as
such ex-
ists as two distinct enantiomeric forms, the R- and S-enantiomers. All
previous
studies on equol appear to have been conducted with the racemic form of equol.
There has in general been a lack of appreciation that two forms of equol exist
and
to our knowledge no previous study has reported on the specific actions or
activity
of the individual enantiomers R- and S-equol specifically bind 5a-DHT. Equol
racemic, R-equol or S-equol does not bind the androgen receptor. R-equol does
not bind the estrogen receptor system. S-equol only binds ERP (with an
affinity
approximately 5-times less than 17(3-estradiol). Thus, R- and S-equol have
SERM-like properties along with having the capability to selectively bind the
most
potent circulating androgen, 5a-DHT.
[0098] Most of the interest in soy and its constituent isoflavone, genistein,
and
to a lesser extent daidzein, has focused on either their estrogenic actions,
or other
non-hormonal actions such as their effects on enzymes, growtll factors or cyto-
kines, or their antioxidant actions. Never previously has there been any
discussion
of the potential antiandrogen actions for isoflavones and rarely is the
enantiomeric
forms of equol even mentioned. The present invention addresses the effects of
the
enantiomeric forms of equol, and specifically, the ability of both S-equol,
the natu-
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14
ral metabolite of daidzein, and R-equol antagonize the actions of the potent
andro-
gen dihydrotestosterone, 5a-DHT. Also, without wishing to be bound by a par-
ticular theory, it is currently believed that equol plays a positive role
acting as a
estrogen-like molecule via the estrogen receptor subtypes. Such effects open
up
novel possibilities for dietary, nutraceutical, and pharmacological approaches
to
prevention and treatment of disease where 5a-DHT plays a detrimental role.
This
includes but is not restricted to prostate cancer, skin diseases, hair loss,
neuropsy-
chiatric diseases or disorders, and neurodegenerative diseases. Additionally,
the
estrogenic actions of S-equol may also be of benefit in treating or preventing
pros-
tate cancer because the combined actions of equol acting at the estrogen
receptor
level and as an antiandrogen.
Equol Binds with 5a-DHT
[0099] Equol (7-hydroxy-3(4'hydroxyphenyl)-chroman) represents the major
metabolite of phytoestrogens daidzin and daidzein, isoflavones found
abundantly
in soybeans and soy-foods, and is an important biologically active molecule.
In
animals fed a phytoestrogen-rich diet, the major circulating isoflavone is
equol,
which accounts for 70-90% of the total circulating isoflavone levels. The
present
invention discloses a novel model of equol's biological properties. In binding
studies, equol enantiomers specifically bind 5a-DHT, but not testosterone,
DHEA
or estrogen. By doing so, equol sequesters 5a-DHT from the androgen receptor
without directly binding the androgen receptor itself. In vivo studies
demonstrate
that equol treatment of intact male rats significantly decreased prostate and
epidi-
dymis but not testes weights. In castrated male rats treated with 5a-DHT after
administering equol, equol blocked 5a-DHT's trophic effects on the prostate
gland
and its negative feedback effects on plasma luteinizing hormone (LA) levels.
[00100] While not wishing to be bound by a particular theory of the mecha-
nism of action, it is currently believed that equol can act as an anti-
androgen, by
specifically binding 5a-DHT and preventing 5a-DHT from binding to the andro-
gen receptor (AR) without itself binding the AR. It also has been shown that
5a-
DHT that has already been bound to the AR will not be competitively bound by
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enantiomeric equol. Therefore, one embodiment of the present invention is a
method of preventing 5a-DHT binding to the AR by contacting 5a-DHT with
equol prior to DHT-AR binding occurs. The enantiomeric equol may be brought
into contact with the 5a-DHT in vitr^o or in vivo.. When the 5a-DHT is to be
con-
tacted in vivo, the equol may be administered by any route that allows
absorption
of equol to the blood stream. Biologically available 5a-DHT is free and
unbound
by any native ligand prior to binding with equol.
[00101] Reproductive organs such as the prostate and epididymis are known
to be under androgenic control. For example, before puberty, when circulating
androgen levels are very low, rats fed a diet containing high levels of soy-
derived
isoflavones have prostate weights that are not altered by the consumption of
this
diet. However, after puberty when androgen levels increase, prostate weights
are
significantly decreased in phytoestrogen-rich-diet fed rats compared to
animals fed
a phytoestrogen-free diet. As shown herein, equol-treated intact rats display
sig-
nificant decreases in prostate and epididymis weights (without alterations in
testes
or pituitary weights during short-term studies). Notably, if the prostate and
epidi-
dymal values are standardized to body weight (per 100 grams) the ratios are
still
significantly different between equol-treated and control values. Equol also
blocked 5a-DHT's androgenic trophic influence on the prostate and epididymis,
without significantly altering testosterone levels.
[00102] 5a-DHT has negative feedback effects on circulating plasma levels
of luteinizing hormone (LH). It is believed that equol significantly increases
LH
levels by binding 5a-DHT and preventing this feedback effect. For example,
equol completely reverses the inhibitory action of 5a-DHT on LH levels in go-
nadectomized (GDX) males, whereas 5a-DHT plus equol-treated male rats display
LH levels similar to that of control values. These data further suggest that
equol
may have a specific ability to bind 5a-DHT, presumably in the blood
circulation
system, and block the hormonal action of 5a-DHT in suppressing LH production
or secretion. Therefore an embodiment of the present invention is a method of
modulating LH levels in an individual by contacting the 5a-DHT of the
individual
with enantiomeric equol. The equol can be administered by any route that
allows
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16
absorption of equol to the blood stream with the amount administered in accor-
dance with the nature of the ailment to be treated and size of the individual.
The Structure of Equol
[00103] Equol is distinct from most isoflavones in having a chiral center due
to the lack of a double bond in the heterocyclic ring. The phytoestrogen
isofla-
vones from soy (daidzein, glycitein and genistein), clover (formononetin and
bio-
chanin A), and kudzu, (peurarin), do not have a chiral center. FIG. 1 shows
the
chemical structures of R-equol and S-equol.
[00104] The R-and S-enantiomers conformationally differ and this is pre-
dicted to influence how an equol enantiomer fits into the binding site in the
cavity
of the dimerized ER complex, and how it binds with 5a-DHT.
[00105] Approximately 50% of equol circulates in the free or unbound form
in humans, and this is considerably greater than the proportion of free
daidzein
(18.7%) or estradiol (4.6%) in plasma. Since it is the unbound fraction that
is
available for receptor occupancy, and presumably for binding 5a-DHT, this
would
effectively contribute to enhancing the overall potency of equol.
Compositions Containing Equol
[00106] The present invention includes a composition having an at least
physiological acceptable quantity of equol that is able to bind and sequester
free
5a-DHT (but not testosterone or DHEA) thereby preventing it binding to the an-
drogen receptor following administration to an individual thereby having impor-
tant ramifications in health and disease and a broad and important use in the
treat-
ment of androgen-mediated pathologies. Specifically, the compositions compris-
ing equol may be used to prevent or treating neuropsychiatric or neurodegenera-
tive diseases or disorders.
[00107] Preferably, compositions containing equol (which may be S-equol,
R-equol, a racemic equol mixture, or a non-racemic equol mixture), are made
for
oral consumption.
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[00108] Preferably, equol is in an amount sufficient to prevent or treat dis-
eases or disorder according to the methods of this invention. Preferably, a
thera-
peutically effective amount of equol is used in the methods of this invention.
The
term "therapeutically effective amount" refers to the amount or quantity of
equol
(S-equol, R-equol, a racemic equol mixture, or a non-racemic equol mixture),
which is sufficient to elicit the required or desired prophylactic or
therapeutic re-
sponse, or in other words, the amount which is sufficient to elicit an
appreciable
biological response when administered to a subject. For example, a therapeuti-
cally effective amount of equol may reduce anxiety or depression in a subject.
[00109] The composition or a product containing the composition may be a
marketed or institutional food product, a pharmaceutical (i.e., drug), and an
over-
the-counter (OTC) medicament (i.e., nutraceutical).
[00110] Preferably, a food composition comprises at least about 0.25 mg,
more preferably, the food composition comprises about 1 mg, and preferably up
to
about 200 mg, enantiomeric equol or equol mixtures, per serving. Other amounts
of the equol in a food composition are also contemplated.
[00111] Preferably, an orally-administered medicament comprises at least
about 0.25 mg, more preferably about 1 mg, and preferably up to about 200 mg,
enantiomeric equol or equol mixture, per dose. Other amounts of the equol in a
medicament composition are also contemplated.
[00112] Preferably, a product for topical application can comprise at least
about 0.1 %, and up to about 10%, by weight S-equol, or R-equol, or
enantiomeric
mixtures. Other concentrations of equol are also contemplated for topical
products
containing equol.
[00113] A composition of the present invention may also include other cos-
metic and pharmaceutical actives and excipients. Such suitable cosmetic and
pharmaceutical agents include, but are not limited to, antifungals, vitamins,
anti-
inflammatory agents, antimicrobials, analgesics, nitric oxide synthase
inhibitors,
insect repellents, self-tanning agents, surfactants, moisturizers, stabilizes,
pre-
servatives, antiseptics, thickeners, lubricants, humectants, chelating agents,
skin
penetration enhancers, emollients, fragrances and colorants.
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[00114] An enantiomeric equol can also be an enantiomeric equol conjugate,
conjugated at the C-4' or the C-7 position with a conjugate selected from the
group
consisting of glucuronide, sulfate, acetate, propionate, glucoside, acetyl-
glucoside,
malonyl-glucoside, and mixtures thereof.
[00115] A composition or preparation comprising enantiomeric or mixture
of equol, for administering to subjects for the treatment and/or prevention
of, or
for reducing the predisposition to, androgen-related diseases and conditions
related
thereto, may also comprise one or more pharmaceutically acceptable adjuvants,
carriers and/or excipients. Pharmaceutically acceptable adjuvants, carriers
and/or
excipients are well known in the art, for example as described in the Handbook
of
Pharmaceutical Excipients, second edition, American Pharmaceutical
Association,
1994 (incorporated herein by reference).
[00116] In one embodiment, the composition of the invention comprises a
non-racemic mixture of S-equol and R-equol, having an EE for S-equol of more
than 0% and less than 90%. A composition that has an EE of 0% is a 50:50 race-
mic mixture of the two enantiomers. The composition can be made directly from
a racemic mixture, by an incomplete separation and removal of either the R-
equol
or S-equol enantiomer from the racemic mixture. The composition can also be
made by combining a first equol component comprising a mixture (either a non-
racemic or racemic mixture) of equol, with a second component comprising a
composition consisting essentially of S-equol or R-equol. This produces a non-
racemic composition that has an excess of S-equol or R-equol. Depending upon
the specific benefit or indication for the R-equol component and the S-equol
com-
ponent in a composition, a composition can be prepared comprising S-equol and
R-equol at a ratio of S-equol to R-equol preferably from greater than about
50:50
to about 99.5: 1, more preferably about 5 1:49 to about 99:1, and preferably
from
less than about 50:50 to about 1:99.5, more preferably about 49:51 to about
1:99.
[00117] The composition may be administered in the form of tablets, cap-
sules, powders for reconstitution, syrups, food (such as food bars, biscuits,
snack
foods other standard food forms well known in the art), or in drink
formulations.
Drinks may contain flavoring, buffers and the like.
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[00118] Compositions suitable for oral administration may be presented in
discrete units, such as capsules, cachets, lozenges, trouche, tablets, buccal
tablets,
sublingual tablet, each containing a predetermined amount of the extract; as a
powder or granules; as a solution or a suspension in an aqueous or non-aqueous
liquid; or as an oil-in-water or water-in-oil emulsion.
[00119] The composition typically does not comprise a significant amount
of any other androgen-receptor binding compound.
Identifying Equol Producers and Non-Equol Producers
[00120] Equol is formed following the hydrolysis of the glycoside conju-
gates of daidzein from soy, and the methoxylated isoflavone formononetin, or
its
glycosidic conjugates found in clever. Once formed, equol appears to be metab-
olically inert, undergoing no further biotransformation, save phase II
metabolism
or a minor degree of additional hydroxylation in the liver. As with daidzein
and
genistein, the predominant phase II reactions are glucuronidation and, to a
minor
extent, sulfation. Following the original discovery that equol's presence in
urine
was a function of soy food ingestion, it was observed that approximately 50-
70%
of the adult population did not excrete equol urine even when challenged daily
with soy foods, for reasons that are unclear. Furthermore, even when the pure
isoflavone compounds are administered, thereby removing any influence of the
food matrix, it has been shown that many people do not convert daidzein to
equol.
This phenomenon has led to the terminology of a person being an `equol-
producer' or `non-equol producer' (or `poor equol-producer') to describe these
two distinct populations.
[00121] Cut-off values have been empirically derived permitting assignment
of individuals to either of these categories. People who have plasma equol con-
centrations of less than 10 ng/mL (40 nmol/L) can be classified as `non-equol
pro-
ducers' and where levels are above 10 ng/mL (40 nmol/L) this defines `equol
pro-
ducers'. This distinction can also be derived from the levels in urine, an
equol
producer being someone excreting greater than 1000 nmol/L. Although the excre-
tion of equol is highly variable among individuals there is a large
demarcation be-
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tween those that can produce equol and those that cannot, consistent with a
precur-
sor-product relationship in enzyme kinetics catalyzing the reaction. There is
con-
sequently an inverse relationship between urinary daidzein and equol levels,
and
thus far no significant gender differences have been defined.
Preparation and Isolation of Equol Enantiomers
[00122] Enantiomeric equol can be prepared per se or as the racemic mix-
ture. Chemical synthesis routes can be used to produce the racemic mixture in
good yields. In a typical synthesis process, standard chemical treatments are
used
to hydrogenate the double-bond of the heterocyclic ring and to remove the car-
bonyl at position C-3. Typical starting materials are isoflavones such as
daidzein,
genistein, glycitein, peurarin, formononetin and biochanin A and their
glucoside
conjugates. Any conjugated form would be reduced to its aglycon by hydrolysis.
Suitable solvents for the reaction include organic acids such as glacial
acetic acid,
lower alcohols such as isopropanol, and mixtures thereof. Reduction catalysts
typically employed include Palladium, such as 10% Pd on charcoal. Reactions
can
run at temperatures from ambient to 60 C., with pressures ranging from
slightly
above ambient, up to 200 psig (14 atm. gauge), and with reaction times of up
to 30
hours or more.
[00123] After reaction completion, the catalyst is removed and any filtrate
evaporated. The crude residue is purified, typically by chromatography
employing
a silica gel column, with an eluent comprising C2-C4 alcohols, C3-C7 alkanes,
and mixtures thereof. The purified residue can be crystallized from n-hexane
to
produce ( )equol as a pure product, typically of at least 99%, with a yield
typically
of at least 75%. The equol crystallized product is colorless, not hygroscopic,
and
stable in air, and does not decompose during the final filtration procedure.
Method for the Isolation of the Individual R- and S-Enantiomers from Racemic
Equol
[00124] A racemic mixture of equol can be separated into its two distinct
enantiomers using a chiral-phase column with a mobile phase comprising a C4-C8
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21
alkyl and a C2-C4 alcohol. A typical example of a chiral-phase column is a
Chiralcel OD column or OJ column, supplied by Daicel Chemical Industries Ltd.
A preferred example of a mobile phase comprises 70% hexane and 30% ethanol.
After a first period of time from passing the racemic mixture into the inlet,
the
time period depending upon the type of column, type of eluent, eluent flow
rate,
temperature, and mass of the racemic mixture, a first effluent is collected
from an
outlet of the HPLC column. The first eluent is typically S-equol. After a
second
period of time from passing the racemic mixture into the inlet, the second
eluent
R-equol is obtained. The elution of an equol enantiomer from the column can be
detected by UV absorbance at 260-280 nm or by a more specific detection system
such as a mass spectrometer and monitoring of ions specific to equol.
Biological Production of S-Equol
[00125] S-equol can be produced biologically in bulk using conventional
food technology. A base solution media, food product or plant extract can be
pro-
vided that comprises daidzein or another related isoflavone from which
daidzein
can be derived. The daidzein or other isoflavone can be converted to S-equol
by a
standard bacterial or enzyme fermentation process, to provide a bulk solution,
food product or plant extract that comprises S-equol.
[001261 Conversion of daidzein to equol involves three major steps: 1) hy-
drolysis of any glucoside conjugate group, 2) conversion of the isoflavone
agly-
cons to a dihydro-intermediate, and 3) conversion of the dihydro-intermediate
to
equol. The metabolic pathway and enzymes for each of the three steps required
may not necessarily be present in one bacterium. Anecdotal evidence from human
studies suggests that there may be one or more bacteria that act in
conjunction to
perform these reactions, as evidenced from the fact that often dihydrodaidzein
can
be present in significant amounts in plasma and urine yet equol may be low or
barely detectable. Although equol may be produced from daidzein by a single or-
ganism it is believed that better or more efficient conversion can be achieved
when
using a mixture of bacterial species, each with its own metabolic profile.
Important
conditions for effective conversion to S-equol include the selection of the
bacterial
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22
organism or mixture of organisms, the temperature of incubation, and the
amount
of oxygen available to the organisms. These conditions can be optimized by
tech-
niques well known to persons skilled in this art. The organisms used to effect
this
change can be inactivated by standard techniques used in the food industry or,
al-
ternately, allowed to remain in an active state in the product.
[00127] Typically, one or more bacterial strains are required to convert the
daidzein (or other related isoflavone) through intermediate products to S-
equol,
which generally involves one or more of the three major reactions: the
conversion
of isoflavone glycone to aglycon isoflavone; the conversion of aglycon
isoflavone
to dihydro isoflavone; and the conversion of dihydro isoflavone to the
product,
equol. For example, a mixed culture of organisms isolated from equine feces or
a
mixed culture of organisms derived from the gastrointestinal tract of a person
known to an `equol producer' can convert, as they do in vivo, the glycone
daidzein
to the final product S-equol.
[00128] Typical bacterial strains that can convert a glycone to an aglycon
(such as daidzein to daidzein) include Enterococcus faecalis, a Lactobacillus
plan-
tarum, Listeria welshimeri, a mixed culture of organisms isolated from the
intesti-
nal tract of an `equol producing' mammal, Bacteriodes fragilis,
Bifidobacterium
lactis, Eubactria limosum, Lactobacillus casei, Lactobacillus acidophilous,
Lacto-
bacillus delbrueckii, Lactobacillus paracasei, Listeria monocytogenes,
Micrococ-
cus luteus, Proprionobacterium freudenreichii and Sacharomyces boulardii, and
mixtures thereof.
[00129] Typical bacterial strains that can convert an aglycon to equol (such
as daidzein to S-equol) include Proprionobacteria freundenreichii, a mixed
culture
containing: Bifidobacterium lactis, Lactobacillus acidophilus, Lactococcus
lactis,
Enterococcus faecium, Lactobacillus casei and Lactobacillus salivarius; and a
mixed culture of organisms isolated from the intestinal tract of an `equol
produc-
ing' mammal.
[00130] The time required for bacterial conversion of the glucosides to agly-
cons, or the aglycons to the equol product, will depend upon bacteria-related
fac-
tors, particularly concentration, the availability of oxygen, and the
temperature and
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23
pH of the incubating system. In most instances it is possible to achieve
substan-
tially complete conversion within 24 hours.
[00131] The pH range for bacterial conversion of the isoflavone glucosides
to aglycon isoflavones is from about 3 to about 9. The optimum pH depends pri-
marily upon the type of bacteria used, and should be selected accordingly.
[00132] The time required for enzymatic conversion of the glucosides to
aglycons, and aglycons to the equol product, depends upon enzyme-related fac-
tors, particularly concentration, and the temperature and pH of the system. In
most instances it is possible to achieve substantially complete conversion
within
24 hours, more preferably within about 2 hours, and most preferably within
about
1 hour.
[00133] S-equol produced in bulk can be separated from the resulting bulk
solution of a bacterial production of S-equol, by methods well known in the
art,
including crystallization, solvent extraction, distillation, and precipita-
tion/filtration. The resulting bulk solution can contain unreacted daidzein or
other
related isoflavone used, by-products,'and any reactants. Such methods can
include
the use of a reverse-phase or straight-phase liquid chromatography column and
these can be combined with chiral-phase chromatography.
[001341 A typical method of removing S-equol from a bulk solution or solid
phase is by extraction. An extractant solution is added to the solution or
solid
phase containing the S-equol. Typically the extractant is a low molecular
weight
alcohol such as methanol, ethanol, isopropyl alcohol, or propyl alcohol, or an
aqueous solution having a pH in the range from 3.5 to 5.5. Typically, if the
aque-
ous alcohol method is being used, sufficient alcohol is added to bring the
alco-
hol/water ratio to between a minimum of 40:60 and a maximum of 95:5. More
typically, the ratio is at least 60:40, and even more typically a ratio
between 65:35
and 90:10.
[00135] If an aqueous acid extraction method is used an aqueous acid solu-
tion is prepared with the pH adjusted to about 3.5 to about 5.5, and more
prefera-
bly within the pH range of about4.0 to about 5Ø Sufficient water is added to
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24
make a dilute liquid with a sufficiently low viscosity to permit separation of
solids
from liquids by centrifugation or filtration.
[00136] The liquid, from which insoluble solid matter has been removed, is
concentrated by conventional methods for removing liquids. Methods used typi-
cally include, but are not limited to, removal of solvent by evaporation,
preferably
under reduced pressure. The residual liquid is concentrated to at least about
15%
solids, and up to about 55% solids, more typically to between 30% and 50% sol-
ids. The concentrate is then diluted with water to reduce the solids content
and
increase the water to alcohol ratio. The amount of water added can be varied
over
a wide range, though a final solid content between 6% and 15%, and more typi-
cally about 13%, is preferred. The pH of the mixture is adjusted between about
pH 3.0 and about pH 6.5, with a preferred value between about pH 4.0 and about
pH 5Ø Typically the temperature is between about 20 C. to about 10 C., and
more typically about 5 C. to 7 C.
[00137] The solid material is then separated from the liquid by standard
separation techniques (centrifugation or filtration) and yields an equol-rich
solid
material.
[00138] The equol-rich material can optionally be purified, typically by
chromatography employing a silica gel column, with an eluent comprising C2-C4
alcohols, C3-C7 alkanes, and mixtures thereof. The purified residue can be
crys-
tallized from n-hexane to produce S-equol as a pure product, typically of at
least
99%, with a yield typically of at least 75%. The equol crystallized product is
col-
orless, not hygroscopic, and stable in air, and does not decompose during the
final
filtration procedure.
[00139] The S-equol product can be authenticated by GC-MS analysis of the
trimethylsilyl ether or tert-butyldimethylsilyl ether derivative, or some
other ap-
propriate volatile derivative of synthesized product as a single pure peak and
a
mass spectrum that is consistent with the published electron ionization
spectrum of
the trimethylsilyl (TMS) ether derivative of authentic equol. Confirmation of
the
product can also be established by direct mass spectrometry using electrospray
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ionization after introducing the sample into the instrument via an HPLC chiral-
phase column.
Treatment of Disease by AdministeringS-Equol, R-Equol, and Mixtures
[00140] This present invention provides a means for an individual subject to
overcome the problem of not being able to produce equol in vivo, or to supply
R-
equol in particular, by providing delivery of equol enantiomers, the S-equol
or R-
equol, or non-racemic mixtures of S-equol and R-equol directly, circumventing
the
need for intestinal bacteria for its production or for the need to consume soy
foods
with equol's precursor isoflavones. The delivery of S-equol can also
supplement
the in vivo production of S-equol in `equol-producers', as well as in `non-
equol
producers'.
[00141] Supplementing the diet of an equol producer with an equol enanti-
omer or mixture, may provide benefits when the ordinary level of S-equol pro-
duced by the equol producer is inadequate because of 1) insufficient
consumption
of isoflavones to produce equol, 2) antibiotic use that ablates the activity
of intes-
tinal bacteria to make equol from precursor isoflavones, or 3) other health
factors
that impact the level of equol production, e.g. short bowel syndrome or
surgical
construction of an intestinal stoma such as ileostomy. In addition, a
supplemental
level of equol is believed to provide enhanced effect on the health and well-
being
of the person.
[00142] This invention provides a method for delivering S-equol, R-equol,
racemic equol, or non-racemic mixtures of equol, in sufficient amounts to have
health benefits toward androgen-related diseases and conditions associated
therewith. The anti-androgenic activity of equol can affect a number of
tissues
throughout the body. In particular, the blocking of androgenic activity of 5a-
DHT
may be beneficial for the treatment and prevention of (A) growth of the
prostate
gland with aging, benign prostatic hyperplasia (BPH) and prostate cancer, (B)
fe-
male- and male-pattern baldness, (C) facial and body hair growth (hirsutism),
skin
health (acne, anti-aging and anti-photo aging), skin integrity (collagen and
elastin
robustness); (D) body weight gain (and loss), reduction in adipose tissue
deposi-
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26
tion and metabolism of lipids, as well as general regulatory behaviors and
effects,
such as food and water intake, blood pressure changes, thyroid, glucose,
leptin,
insulin and the influence on the immune system; and (E) Alzheimer's disease
and
emotional, mental health issues, such as, mood, depression, anxiety and
learning
and memory by reducing the 5a-steroid metabolites (covering androgens and pro-
gesterone) that are potent modulators of the GABAA receptor in the brain that
in-
fluences all of the brain characteristics above.
[00143] Typically, the amount of composition comprising the equol is ad-
ministered in an amount sufficient to produce a transient level of
enantiomeric
equol in the blood plasma of the mammal of at least 5 nanograms per milliliter
(ng/mL), more typically at least 10 ng/mL or greater, or transient levels of
enanti-
omeric equol in urine of greater than 1000 nmol/L. Preferably, the composition
is
administered orally in a dose amount of at least about 0.25 mg, more
preferably
about 1 mg, more preferably of at least about 5 mg, and of up to about 200 mg,
more preferably, up to about 50 mg; of enantiomeric equol. A typical level of
bioavailability of R-Equol in plasma after oral administration of 20 mg of R-
equol
enantiomer to a healthy adult is shown in the appearance/disappearance plots
of R-
equol in FIGS. 2 A and B.
[00144] The ability to deliver R- and/or S-equol in sufficient amounts is be-
lieved to provide several advantages over delivery of a racemic mixture of
equol.
First, the potency of R-equol or S-equol alone would typically be at least
twice
that of the racemic mixture. Second, the human body only produces the S-equol,
and therefore, a composition comprising only S-equol represents a "natural"
prod-
uct with an ingredient, S-equol, with which the body is familiar. Third, since
the
R-equol enantiomer has unique properties, a treatment composition comprising
only, or substantially only, the R-enantiomer can produce beneficial and/or
thera-
peutic effects. And fourth, administration of R-equol would supplement any en-
dogenous S-equol present and allow for both estrogenic and anti-androgenic ac-
tions to occur in the body.
[00145] The invention includes the use of enantiomeric equol to treat and
prevent diseases and conditions related to male- and female-pattern baldness.
5a-
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27
DHT is a known cause of scalp hair loss. An androgen, specifically the
principal
circulating androgen, testosterone, is converted to the more potent androgen,
5a-
DHT (in the hair follicle), and the hormonal action of 5a-DHT on scalp hair
folli-
cles cause hair loss. Thus, if the hormonal action of 5a-DHT can be blocked,
such
as by the use in the present invention of equol to bind 5a-DHT in the
circulation
(within blood vessels) and within the hair follicle, then scalp hair loss can
be de-
creased or prevented.
[00146] The invention includes the use of enantiomeric equol to treat and
prevent diseases and conditions related facial and body hair. Facial and body
hair
are regulated by androgens, but oppositely to that of the regulation of scalp
hair.
Specifically, the more potent androgen, 5a-DHT, increases facial and body
hair.
5a-DHT also increases the production of sebum (oil) from the sebaceous gland,
which can contribute to an increase in acne. Thus, the binding of 5a-DHT by
equol can cause a decrease in facial and body hair and in secretion of sebum
(oil),
and a reduction or prevention of acne.
[00147] The invention includes the use of enantiomeric equol to treat and
prevent diseases and conditions related to skin effects, skin quality and
integrity,
skin aging, skin photoaging, and skin pigmentation and lightening. Estrogens,
be-
fore but especially after menopause, improve skin health by increasing elastin
and
collagen content to improve skin characteristics or robustness. Also, when
skin is
damaged by acne or other skin disruptions (scratches, popping pimples or minor
cuts, etc.), the repair mechanism is faster and the skin heals better if
estrogen or
estrogen-like compounds, such as equol, are present. It is believed that an
equol
enantiomer mixture, and particularly S-equol, is a good stimulator of elastin
and
collagen and also can protect against photo-aging. Equol's blocking the
hormone
action of 5a-DHT can decrease sebum oil production from the sebaceous gland,
which can decrease or eliminate acne. Since S-equol (though not R-equol) binds
estrogen receptor(s) (mainly ER(3), the protective effects of this estrogen-
like
molecule would stimulate elastin and collagen in the skin. Additionally, since
equol is a strong antioxidant, it can protect the skin from aging, including
photo-
aging.
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28
[00148] The invention includes the use of enantiomeric equol to treat and
prevent diseases and conditions related to improved prostate health. The
conver-
sion of testosterone to the more potent androgen 5a-DHT is the result of the
action
of the enzyme 5a-reductase within the prostate. 5a-DHT causes benign prostatic
hyperplasia (BPH), increases in the prostate weight, and can result in the
need for
prostatectomies and radiotherapy to treat these conditions. Finally,
consumption
of soy foods has received increased attention due to their `health benefits'
of de-
creasing hormone dependent cancers such as prostate and breast cancer. Thus,
blockage of 5a-DHT by equol decreases prostate weight in animal models and
presumably will block BPH to prevent prostate cancer.
[00149] The invention includes a method of preventing or ameliorating neu-
ropsychiatric and neurodegenerative diseases or disorders by administering
equol
in an amount sufficient to prevent or ameliorate the neuropsychiatric or
neurode-
generative disease or disorder.
[00150] Neurodegenerative disorders, which are often associated with aging,
such as Alzheimer's disease and minor depression, as well as Parkinson's
disease,
are prevalent in the general population and are expensive to treat. About 4
million
people in the U.S. now are thought to have Alzheimer's disease resulting in
just
over 100 billion dollars in cost a year, according to the Lasker Foundation.
The
risk of developing Alzheimer's doubles every five years after age 65 and
almost
40 percent of people over age 85 have the disease (J.M. Lyness et al., Ann.
Inter.
Med., 144: 496-504, 2006; and Lephart et al., Curr Neurovas. Res., 1: 455-464,
2004). Minor depression is more frequent than major depression, with almost
50% percent of older community residents (60 years or older) displaying symp-
toms that are associated with minor depression (J.M. Lyness et al., Ann.
Inter.
Med., 144: 496-504, 2006).
[00151] In one embodiment, the invention includes the use of equol, includ-
ing enantiomeric equol to treat and prevent diseases and conditions related to
brain
function and mental health, including brain disorders, such as Parkinson's dis-
eases, dementia of the Alzheimer type, as well as other reduced or impaired
cogni-
tive functions associated with advancing age and with short- and long-term mem-
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29
ory loss. Brain mechanisms are more complex, and attempting to define what
molecules and factors regulate, influence, etc., mood, depression, anxiety and
so
on, can be difficult. However, there are some data to support the concept that
es-
trogens or estrogen-like molecules like isoflavones can assist cognitive
function in
conditions such as Alzheimer's disease, and may help to prevent the onset of
such
disorders, especially in postmenopausal women.
[00152] It is well established that neuropsychiatric disorders represent the
second largest cause of morbidity and premature mortality worldwide. The World
Health Organization estimates, collectively, that neuropsychiatric disorders
com-
prise 13 % of all reported diseases. These disorders include depression,
anxiety,
schizophrenia, bipolar disorder, obsessive-compulsive disorder, panic
disorder,
bulimia, alcohol and substance abuse, overeating and obesity, and attention-
deficit
hyperactivity disorder (World Health Organization. The world health report
2000
- health systems: improving performance. Geneva: WHO 2000). Approximately
one out of five Americans will experience an episode of a psychiatric illness
such
as depression or anxiety in any given year (Mental Health Report of the U.S.
Sur-
geon General, 1999). Notably, the most common co-morbid psychiatric diagnoses
with alcoholism are other substance abuse-related disorders such as,
depression
and anxiety disorders (R. Hitzemann, Alcohol Res. & Health 24: 149-158, 2000).
[00153] Depression is an effective disorder, or a disorder of mood. Symp-
toms include feelings of sadness, worthlessness, despair and loss of interest
in
life's activities or all pleasures. Most individuals suffering from depression
also
experience mental slowing, confusion, loss of energy and an inability to make
de-
cisions or concentrate. The symptoms can range from mild or minor to severe
and
are often associated with anxiety and/or agitation (Diagnostic Criteria form
DSM-
IV, American Psychiatric Association, Washington, D.C., 1994).
[00154] Depression has been referred to as the "common cold" of mental
illness. It is a prevalent disorder with approximately 18 million adults in
the U.S.
population afflicted on an annual basis (National Institutes of Healtlz,
Invisible
Disease: Depression. Bethesda, M.D., NIH publication 01-4591, 2001). It is es-
timated to be the second leading cause of disability, surpassed only by heart
dis-
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ease (C. J. Murray & A.D. Lopez (eds.), in The Global Buf-den of Disease, Har-
vard Univ. Press, Boston, 1996). Worldwide, its prevalence is 21 % in women
and 13 % in men (C.B. Nemeroff and M.J. Owens, Nature Neurosci., 5: 1068-
1070, 2002).
[00155] Although research continues as to the underlying causes of depres-
sion, it is generally believed that the disorder stems from decreases in
synaptic
concentrations of neurotransmitters or associated mechanisms of
neurotransmitter
action. Antidepressants are the current mode of therapy. Unfortunately, most
an-
tidepressants typically require several weeks of administration before
clinical ef-
fects are apparent, despite the fact that their biochemical actions occur
almost im-
mediately. Finally, there can be concerns about side effects, contra-
indications,
and safety of antidepressants.
[00156] Anxiety disorders are often characterized by excessive worry, fear
and apprehension, occurring frequently and causing a significant impairment of
functioning in a daily routine. Symptoms can include restlessness, fatigue,
diffi-
culty concentrating, irritability, muscle tension and sleep disturbances. The
life-
time prevalence of anxiety disorders in the general population is
approximately 5
to 6% (D.J. Nutt et al., Int. J. Neuropsychopharm., 5: 315-325, 2002). Women
are
affected more frequently than men, with a prevalence of 10% in women over the
age of 35, anxiety rates tend to increase in midlife and in older adults (H.W.
Wit-
tchen, Depress. Anxiety, 16: 162-171, 2002), that are similar to that seen for
de-
pression.
[00157] Current methods of treatment of anxiety includes pharmacological
treatment witli compounds, including anxiolytics, such as benzodiazepines
(e.g.,
diazepam, valium). Benzodiazepines possess anxiolytic, sedative-hypnotic, anti-
convulsant and muscle relaxant properties. However, if the circulating plasma
levels exceed the anxiolytic range, benzodiazepines may cause impairments of
mental and motor function, confusion and amnesia. Benzodiazepines may also be
subject to dose escalation and abuse by some patients. Withdrawal from long-
term use can precipitate anxiety, insomnia, restlessness and agitation (R.M.
Julien,
Prifner ofDrugAction, 7th ed., W.H. Freeman Co., New York, 1995; and M.E.
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31
Likey and B. Gordon, Medicine and Mental Illness, W.H. Freeman Co., Boston,
1991).
[00158] In reference to mood, anxiety, depression, and other mental health
conditions, there are two basic viewpoints. One line of research supports the
view
that estrogens (especially in women) regulate anxiety and help to decrease
anxiety
levels. Both estradiol and progesterone alter anxiety-related behaviors as
well as
the testicular androgen, testosterone (Imhof J. T. et al., Behav Brain Res,
56:177-
180 (1993)). The anti-androgenic activity of equol acts in the brain by
enhancing
neurotransmission and restoring synaptic density. Without being bound by any
particular theory, we believe that R- and/or S-equol are active in the brain
at the
same site(s) as estrogen, exerting an estrogenic response.
[00159] The second line of research is more complex and supports the view
that 5a-reduced steroids, especially progesterone, have the ability to bind
GABAA
receptors in the brain and cause sedation. GABAA is the major inhibitory neuro-
transmitter in the brain and its receptors are abundant in brain areas that
control
mood/emotion. By causing sedation, individuals express less anxiety. For exam-
ple, most women during pregnancy, report that they feel OK but they are
usually
tired or sleepy. This is due to progesterone being converted by the 5a-
reductase
enzyme in the body, but especially in the brain to 5a-dihydroprogesterone (5a-
DHP) and further metabolism of this molecule results in the most potent
`neuros-
teroid' that can bind the GABAA receptor and enhance the action of GABAA. Ad-
ditional supporting evidence that this 5a-dihydroprogesterone molecule (and
its
metabolite) can decrease brain activity is seen in epileptic women (who
experience
epilepsy and hence seizures) where these individuals almost never experience
sei-
zures during pregnancy due to the high circulating levels of progesterone. It
should be noted that 5a-reduced androgens like 5a-DHT also have a similar
effect
on GABAA receptors to cause sedation (in men) but at much lower levels com-
pared to 5a-DHP.
[00160] Putting these two views together, estrogen on the one hand de-
creases anxiety and hence increases activity. Conversely, blocking the action
of
5a-DHP also increases activity and thus in behavioral tests is interpreted as
de-
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32
creasing anxiety. For example, when the conversion of progesterone to 5a-DHP
in pregnant rats has been blocked, this results in a significant increase in
their lo-
comotor activity levels.
[00161] Taking a similar perspective on this in reference to equol, equol has
the ability to bind 5a-DHP (mainly seen in women) and 5a-DHT (mainly seen in
men). This would decrease the potent `neurosteroid' effects at the GABAA recep-
tor and decrease sedation and thus increase activity or decrease anxiety.
Moreover,
the ability of S-equol to bind the estrogen receptor(s) beta would also
increase ac-
tivity. Finally, our studies using young- or mid-aged adult rats, in males or
fe-
males have shown that dietary phytoestrogen consumption (Lund T. D. et al.,
Bt=ain. Res, 913:180-184 (2001); Lephart, E. D. et al., Neurotoxicology
Teratology,
24:1-12 (2002)), or injections with equol significantly decrease anxiety
levels as
expressed in the elevated plus maze test.
[00162] The elevated plus maze is a behavioral test used to quantify anxiety-
related behavior and identify anxiolytic drugs (Pellow S. et al., JNeurosci
Meth-
ods, 14:149-147 (1985); Current Protocols In Neuroscience (1997) 8.3.1-8.3.15,
John Wiley & Sons. NY, N.Y.). The test relies on the inherent conflict between
exploration of a novel environment and avoidance of its aversive features. Nor-
mally, animals spend little time and make few entries into the open arms of
the
maze compared to the closed arms of the maze (Imhof J. T. et al., , Behav
Brain
Res, 56:177-180 (1993). However, animals treated with anxiolytics, such as ben-
zodiazepines (valium), spend more time in the open arms and the number of en-
tries into the open arms reflects a decrease in anxiety-related behaviors
(Pellow S.
et al., JNeurosci Methods, 14:149-147 (1985); Current Protocols In
Neuroscience
(1997) 8.3.1-8.3.15, John Wiley & Sons. NY, N.Y.; Chopin P. et al., Psycho-
pharm, 110:409-414 (1993).
[00163] The invention also includes the use of equol (R- and S-enantiomeric
mixtures, etc.) to treat or prevent peri- or postmenopausal symptoms. These
symptoms include mood alterations, depression, anxiety, fatigue and emotional
and mental health, weight gain and obesity, and polycystic ovarian disease
among
others
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33
[00164] In another embodiment, the invention is a method of use of equol to
prevent or treat neuropsychiatric diseases, including depression, anxiety, and
oth-
ers mentioned above.
[00165] The invention includes the use of enantiomeric equol to treat and
prevent diseases and conditions related to body weight and body fat formation.
Weigh gain and obesity represent a huge health concern in the U.S. and
developed
countries throughout the world. For example, in the U.S., about 55 % of adults
are
overweight by international standards, about 23 % of American adults are
consid-
ered obese; and one in five American kids are considered overweight. Obesity
in
the U.S. cost the United State Government 12 % of the national health care
budget
in the late 1990s or $118 billion (World Watch Institute, March, 4, 2000).
[00166] Phytoestogens including equol, have the ability to decrease the for-
mation of white adipose (fat) tissue and increase white adipose tissue
breakdown,
thus decreasing body weight. Also, the estrogen-like nature of phytoestrogen
molecules decreases LDL (so-called "bad" cholesterol), blood pressure, and pre-
vents insulin resistance (or in other words, provides beneficial effects to
the dia-
betic condition). Since equol is a more potent isoflavone inolecule compared
to
the other phytoestrogeiis, it presumably provides the health benefits and
protects
against the conditions outlined above.
[00167] Since equol binds 5a-DHT, equol can also block the actions of 5a-
DHT that promote body weight gain. Thus, equol, and particularly S-equol, com-
bined anti-androgenic but at the same time an estrogenic hormone action from
the
same molecule (equol) would further improve the health benefits of body weight
loss (and weight management), decrease LDL cholesterol, decrease blood pres-
sure, and help prevent the devastating effects of diabetes.
[00168] The invention includes the use of equol to prevent or treat increase
in body weight and obesity. The term "increase in body weight" refers to any
in-
crease in body weight above an optimal healthy level that would be dependent
upon age, sex and statute of the subject. A person is considered to be obese
if they
are more than about 20 % over their ideal weight. That ideal weight must take
into account the person's height, age, sex, and build. Overweight or obesity
may
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34
also be defined using the body mass index, for instance. For adults,
overweight
and obesity ranges are determined by using weight and height to calculate a
num-
ber called the "body mass index" (BMI). BMI is used because, for most people,
it
correlates with their amount of body fat. An adult who has a BMI between ap-
proximately 25 and 29.9, is considered overweight. An adult who has a BMI of
around 30 or higher, is considered obese. Being overweight or obese increases
the
risk of many diseases and health conditions, including the following:
hypertension,
dyslipidemia (for example, high total cholesterol or high levels of
triglycerides),
type 2 diabetes, coronary heart disease, stroke, gallbladder disease,
osteoarthritis,
sleep apnea and respiratory problems and some cancers (endometrial, breast,
and
colon) (U.S. Department of Health and Human Services, Center for Disease Con-
trol (CDC) and Prevention, 2006).
[00169] The invention includes the use of equol, as enantiomeric equol or
mixture thereof, to treat and prevent lipid disorders such as high cholesterol
(hy-
percholesterolemia), lipidemia, lipemia and dyslipidemia (disturbances in
lipids).
A study has shown that plasma total cholesterol concentrations decreased 7.2%
(p=0.04) in equol producers compared with baseline levels and 3.0% (p NS) in
non-equol producers. The failure of soy protein to have significant
cholesterol-
lowering effects in adults with normal blood cholesterol levels, is, with few
excep-
tions, probably because of heterogeneity in the study populations with regard
to
the metabolism of soy isoflavones and the failure to recognize the relevance
of
equol formation (and specifically, non-formation in non-equol producers).
These
data suggest that enantiomeric equol influences lipids in a favorable manner,
and
that the effect is mediated by androgens. The composition comprising equol is
administered in an amount sufficient to reduce the level of lipids in the
blood
stream.
[00170] The invention further includes the use of R- and/or S-equol to im-
prove diminished blood vessel quality, by increasing reactivity or flexibility
in re-
sponse to acute changes in blood pressure, improving blood flow, and reducing
blood pressure. The invention also includes the use of R- and/or S-equol to
treat
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and prevent cancer, including benign prostate cancer, prostate cancer, and
skin
cancer.
[00171] Another embodiment of the present invention is the use of equol to
treat enlarged prostate or epididymis. Equol may also be used to prevent
enlarged
prostate or epididymis in individuals believed to be at risk for development
of
these pathologies, without alterations in testes, pituitary or body weights.
The
equol may be administered by any route that allows absorption of equol to the
blood stream.
DHT-Androgen Receptor
[00172] Other embodiments of the present invention include the use of equol
as a diagnostic agent in androgen-related disorders as well as disorders
arising
from disturbances in estrogenic/androgenic balance. In these embodiments,
equol
is administered to an individual to bind 5a-DHT and thereby prevent 5a-DHT
binding to androgen receptors. The changes in estrogenic balance are then meas-
ured or the change in androgen-binding is assessed to diagnose or further
elucidate
androgen-related anomalies.
Binding to 5a-DHT
[00173] Equol can be administered to bind 5a-DHT prior to or along with
other therapeutic moieties in order to assess the binding capacity of 5a-DHT
with
respect to the therapeutic moiety in question. Also, androgen-binding moieties
can be administered following administration of equol to assess the efficacy
of the
androgen-binding moiety to restore androgen activity and balance estrogenic ac-
tivity in the absence of 5a-DHT binding. Further, equol can be administered in
the presence of 5a-DHT-binding moieties in order to displace these naturally-
occurring or xenobiotic 5a-DHT-binding moieties from 5a-DHT.
Administration of Equol
[00174] In each of the embodiments of the present invention described
herein, if the administration of equol is to be oral, the equol may be
administered
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36
by supplying an oral dosage form of equol to either an "equol-producing"
mammal
or a "non-equol producing" mammal, or an oral dosage of daidzein, daidzin,
isoflavone mixtures containing daidzein, or soy protein preparations to an
"equol-
producing" mammal, wherein the administration of the oral dosage form results
in
effective absorption of equol to the blood stream. Administration of equol may
be
made by routes other than oral, if desired. For example, it is contemplated
that
rectal or urethral administration may be used to administer equol for the
treatment
of enlarged prostate or to prevent prostate enlargement. Additionally, it is
con-
templated that the active ligand binding site of the equol molecule may be
isolated
and synthesized for administration, which may provide 5a-DHT binding without
the full equol molecule. The dose of the equol molecule or fragment thereof
hav-
ing 5a-DHT-binding abilities is dependent upon the route of admiration and the
condition to be treated. Based on our in vivo studies it is apparent that
relatively
low doses of equol antagonize much higher doses of 5a-DHT, and this may be ex-
plained by the marked differences in the binding of equol to serum protein com-
pared with 5a-DHT. The latter circulates mostly bound to proteins, while equol
is
50% free. Generally, a dose sufficient to produce a concentration of equol or
ac-
tive fragments thereof in the bloodstream of the recipient of at least about
0.2 mg
equol per kg weight of the recipient and preferably at least about 0.5 mg/kg.
The
dose may be increased dramatically without incurring significant dose-limiting
side effects to greater than about 10 mg/kg. Oral administration can be
effected in
microencapsulated forms that can provide delayed or sustained release of the
me-
dicament.
[00175] Equol can be administered topically, transdermally, and subder-
mally in a variety of forms, including lotions, ointments, foams (including
shaving
creams), nasal sprays, skin patches (such as described in U.S. Pat. No.
5,613,958,
incorporated herein by reference in its entirety), electromechanical devices,
includ-
ing micropumps systems (such as described in U.S. Pat. No. 5,693,018 and U.S.
Pat No. 5,848,991, incorporated herein by reference in their entirety), and
subder-
mal implants (such as described in U.S. Pat. No. 5,468,501, incorporated
herein by
reference in its entirety).
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37
Experiments
(a) Determination of Equol Enantiomer in `Equol-Producing' Adults
[00176] The urine samples from adults consuming soy foods previously
identified as being `equol-producers' were analyzed. Equol was isolated from
urine (25 mL) by passage of the sample through a solid-phase Bond Elut C 18
car-
tridge. After washing the cartridge with water, the isoflavones were recovered
by
elution with methanol (5 mL) and the methanolic phase was taken to dryness un-
der a stream of nitrogen. The sample was subjected to enzymatic hydrolysis
with
Helix pomatia and then re-extracted on a Bond Elut C18 cartridge. The methano-
lic extract was taken to dryness under nitrogen gas and redissolved in HPLC mo-
bile phase (100 L). Equol enantioiners were identified by HPLC using a Chiral-
cel OJ chiral phase column as described herein above. The detection of equol
was
achieved by selected ion monitoring electrospray ionization mass spectrometry
(ESI-MS). Mass chromatograms of a pure standard of S-equol, and of urine from
an adult consuming soy food are shown in FIG. 3. Similar studies have demon-
strated that soy-derived isoflavones are converted to equol in rats, as well,
thus
validating rodent models of isoflavone metabolism.
[00177] The retention index and mass chromatogram establish that it is ex-
clusively the S-enantiomer of equol that is excreted in human urine as no
detect-
able R-enantiomer of equol could be found. Analysis of the plasma from the
same
`equol-producer' also revealed only the S-enantiomer of equol.
(b) Chemical Synthesis of Racemic Equol
[00178] Daidzein (200 mg, 0.8 mmol) is dissolved in a mixture of glacial
acetic acid (20 mL) and isopropanol (20 mL), and is reduced with 10% Pd on
charcoal (150 mg) at 55 p.s.i.g. (3.7 atm gauge). At the end of the reaction
(2
hours, TLC:isopropanol/n-hexane'/4) the catalyst is filtered off, and the
filtrate is
evaporated. The crude residue is purified by chromatography on a silica gel
col-
umn using as eluent a mixture of isopropanol and n-hexane (1:4 v/v), to give
( )equol as a pure product (160 mg, yield: 82%) crystallized from n-hexane.
The
product, colorless crystals, is not hygroscopic, is stable in air, and does
not de-
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38
compose during the final filtration procedure. The product of this chemical
syn-
thesis was in all respects identical with an authentic sample of ( )equol
(racemic
equol). FIG. 4 shows the GC-MS analysis of the trimethylsilyl ether derivative
of
synthesized product as a single pure peak and a mass spectrum that is
consistent
with the published electron ionization spectrum of the trimethylsilyl (TMS)
ether
derivative of authentic equol. The molecular ion as expected is at m/z 470 and
the
base peak at m/z 234. The purified equol product had a purity of greater than
99%, as confirmed by HPLC and mass spectrometry.
(c) Elution Order of S- and R-enantiomer by Optical Dichroism
[00179] A racemic mix of S-equol and R-equol were separated by chiral
chromatography on a Chiralcel OJ Column using a flow-rate of 1.0 mL/min and
with a gradient elution consisting of an initial mobile phase of 10% ethanol
in
hexane and increasing to 90% ethanol in hexane over a time period of 15
minutes
according to the program shown in Table 1:
[00180] TABLE 1:
Chiral Separation Gradient Eluent of Hexane and Ethanol
Time (min.) % Hexane % Ethanol
0 90 10
1.0 90 10
15.0 10 90
16.0 90 10
17.0 90 10
[00181] FIG. 5 shows the mass chromatogram of the ions recording (m/z
241) for a racemic mixture of S- and R-equol.
[00182] The first eluting material, designated as Enantiomer- 1, and the sec-
ond eluting material, designated as Enantiomer-2, were collected separately.
Each
enantiomer was weighed and the weighed samples dissolved in 1 mL of spectro-
scopic grade ethanol. Measurement of the optical rotation of each enantiomer
was
carried out at 200 C. using the light of wavelength in the line D of sodium.
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[00183] Enantiomer-1 material (1.6 mg exact weight) had first and second
measurements of -0.023 and -0.022, resulting in an optical rotation of -14[-
0.0225 X 1000/1.6], wllich corresponds with the S-enantiomer of equol. Enanti-
omer-2 material (1.7 mg exact weight) had first and second measurements of
+0.023 and +0.023, resulting in an optical rotation of +13.5 [+0.023 x
1000/1.7],
which corresponds with the R-enantiomer of equol.
d) Deter=mination of Receptor Binding Capacity of S- and R-Enantiomers
[00184] In vitro binding studies were performed to examine the relative af-
finities of S- and R-enantiomeric equol witli the estrogen receptors ERa and
ER(3.
[00185] Synthesis of Hormone Receptor Proteins: Full length rat ERa ex-
pression vector (pcDNA-ERa; RH Price UCSF) and ER(3 expression vector
(pcDNA-ER(3; T A Brown, Pfizer, Groton, Colo.) were used to synthesize hor-
mone receptors in vitro using the TnT-coupled reticulocyte lysate system
(Promega, Madison, Wis.) with T7-RNA polymerase, during a 90 min reaction at
30 C. Translation reaction mixtures were stored at -80 C. until further use.
[00186] Saturation isotherms: In order to calculate and establish the binding
affinity of the S-equol and R-equol enantiomers for ERa and ER(3, 100 L
aliquots
of reticulocyte lysate supernatant were incubated at optimal time and
temperature;
90 min at room temperature (ER(3) and 18 hrs at 4 C. (ERa), with increasing
(0.01-100 nm) concentrations of [3H] 17(3-estradiol (E2). These times were de-
termined empirically and represent optimal binding of receptor with estrogen.
Nonspecific binding was assessed using a 300-fold excess of the ER agonist
dieth-
ylstilbestrol, in parallel tubes. Following incubation, bound and unbound
[3H]E2
were separated by passing the incubation reaction through a 1 mL lipophilic
Sephadex LH-20 (Sigma-Aldrich Co., Saint Louis, Mo.) column Columns were
constructed by packing a disposable pipette tip (1 mL; Labcraft, Curtin
Matheson
Scientific, Inc, Houston, Tex.) with TEGMD (10 mm Tris-Cl, 1.5 mm EDTA,
10% glycerol, 25 mm molybdate, and 1 mm dithiothreitol, pH 7.4)-saturated
Sephadex according to previously published protocols (Wanda et al., 1986;
O'Keefe and Handa, 1990). For chromatography, the columns were re-
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equilibrated with TEGMD (100 L), and the incubation reactions were added in-
dividually to each column and allowed to incubate on the column for an
additional
30 min. Following this incubation, 600 L of TEGMD were added to each col-
umn, flow-through was collected, 4 mL scintillation fluid was added, and
samples
were counted (5 min each) in an 2900 TR Packard scintillation counter (Packard
Bioscience, Meriden, Conn.).
[00187] ' Competition binding studies were used to assess the estrogenic
properties of equol's S-equol and R-equol enantiomers. Based on the ability of
S
and R to compete with [3H] E2 for ER binding, the affinities for in vitro
translated
ER were shown to be very different for the two enantiomers. The S-equol enanti-
omer showed greatest affinity for ER(3 [Kd (nm)=0.73 0.2], while its affinity
for
ERa was relatively low by comparison [Kd(nm)=6.41 1.01. The R-equol enanti-
omer possessed a much lower affinity for both ER(3 [Kd (nm)=15.4 1.31 and ERa
[Kd (nm)=27.38 3.8]. For reference 17(3-estradiol binds ERa with a Kd
(nm)=0.13 and RE(3 with a Kd (rum)=0.15 in this system.
[00188] The study shows that only the S-equol enantiomer binds ER with
sufficient affinity to have potential relevance to circulating equol levels
reported in
humans. Compared with 17p-estradiol the relative binding affinities of the S-
equol and R-equol enantiomers for ERa were 49-fold and 211-fold less, respec-
tively. However, the S-equol enantiomer seems to be largely ER(3-selective
with a
relatively high affinity for ER(3, while the R-equol enantiomer binds with ap-
proximately 100-fold less affinity. The separate and associated determination
that
exclusively S-equol is found in human plasma and urine is significant in view
of
the specificity in binding of the two enantiomers.
e) Bioavailability of R-Equol
[00189] 20 mg of pure R-equol was administered orally to a healthy adult
after an overnight fast. Blood samples were collected at timed intervals over
the
next 24 hours and the plasma concentration of equol was determined by isotope
dilution gas chromatography-mass spectrometry with selected ion monitoring.
Rapid appearance of equol is observed in the plasma with peak concentrations
ob-
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41
served after 8 hours. The terminal elimination half-life of R-equol was
approxi-
mately 8 hours. Electrospray ionization mass spectrometry confirmed that the
equol present in plasma was the R-equol enantiomer (data not shown but
available
on request), thereby establishing that it is stable and does not undergo any
racemi-
zation or further biotransformation in the intestine. FIG. 2A shows an appear-
ance/disappearance plot of R-equol. FIG. 2B. shows an appearance/disappearance
plot of S-equol and R-Equol in plasma (mean levels) after oral administration
of
S-equol or R-equol in healthy human adults (from: K.D. Setchell et al., Am J.
Clin.
Nutr., 81: 1072-1091, 2005).
[00190] These results establish that R-equol if administered as a pharma-
cologic or nutraceutical preparation is extremely bioavailable.
EXAMPLES
[00191] Where appropriate, data were analyzed by analysis of variance sta-
tisti6s (ANOVA) followed by Newman-Keuls post hoc tests. Significance was set
at p<0.05. Curve fitting, scientific graphing, and analysis were completed
using
GraphPad Software (GraphPad Prism 3.0, San Diego, Calif.).
EXAMPLE 1
[00192] This example demonstrates the in vivo effects of equol on prostate
size and hormone secretion. Male Sprague-Dawley rats (400-500 grams) are ob-
tained from Charles Rivers Laboratories (Wilmington, Mass., USA). Rats are
caged in pairs and maintained on a 12-hour dark 12-hour light schedule (lights
on
at 0700 h) with ad libitum access to food and water.
[00193] One week following arrival, animals are given subcutaneous (sc)
injections (1/day for 4 days) of either dimethylsulfoxide (DMSO) (vehicle
control)
or racemic equol (0.25 mg/kg). Eighteen hours after the final injection,
animals
are killed via decapitation and trunk blood and prostate are collected for
analysis.
[00194] A significant reduction in prostate weight is observed in intact
males injected subcutaneously with equol in comparison to intact control
males.
Additionally, luteinizing hormone (LH) in these same intact males is
significantly
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42
increased in equol compared to control treated males. These finding are shown
in
FIGS. 6A and 6B, respectively. These effects are observed with relatively low
levels of equol compared to 5a-DHT and this can be explained by the marked dif-
ferences in the protein binding of equol, which circulates about 50% free, and
5a-
DHT which is mostly bound to serum protein.
EXAMPLE 2
[00195] In addition to equol's effects on prostate racemic equol blocks the
effects of DHT in other tissues, and decreases body weight. One week following
arrival, intact'males are given subcutaneous injections of either DMSO
(control) or
equol (0.5 mg/kg) once/day for 7 days. Following treatments animals are
weighed
and then killed via decapitation and tissue collection (prostate, testes,
epididymis,
and pituitary).
[00196] A significant weight decrease in the 5a-DHT-sensitive epididymis is
observed in racemic equol-treated males compared to controls. However, racemic
equol did not affect testes weight or pituitary weight. Body weight during
this
relatively brief treatment period does not differ significantly between
racemic
equol and control treatments. These results are presented in Table 2.
[00197] TABLE 2
Tissue Weights from Intact Males Given /Subcutaneously Injections
of Either DMSO (Control) or Racemic Equol
Prostate (g) Epididymis (g) Testes (g) Pituitary (g) Body (g)
DMSO (Con- 0.58 (:L0.05) 1.91 ( 0.10) 3.52 (:L0.10) 0.011 (:L0.003) 428.23
(14.04)
trol)
Racemic Equol 0.38 (10.06)* 1.59 (_L0.06)* 3.48 (:L0.08) 0.014 (10.002) 418.0
(+-7.52)
(0.25 mg/kg)
EXAMPLE 3
[001981 Adult male Sprague-Dawley rats are randomly assigned to three
groups and receive daily injections of either DMSO, a racemic mixture of equol
at
0.250 mg/kg/day), R-equol at 0.250 mg/kg/day, or S-equol at 0.250 mg/kg/day.
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The total volume of each injection is 0.3 cc, administered sc at the nape of
the rat's
neck. After seven consecutive days of treatment, the rats are killed and the
body
weight gain during the injection period is determined. Rats injected with R-
equol
have a significant decrease in body weight gain compared to control rats, as
shown
in Table 3.
[00199] TABLE 3
Body Weight Gained in Male Sprague-Dawley Rats Treated with Equol
Treatment Group
Prostate (g) Epididymis (g) Body Wt Gain (g)
Control 0.38 ( 0.01) 0.96 (10.03) 59.4 (:L3.4)
Racemic Equol (0.25 0.38 ( 0.02) 0.89 (:L.03) 56.1 ( -3.7)
mg/kg)
R-equol (0.25 mg/kg) 0.31 (-+0.02)* 0.85 (.1:0.04)* 45.6 ( 5.5)*
S-equol (0.25 mg/kg) 0.35 ( 0.03) 0.86 ( 0.05) 56.1 ( 3.1)
*Significant reduction compared to control
[00200] Testis and pituitary gland weights are not significantly altered by
the treatments (data not shown). The slight decreases in body weight (around
10%) from the equol experiments are very similar to those seen between animals
fed a Phyto-Free diet (a diet containing very low levels of phytoestrogens)
vs. a
Phyto-600 diet (a phytoestrogen-rich diet containing 600 ppm of isoflavones).
The
significant reduction in white adipose tissue deposition with the racemic
equol in-
jections (around 36%) is also comparable with that seen with the data sets
derived
from the dietary treatment studies. These findings suggest that R-equol can
regu-
late body weight and significantly decrease white adipose deposition.
EXAMPLE 4
[00201] This Example demonstrates equol binding to 5a-DHT. In initial
binding competition studies conducted to determine and establish equol's
binding
affinity for AR, we repeatedly observed that the apparent binding of [3H]DHT
was greater in the presence of equol than in its absence. Slight modifications
in
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44
the protocol where AR was removed from the incubation tube (leaving only
[3H]DHT and equol) resulted in the elution of [3H]DHT into the eluate
containing
[3H]DHT reaction complex
[00202] To further investigate this phenomenon, a 30 cm long Sephadex
LH-20 columns are used in order to identify elution peaks establishing the
binding
of [3H]DHT to equol. As shown in FIG. 7, a peak of [3H]DHT is apparent in the
elution fractions between 5 and 9 mL when the [3H]DHT+equol column incubate
is applied. This peak is not present when [3H]DHT alone is applied to the col-
umn. Furthermore, when 5a-DHT or 5a-DHT+equol are incubated with prostate
supernatant and then passed through the 30 cm column (FIG. 8A) two distinct
binding peaks are identifiable. The first peak of [3H]DHT represents that
bound
to the AR in prostate. This is found in the elution fractions between 4 and 5
ml.
In addition there is a later peak (between 5 and 9 ml), consistent with the
binding
of [3H]DHT to equol. However, when [3H]DHT is allowed to incubate with the
prostate supernatant for 36 hours (until equilibrium) prior to the
introduction of
equol there is no apparent binding of [3H]DHT (FIG. 8B). Both [3H]DHT and
[3H]DHT+equol (equol added 36 hours later) show a single peak in the elution
between 4 and 5 ml, suggesting that equol does not compete with 5a-DHT for the
AR nor does it bind [3H]DHT that is already bound to the receptor.
Furthermore,
it should be noted that the binding of equol to 5a-DHT appears to be specific,
since similar competition and binding studies have been conducted using
[3H]E2,
[3H]T, [3H]DHEA, [3H]CORT and [3H]progesterone without any occurrences of
binding to equol (data not shown). Saturation analysis of equol binding to
[3H]DHT shows an apparent Kd calculated at 1.32 0.4 nM (FIG. 9).
EXAMPLE 5
[00203] This example demonstrates that, in addition to modulating the effect
of 5a-DHT on prostate size, equol binds to 5a-DHT in vivo and blocks the nega-
tive effects on LH secretion. GDX males treated with a long-lasting analog of
5a-
DHT, DHT propionate (DHTP), show a significant increase in prostate weight
compared to vehicle-treated GDX control rats. Concomitant treatment with equol
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(DHTP+equol) blocks the effects of DHTP, while equol has no effect alone on
prostate size as shown in FIG. 10A. Equol also blocks 5a-DHT's negative feed-
back effects on LH. In GDX males LH is significantly decreased by DHTP treat-
ment compared to treatment with DMSO. Treatment with equol in combination
with 5a-DHT blocks the negative feedback effects of DHTP on LH secretion.
Equol alone has no effect on LH levels, shown in FIG. IOB.
EXAMPLE 6
[00204] This example demonstrates the effects of racemic equol on andro-
gen-sensitive tissues. One week following arrival, animals are gonadectomized
(GDX) under isoflurane anesthesia and allowed to recover for 7 days. Following
recovery, animals are assigned to the following groups 1) DMSO, 2) DHTP
(2mg/1 kg), 3) racemic equol (0.25 mg/kg), or 4) both DHTP and racemic equol.
Injections are given subcutaneously daily for 4 days. Animals are killed via
de-
capitation and trunk blood and tissues are collected for analysis. Plasma 5a-
DHT
is measured, shown in FIG. 11.
[00205] As expected there were significant elevations of plasma 5a-DHT in
animals treated with DHTP (GDX+DHTP, GDX+equol+DHTP groups). Plasma
5a-DHT was further elevated, although, not significantly, by co-treatment with
equol. Tissues, including prostate, testes and epididymis, are removed from
the
animal, dissected free of fat and connective tissues, weighed, fixed by
immersion
in 4% paraformaldehyde, and then sectioned at 15 m on a cryostat. Tissue sec-
tions are mounted on charged slides (Superfrost Plus, Fisher Scientific,
Pittsburgh,
PA) prewarmed to 23 C, and stained with hematoxylin and eosin (H&E), dehy-
drated in ascending alcohol and cleared with xylene. Histological sections are
shown in FIGS. 12 and 13. H&E stained prostates reflect a change due to both
GDX and treatments. The prostate glands of control, equol, and DHTP plus equol
treated groups show similar histology (FIG. 12 A, B, D). In these animals pros-
tates are characterized by very small atrophic glands with little volume in
the
gland lumen. In DHTP-treated animals (FIG. 12C), the glands show signs of cell
proliferation. Lumen size is increased compared to GDX animals; the epithelium
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46
is of a tall columnar type (FIG. 12C). In comparison to intact control animals
(FIG. l2E) the prostate of equol treated males show involution and consist of
more
closely spaced, atrophic glands (FIG. 12F). In comparison to control males,
the
epididymal histology of equol-treated intact males shows overall smaller
ducts, as
evidenced by shrunken lumen (FIG. 13).
EXAMPLE 7
[00206] Long-Evans male rats are raised (life long from conception to time
of sample collection) on either a phytoestrogen-rich diet containing 600 micro-
grams of isoflavones per gram of diet or 600 ppm of isoflavones (referred to
here-
after as the "Phyto-600" diet) or a diet containing very low levels of
isoflavones
(referred to hereafter as the `Phyto-Free' diet; containing approximately 10
ppm of
isoflavones).
[00207] As shown in FIG. 14 male Long-Evans rats fed the Phyto-600 diet
display significantly lower body weights at 33, 55 or 75 days of age compared
to
animals fed the Phyto-Free diet Adipose tissue (dissected from just below the
kid-
neys to just above the testes in the abdominopelvic cavity) is measured in the
55 or
75 day-old males. At both ages, white adipose tissue mass is significantly
greater
in the Phyto-Free-fed males compared to Phyto-600-fed animals, FIG. 15. It
should be noted that the reductions in body weight of Phyto-600-fed males are
modest, at approximately 10 to 15% percent, whereas, the reductions in white
adi-
pose tissue from the same animals are approximately 50-60% compared to Phyto-
Free-fed males. This greater reduction in white adipose tissue compared to
body
weight in soy fed animals is also a general characteristic seen in humans
consum-
ing soy-based diets (D. B. Allison et al., Eur J Clin Nutr, 2003, 57:514-522).
This
particular result is repeatedly seen throughout the various experiments
present in
these data sets, regardless of age, sex, rat strain or whether female rats
have their
ovaries removed (simulating the postmenopausal condition in humans).
[00208] When food and water intake is measured to determine whether these
parameters might influence body and adipose tissue weights, Phyto-600-fed
males
display slight but significantly higher food (FIG. 16A) and water (FIG. 16B)
in-
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47
takes compared to Phyto-Free-fed animals. Thus, the reductions in body and adi-
pose tissue weights cannot be explained by alterations in food/water intake be-
tween the diet treatments.
[00209] Since leptin is produced by adipose tissue (D. A. Sandoval et al., J
Diabetes Complications, 2003, 17:108-113.), serum leptin levels are measured,
along with insulin levels, to determine the alterations in these metabolic
hormones.
As shown in FIG. 17A, leptin levels at 33, 55 or 75 days of age are
significantly
decreased in Phyto600-fed males (which corresponds with the reductions seen in
adipose tissue weights from these same animals) compared to Phyto-Free-fed
males. Also, insulin levels are significantly decreased in Phyto-600-fed male
vs.
Phyto-Free-fed males (FIG. 17B), consistent with the benefits of protecting
against
insulin resistance associated with type-2 diabetes (V. Jayagopal et al.,
Diabetes
Care, 2002, 25:1709-1714.).
[00210] To demonstrate that circulating isoflavone levels are different in
Phyto600- vs. Phyto-Free-fed male and female (75 day-old) rats, serum
isoflavone
levels are determined by GC/MS as previously performed by our laboratories
(see
methods in K. D. R. Setchell, Am J Clin Nutr 129:1333S-1346S, 1998; and K. D.
R. Setchell et al., JNutr 132:3577-3584, 2002.). In each case for the
different
classifications of isoflavones Phyto-600-fed males display significantly
higher
isoflavone levels compared to Phyto-Free-fed values, shown in Table 4. More im-
portantly, equol levels in the Phyto-600-fed rats account for approximately
78% of
the total phytoestrogen levels.
[00211] TABLE 4
Isoflavone Concentrations in Adult Male and Female Rats
Genistein Daidzein Equol Total
(ng/ml)
Males:
Very low Isofla- 9.6-+0.3 10.8 0.6 23.2+0.4 43.5 1.0
vone Diet
High Isoflavone 413 67 394 58 1,161 325 1,967 45
Diet
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48
Females:
Very Low 3.9 0.2 5.3 0.8 21.6 1.2 30.8 2.2
Isoflavone Diet
High Isoflavone 99 9 117 7.4 931 21 1,147 5
Diet
[00212] To determine if other metabolic hormones were altered by the diet
treatments or by age, serum glucose and tliyroid (T3) levels are assayed.
Glucose
levels are slightly (but not significantly) higher in the Phyto-600-fed males
com-
pared to Phyto-Free-fed values, independent of age or source of the blood
samples
[either arterial (ART) or venous (TRUNK)], shown in FIG. 18. However, when
T3 levels are quantified, there is a significant increase in T3 serum levels
in 80 or
110 day-old male Long-Evans rats fed the Phyto-600 diet compared to Phyto-
Free-fed animals, shown in FIG. 19. This suggests that thyroid levels are en-
hanced with soy consumption consistent with anecdotal evidence of individuals
that decreased their thyroid medication or went off of thyroid treatment com-
pletely with the consumption of soy based foods in their diets. This is also
consis-
tent with reports of a similar increase in T3 levels in humans following
consump-
tion of soy foods (Watanabe, S. et al., Biofactors 2000: 12(14):23341).
EXAMPLE 8
[00213] In this experiment female Long-Evans rats are raised (life long from
conception to time of sample collection) on either Phyto600 or Phyto-Free
diets.
As shown in FIG. 20, rats fed the Phyto-600 diet display significantly lower
body
weights at 80 days of age compared to animals fed the Phyto-Free diet,
represent-
ing about a 12% reduction in body weight in the Phyto-600-fed animals.
[00214] As seen in the male Long-Evans Phyto-600-fed rats previously, fe-
males fed the Phyto-600 diet also displayed significant reductions in adipose
tissue
weight (by about 68%) compared to females fed the Phyto-Free diet, shown in
FIG. 21.
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49
[00215] Similar to results with male rats, serum glucose levels are slightly
but not significantly higher in Phyto-600-fed female rats at 80 or 10 days of
age
compared to animals in the Phyto-Free diet treatment group, shown in FIG. 22.
However, T3 levels are significantly higher in females fed the Phyto-Phyto-600
diet compared to Phyto-Phyto-Free fed animals at 110 days of age, shown in
FIG.
23. The T3 and glucose results in females are very similar to those obtained
in
male rats exposed to the same diet treatments, and thus, suggest similar
health
benefits for both genders. Samples collected at 100 days of age yield similar
re-
sults (i.e., significant reductions in body weight and adipose tissue weights
with
the consumption of the Phyto-600 diet vs. the Phyto-Free diet) in female Long-
Evans rats (data not shown).
EXAMPLE 9
[00216] Adult female rats are placed on the Phyto-600 or Phyto-Free diet
treatments from 50 to 215 days of age. Prior to 50 days of age the animals can
be
raised on a diet that contains approximately Phyto-200 ppm of isoflavones, or
similar a diet such as those used by animal suppliers. At 215 days of age,
Phyto-
600-fed females weigh significantly less than Phyto-Free-fed females,
represent-
ing about a 7% reduction in body weight, shown in FIG. 24. White adipose
tissue
in Phyto-600-fed females at 215 days of age is significantly reduced by about
30%
compared to that of females fed the Phyto-Free diet, shown in FIG. 25. Corre-
spondingly, serum leptin levels in the Phyto-600-fed females were
significantly
lower than those of Phyto-Free-fed, shown in FIG. 26. Insulin levels were
reduced
in the Phyto-600-fed vs. Phyto-Free-fed females to a similar degree seen previ-
ously, but did not reach statistical significance, shown in FIG. 27.
EXAMPLE 10
[00217] This example demonstrates the effects of a Phyto-600 or Phyto-Free
diet on adult ovariectomized (OVX) rats. The OVX rat is a well-established ani-
mal model of postmenopausal human females. In addition, OVX permits the sub-
cutaneous injections of estrogen and progesterone to stimulate behavioral
estrus in
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rats, to determine the effects of a Phyto-600 or Phyto-Free diet. Adult
ovariec-
tomized rats are fed a phytoestrogen diet of approximately 200 ppm of
isoflavones
("Phyto-200") until 50 days of age (all animals are ovariectomized at approxi-
mately 40 days of age). The female rats are age and weight matched at 50 days
of
age and placed into one of two diet treatments: either the Phyto-600 (black
bars) or
Phyto-Free (white bars) until 94 days of age. Baseline body weights are taken
at
50 days of age before the animals are placed on the diet treatments, again at
58
days (8 days of diet treatment), at 92 days of age (before injection of
estradiol),
and at 94 days of age (before injection of progesterone, and 6 hours later at
94
days of age (after chemical induction of behavioral estrus), shown in FIG. 28.
[00218] After consuming the diets for 8 days, the Phyto-600-fed rats display
a slight but significant reduction in body weight (of about 7%) compared to
Phyto-
Free-fed. This reduction in body weight is maintained before and during the
chemical induction of behavioral estrus by the estrogen and progesterone
(steroid)
injections.
[00219] White adipose tissue is measured at 94 days of age after the chemi-
cal induction of behavioral estrus, Phyto-600-fed OVX rats have approximately
50% less white adipose tissue mass compared to Phyto-Free-fed OVX rats, shown
in FIG. 29, consistent with findings in Examples 9 and 10.
[00220] Serum leptin levels in Phyto-600-fed OVX rats are decreased by
approximately 30% compared to Phyto-Free-fed rats, shown in FIG. 30,
reflecting
the decreased white adipose tissue mass.
EXAMPLE 11
[00221] Male and female Long-Evans rats are purchased from a supplier at
50 days of age. All animals are raised (from conception to 50 days of age) on
the
Phyto-200 diet At 50 days old the male and female rats are randomly assigned
to
one of four diet treatment groups: 1) AIN-76 diet containing approximately <5
ppm isoflavones, 2) the Phyto-Free, 3) Phyto-200, or 4) Phyto-600 diet,
described
in previous examples. The AIN-76 diet contains extremely low concentrations of
isoflavones, its formulation is quite different compared to the other three
diets.
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For example, the sucrose content is very high (almost approaching 50% of the
to-
tal diet formulation) and has a dense white consistency that the rats may not
enjoy
consuming as much as the regular plant-based ingredient diets (i.e., the Phyto-
Free
diet uses corn and wheat in its formulation which contains very low levels of
isoflavones); the Phyto-200 or Phyto-600 diets use varying amount of soy meal
in
their formulations. The male rats are maintained on their assigned diets until
350
days of age (equivalent to middle-age in humans). The female rats are
maintained
on the diets until 279 days of age (approaching middle-age in humans). Food
and
water intake is measured to determine the potential influence of these
parameters
on body weight changes. In each case these factors do not contribute to the
reduc-
tions in body weight with consumption of the isoflavone-containing diets
(i.e.,
Phyto-200 and Phyto-600 diets; data not shown).
[00222] Males-
[00223] At 112 days of age (on the diets for approximately 62 days), body
weights are recorded, shown in FIG. 31. The males fed the Phyto-Free diet have
the heaviest body weights and the Phyto-600-fed males have the lowest, while
the
males on the AS-76 and the Phyto-200 diets fall in between these two group val-
ues. The Phyto-600-fed body weights are significantly lower, by approximately
14%, than the Phyto-Free-fed males.
[00224] Correspondingly, at 279 and 350 days of age the male rats have a
similar profile to that observed at 112 days of age, shown in FIGS. 32 and 33,
re-
spectively. The males fed the Phyto-Free diet display the heaviest body
weights
and the Phyto-600-fed males display the lowest body weights, while the males
on
the AN-76 and Phyto-200 diets fall in between these two group values.
[00225] Males fed the AIN-76 or the Phyto-Phyto-Free fed males display the
highest white adipose tissue weights, measured at 350 days of age. The Phyto-
200-fed males show a 19% non-significant reduction in white adipose tissue
weight compared to AIN-76 or Phyto-Free-fed rats. Male rats fed the Phyto-600
diet have significantly less adipose tissue mass, an approximate 40%
reduction,
compared to AIN-76 or Phyto-Free-fed rats, shown in FIG. 34.
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[00226] Both serum insulin and leptin levels are significantly reduced as a
function of increasing concentrations of isoflavones in the diet treatments,
shown
in FIGS. 35 and 36, respectively. For example, males fed the Phyto-200 or
Phyto-
600 diets have significant reductions in insulin levels compared to AIN-76 fed
males. Also, Phyto-600-fed males show an approximate 50% reduction in insulin
levels compared to Phyto-Free-fed male Serum leptin profiles display a similar
pattern to that of the insulin results, where Phyto-200- or Phyto-600-fed
males
have significant reductions in serum insulin levels compared to either AIN-76
or
Phyto-Free-fed males. Insulin levels in the Phyto-600-fed males are 46% lower
compared to the Phyto-200-fed males. However, the difference between these two
diet groups do not reach significance (p<0.065).
[00227] Females-
[00228] To determine the influence of the four diet treatments on body
weight in female rats, the body weights are measured at 112 and 279 days of
age.
At 112 days of age, females fed the Phyto-Free and the Phyto-200 diets have
the
heaviest body weights and the Phyto-600-fed females have the lowest, while the
AIN-76 diet group fall in between the values of the other three groups, shown
in
FIG. 37. Body weights of the Phyto-600-fed groups are significantly lower, by
approximately 10%, compared to the Phyto-Free- and the Phyto-200-fed females.
[002291 At 279 days of age, female rats have a similar profile to that of age-
matched males for changes in body weight as influenced by the diet treatments,
shown in FIG. 38. Females fed the Phyto-600 diet display the lowest body
weights compared to AIN-76 or Phyto-Free-fed groups. This significant
reduction
in body weight in Phyto-600-fed females is approximately 15% between the diet
treatment groups tested.
EXAMPLE 12
[00230] Noble rats were used to determine whether an inbred strain of rat
has body and adipose tissue changes similar to those of out-bred strains of
rats
such as the Long-Evans animals when placed on isoflavone-rich diets. Due to in-
breeding, Noble rats are more fragile animals. For example, pregnant dams do
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not always carry their litters to term and frequently have smaller litters.
Noble rats
have been used for more than twenty years because they spontaneously generate
tumors with aging, especially in hormonal-dependent organs of the reproductive
tract. Thus, Noble rats have been extensively studied in the area of cancer re-
search (e.g., R. L. Noble, "Prostate carcinoma of the Nb rat in relation to
hor-
mones," Int Rev Exp Pathol, 1982, 23:113-159).
[00231] Male and female Noble rats are fed either the Phyto-Free or Phyto-
600 diets from conception until 145 days of age. Male Noble rats fed the Phyto-
600 diet have significantly lower body weights at 145 days of age compared to
age-matched males fed the Phyto-Free diet, shown in FIG. 39. As previously ob-
served for Long-Evans rats, the significant reduction in body weight
represents a
modest but consistent decrease of approximately 8% compared to Phyto-Free-fed
males. In addition, white adipose tissue mass is significantly decreased in
Phyto-
600-fed males compared to Phyto-Free-fed, shown in FIG. 40.
[00232] Female Noble rats fed the Phyto-600 diet have a 6% reduction in
body weight compared to Phyto-Free-fed females, shown in FIG. 41. White adi-
pose tissue mass is markedly decreased female Noble rats fed the Phyto-600
diet,
shown in FIG. 42. The Phyto-600 diet group has a 61% reduction in adipose tis-
sue compared to Phyto-Free- fed rats. The decrease in white adipose tissue is
similar to that seen in Long-Evans rats.
EXAMPLE 13
[00233] Prior to initiation of a Phyto-Free diet period Male Long-Evans rats
are fed a Phyto-200 diet, as described in previous examples. The rats are
placed
on a diet containing the Phyto-Free diet at approximately 52 days of age and
ran-
domly assigned to three groups. Baseline body weights after 14 days and 21
days
on the Phyto-Free diet for all rats are similar, shown in FIGS. 43 and 44,
respec-
tively. Beginning at 73 days of age, rats receive daily subcutaneous 0. 1 cc
injec-
tions of vehicle (peanut oil), 1 milligram of a racemic mixture of equol in
vehicle
(0.83 mg/kg body weight/day), or 5 milligrams of a racemic mixture of equol in
vehicle (4.2 mg/kg body weight/day) once every three days.
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[00234] At 80 and 88 days of age, there are slight decreases in body weights
and average body weight gains in both equol-injected groups compared to con-
trols, however, these values are not significantly different from controls,
shown in
FIGS. 45 and 46, respectively.
[00235] By the time the animals are 95 and 101 days of age, body weights
are only slightly decreased, ranging from 5 to 9% in equol-treated groups,
shown
in FIGS. 47 and 48. However, the average body weight gains in equol-injected
animals at both 95 and 101 days are significantly reduced compared to control
values. Though body weight differences are not significant, adipose tissue
deposi-
tion is strikingly lower in equol-treated groups. Adipose tissue mass in 101-
day-
old rats injected with equol is reduced by approximately 33% compared to con-
trols, shown in FIG. 49.
[00236] To determine whether equol injections have an adverse effect on
male reproductive organs, testis weights are quantified in these animals.
There are
no significant alterations in testes weight with the equol injections, with
testicular
weight essentially the same among the injection treatment groups, shown in
FIG.
50.
EXAMPLE 14
[00237] Fifty day-old Long-Evans males and females are caged individually
and maintained on a 10-hour dark 14-hour light schedule (lights on 1400-0400).
Animals are randomly assigned to diet groups, and allowed ad libitum access to
one of four diet treatments: 1) AIN-76, 2) Phyto-Free, 3) Phyto-200, or 4)
Phyto-
600 diet. The rats remain on the diets until mid-aged (at approximately 300
days
of age in males and at approximately 330 days of age in females) when the ani-
mals are tested in the elevated plus maze and anxiety-related behaviors were
quan-
tified. Thereafter, serum phytoestrogen levels are quantified by GC/MS
according
to the method described by Coward L et al., JAgric Food Chenz, 41:1961-1967.
The behavioral patterns of anxiety are compared to the serum profiles of
circulat-
ing isoflavone levels in the diet treatment groups by sex.
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[00238] In males, there is a dose-dependent expression of anxiety-related
behaviors where animals fed the highest concentration of isoflavones display
the
lowest anxiety parameters. In contrast, animals fed the AIN-76 diet display
the
highest levels of anxiety, shown in FIG. 51. When the percent of time spent in
the
open arms is analyzed a similar pattern is seen to that of the nunlber of
entries into
the open arms. Notably, the Phyto-600 fed males display the highest percentage
of time spent in the open arms, while the lowest percentage of time spent in
the
open arms is display by animals fed the AIN-76 diet, with Phyto-free and Phyto-
200 values falling in between these maximal responses in a dose-dependent fash-
ion, shown in FIG. 52.
[00239] Prior to testing in the elevated plus maze, females are monitored by
vaginal smears for 12 consecutive days to verify that none are cycling to
minimize
effects of the estrous cycle. Female rats have a similar pattern of anxiety-
related
behaviors as those observed in the male rats. However, the influence of
dietary
isoflavones is not as robust as that seen in males. Although, the highest
percent-
age of the number of entries into (FIG. 53) or time spent on the open arms
(FIG.
54) is seen in Phyto-600-fed females, with a stair-step pattern of decline
until the
lowest percentage of entries is seen in the AIN-76-fed females.
[00240] When behavioral testing is complete, the serum phytoestrogen lev-
els are determined and compared to the patterns of anxiety-related behaviors.
In
both males (FIG. 55) and females (FIG. 56), the circulating isoflavone levels
cor-
respond to the expression of anxiety-related behaviors, demonstrating an
associa-
tion between circulating isoflavone molecules and anxiety. These data demon-
strate that the isoflavone content of a diet can have significant effects on
anxiety.
EXAMPLE 15
[00241] Adult male Sprague-Dawley rats receive daily injections of either
DMSO, racemic equol (0.250 mg/Kg/day), R-equol (0.250 mg/Kg/day), or S-
equol (0.250 mg/Kg/day) in a total volume of 0.3 cc DMSO by subcutaneous in-
jection. At the end of seven consecutive days of treatment the animals are
tested
in the elevated plus maze in order to quantify anxiety-related behaviors. As
shown
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in Table 5 below, males injected with racemic equol or R-equol display a
signifi-
cant decrease in anxiety levels compared to control rats.
[00242] TABLE 5
Anxiety-Related Behaviors in the Elevated Plus Maze of Equol Injected Male
Rats
Injection Center Area Open Arm Time Open Arm Entries
Treatment Groups (in seconds) (in seconds) (in seconds)
DMSO 17.5+4.1 16.3+4.0 1.0+0.03
Equol (racemic) 37.1 + 6.3* 40.9 + 7.0* 2.3 + 0.4*
R-Equol 36.4+7.6 50.0 + 10.0* 2.0+0.3*
S-Equol 27.5 + 4.1 33.6 + 7.0 2.0 + 0.4**
*= Significant decrease in anxiety-related parameters (i.e., center area of
maze
time or time spent in the open arms or number of open arm entries) vs. control
values
**= Significant decrease in anxiety-related behavior (i.e., number of open arm
en-
tries ) vs. control values.
n = 8 animals per group
[00243] These findings are consistent with those obtained utilizing the 4 die-
tary treatments containing different concentrations of isoflavones, and
demonstrate
that equol is a major factor in regulating anxiety and other neurological
states such
as mood and depression that have obvious potential for broad health benefits.
EXAMPLE 16
[00244] Twenty-nine (29) adults with hypercholesterolemia are fed a diet
containing 33 mg of total isoflavones daily for 5 weeks. FIG. 57 shows the ob-
served change in BMI for each of the 29 individuals after 5 weeks of strict
adher-
ence to the diet containing isoflavones. The average reduction in BMI over
this
period, although small, is nevertheless significant (p=0.01). These results
suggest
that phytoestrogen-rich diets can influence weight control in humans. The
study
did not identify the component(s) responsible or the mechanism of weight
control.
Average baseline BMI (n=29) is 26.6+-0.8, and the average BMI (n=29) at 5
weeks
is 26.2 0.7.
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[00245] While various embodiments of the present invention have been de-
scribed in detail, it will be apparent that further modifications and
adaptations of
the invention will occur to those skilled in the art. It is to be expressly
understood
that such modifications and adaptations are within the spirit and scope of the
pre-
sent invention.
EXAMPLE 17
[00246] Equol Decreases Depression and Body Weight in Mid-Aged Female
Rats:
[00247] Isoflavones have been shown to possess beneficial effects on car-
diovascular and hormone-dependent diseases, however, little is known about
their
influences on brain and behavior, especially behaviors such as depression and
de-
spair. In this study, mid-aged Long-Evans female rats were examined in this
con-
text.
[00248] Age-associated morphological and physiological changes in rodents
are equivalent to humans using the following age ranges: Mid-aged rats (10 -
12
months of age) are roughly equivalent to humans 45 - 55 years of age, while
aged
rats (21-23 months of age) are roughly equivalent to humans 65 - 75 years of
age
(D. Mileusnic et al., Neurobiol. Aging, 20: 19-35, 1999). Additionally, mid-
aged
rats (at 12 months old) are equivalent in age to postmenopausal women at ap-
proximately 55 years of age. The average age of menopause in the United States
is approximately 51 years of age (National Institutes of Health, USA).
[00249] All female rats in this study were examined for hormonal patterns of
estrus cycling characteristics. All animals in this study were not cycling
which is
consist with scientific reports using this aging animal model (C.R. Anzalone
et al.,
Biol. Reprod., 64: 1056-1062, 2001).
[00250] The female rats were exposed (from time of conception) to either a
phytoestrogen-rich (Phyto-600) or a phytoestrogen-free (Phyto-free) diet.
[00251] At 12 months of age, the animals were analyzed using the Porsolt
forced swim test that is a standard preclinical test with applications to
humans for
investigating the neurobiology of depression and of action of antidepressant
agent
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58
(R.D. Porsolt, M. Pichon and M Jalfre, Nature 266: 73 0-732, 1977; and M.A.
Geyer & A. Markou, in Psychopharmacology: The Fourth Generation of Pro-
gress, NY, Raven Press, pp. 787-798, 1995).
[002521 The test is designed to force rats to swim vigorously. Over time,
some of the animals may become immobile which is considered a condition of be-
havioral despair. The total time an animal is immobile is an index of despair
or
depression. Other parameters include: a) total distance traveled, b) total
time mo-
bile (the opposite of the immobile index), c) number of dives the animal
attempts
demonstrating escape behaviors and d) the number of defecations or boli
excreted
during the test which indexes the level of emotionally (C.S. Hall, J. Cornp.
Psych.,
18: 385-403, 1934), increased boli numbers during the test would indicate
heighten emotional behavior. All of these parameters were recorded in this
study
and quantified by AnyMaze computer software and the total time of the Porsolt
forced swim test was 480 seconds or 8 minutes.
[00253] Thus, animals displaying increased levels of total distance traveled,
mobility times and dives along with low numbers for total time immobile and
boli
excreted would exhibit low levels of despair or depression. Animals exhibiting
behaviors opposite to the parameters above would display increased levels of
de-
spair or depression.
[00254] The animals were tested twice.
[00255] Test 1 compared diet exposure alone, shown as A in FIGS. 58-69
and labeled as "before equol injections."
[00256] In the second stage of testing the Phyto-free fed females were in-
jected with equol (@ 5 mg/day or 2.5 mg/kg via subcutaneous administration)
for
8 days while the Phyto-600 fed females received dimethylsulfoxide (DMSO) vehi-
cle control injections. (DMSO has been used in medicine due to its ability to
penetrate deeply through the skin and other membranes without damaging them
and ability to carry compounds into the circulation).
[00257] Test 2 assessed depressive behaviors following the equol treatments,
shown as B in FIGS. 58-69 and labeled as "after equol injections."
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[002581 In the first Porsolt test - labeled A: Phyto-600 fed females dis-
played significantly greater a) distance traveled (meters), see FIG. 58 A; b)
overall
average speed (meters/second), see FIG. 59 A; c) total time mobile (seconds),
see
FIG. 60 A; and d) number of dives compared to.Phyto-free females, see FIG. 61
A. Conversely, the Phyto-free females displayed significantly greater a) total
time
immobile (seconds), see FIG. 62 A and b) boli excreted, see FIG. 63 A,
compared
to Phyto-600 females.
[002591 Following the equol treatments for 8 days, the Phyto-free females
displayed values that were similar to those exhibited by the Phyto-600 females
for
all of the parameters tested above. For example, a) total distance traveled
was in-
creased and similar to control values, see FIG. 58 B; b) increased average
speed
was also similar to controls, see FIG. 59 B; c) along with increased total
time mo-
bile, see FIG. 60 B (the total time mobile significantly increased in the
Phyto-free
rats after the equol injections compared to the before equol injections levels
(see
* *)); d) and the number of dives was significantly reduced and comparable to
con-
trol levels, see FIG. 61 B. Furthermore, the total time immobile was
significantly
reduced and similar to controls, see FIG. 62 B. Finally, as illustrated in
FIG. 63 B,
the number of boli was reduced after equol treatments and this was essentially
the
same to that of control values.
[00260] Also, the average body weight loss (in grams) at the end of the 8
day equol treatments revealed that equol significantly decreased body weights
compared to controls, see TABLE 6, representing a 2.6-fold decrease in body
weight over controls after the 8 day treatment interval.
[00261] TABLE 6. Body Weight Loss in Mid-Aged Female Long-Evans Rats
Treated with Equol
Treatment Group Body Weight Loss (grams)
Phyto-free plus EQUOL - 31.2( 4.8*)
Phyto-600 plus vehicle - 11.7 ( 2.1)
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*= significant decrease in body weight in equol-treated mid-aged female rats
vs.
control values (SEM = standard error of the mean)
[00262] These data suggest that exposure to equol, including short term ex-
posure, has beneficial antidepressant effects in mid-aged female rats. It is
believed
that the positive anti-anxiety parameters seen in the Phyto-600 fed animals
was
due to consuming the enriched isoflavone (soy) diet that in turn would then be
converted to equol in these rats. It is well established that rats generate
very high
levels of equol when fed an enriched soy diet (i.e. the Phyto-600 diet) and
that a
majority (70 to 90 % of the total) of the circulating isoflavone/isoflavone
metabo-
lite levels are represented by equol. Notably, when equol was administered
(short-
term) to mid-aged female rats fed a Phyto-free diet all of the Porsolt test
depres-
sant parameters (shown in B in FIGS. 58-63) were reversed and were similar to
the Phyto-600 group. These data sets are strengthened by the fact that the
Phyto-
600 and Phyto-free animals served as their own controls, before and after the
equol injections. Thus, the results of this study indicate that equol alone ac-
counted for all or at least the majority of the positive effects of
significantly reduc-
irig depressive-like behaviors. As such, the use of equol has great promise in
im-
proving mood, despair and depressant-related behaviors with potential food,
nutri-
tional and pharmaceutical applications.
EXAMPLE 18
[00263] Equol Decreases Anxiety-Related Behaviors and Body Weight In
Adult Male Rats, But Does Not Alter Hormone Sensitive Brain Regions
[00264] Hypothalamic structures such as the sexually diinorphic nucleus
(SDN) and anterioventroperiventicular nucleus (AVPV) are important regions in-
volved in reproductive function/regulatory behaviors that are sensitive to
steroid
hormonal influences during perinatal and subsequent postnatal development in
rats. It was previously shown that SDN and AVPV structures are sensitive to
isoflavone influences in adult rats and, in general, many reports have
demonstrated
that phytoestrogens are neuroprotective (E.D. Lephart et al., J. Steroid
Biochem.
Mol. Biol., 85: 299-309, 2003; E. D. Lephart et al., Curr. Neurovas. Res., 1:
455-
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61
464, 2004; and L. Bu & E.D. Lephart, Neurosci. Lett., 385: 153-157, 2005). How-
ever, equol, the major isoflavone metabolite in rodents (and many other animal
models) has not been examined in brain studies.
[00265] In this study, anxiety-related behaviors were examined along with
hormone levels in adult Long-Evans male rats exposed to equol. A known and
accepted animal behavioral test for psychiatric disorders in humans, such as
ele-
vated plus maze (EPM) was used for this purpose (P. Willner, in Behavioral Mod-
els in Psychopharmacology: Theoretical, Industrial and Clinical Perspectives,
New York, Cambridge Univ. Press, 1991; M.A. Geyer & A. Markou, in Ps,y-
chopharmacology: The Fourth Generation of Progress, NY, Raven Press, pp.
787-798, 1995; and R. Hitzemann, Alcohol & Res. Health, 24: 149-158, 2000).
[00266] The EPM test relies on the inherent conflict between exploration of
a novel environment and avoidance of its aversive features. Typically,
aiiimals
spend little time and make few entries into the open arms (new environment)
compared to the closed arms of the maze (secure environment). For example, an
anti-anxiety agent or drug would increase the number of entries into and total
time
spent in the open arms of the maze.
[00267] Male rats 50 days of age were given a diet low in phytoestrogens
(10 ppm of isoflavones) and remained on this diet until 215 days of age. At
150
days of age the rats were age and body weight matched and then divided into
con-
trol or equol treatment groups. Starting at 190 days, the male rats received
daily
subcutaneous (0.1 cc) injections of control vehicle (DMSO) or equol (2.5
mg/Kg)
for 25 consecutive days.
[00268] After 15 days on the treatments the animals were tested in the EPM
that was video-taped for 3 minutes and later recorded for the number of
entries
into- and the percentage of total time spent - on the open arms via AnyMaze
computer software. `
[00269] At 215 days of age the animals were sacrificed and body/prostate
weights, serum hormone and isoflavone levels were determined along with deter-
mining the volume of hypothalamic regions that are sensitive to hormones.
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[00270] The significant reductions in prostate weight and 5a-DHT, along
with no alteration in serum testosterone, luteinizing hormone (LH) or 17(3-
estradiol levels in equol-treated male rats compared to control values, were
previ-
ously reported in U.S. Publication No. 2005/0245492 A1, contents of which are
incorporated by reference in its entirety.
[00271] The brains were processed via standard staining and analyzed via
Bioquant for morphometric SDN and AVPV parameters by treatments. The se-
rum equol levels in equol-treated animals were equivalent to consuming a phy-
toestrogen-rich soy diet.
[00272] As illustrated in FIG. 64, body weights were significantly decreased
in the equol-treated males as compared to control animals. In the EPM, equol-
treated animals displayed significantly less anxiety-related behaviors
compared to
control values (i.e., the number of entries into- and time spent on- the open
arms of
the maze were significantly greater vs. controls), see FIGS. 65 and 66, respec-
tively.
[00273] No significant alterations in SDN or AVPV volumetric morphomet-
ric parameters were seen with the exposure to equol for 25 consecutive days,
see
FIG. 67 and TABLE 7.
[00274] These results suggest that equol does not alter hormone-sensitive
hypothalamic volumes in rodents during adulthood which is opposite to the con-
sumption of soy (phytoestrogens) via dietary routes for an equivalent exposure
in-
terval previously reported by our laboratory. This further suggests that equol
ap-
pears may be a safe and nontoxic agent with potential neural applications to
ad-
dress brain/behavior disorders and men's/women's health issues. Finally, these
data suggest that the presence of equol in nutritional food products, dietary
sup-
plements or pharmaceutical applications would be an effective treatment in
weight
control and to modulate or reduce anxiety.
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[00275] TABLE 7: Morphometric Volumes (expressed in mm3 x 10 "3) of the
Hypothalamic Structures- The Sexually Dimorphic Nucleus (SDN) and The Ante-
rioventroperiventicular Nucleus (AVPV) of Male Rats Treated with Equol.
Treatment Group SDN Volumes AVPV Volumes
Control 53.2+2.9 10.58+2.4
Equol 46.3 3.1 10.59 + 1.6
Regional brain volumes were expressed in mm x 10 ". There were no significant
differences in the SDN or AVPV volumes between the treatment groups, n = 4 per
group.
EXAMPLE 19
[00276] Equol Is More Potent Compared To Genistein In Decreasing Anxi-
ety Levels Via Pre- and Early Postnatal Treatments In Male Rat Offspring
Tested
As Adults
[00277] In recent years isoflavones are reported to have beneficial health
effects regarding cancers (breast and prostate), cardiovascular disease,
osteoporo-
sis and symptoms associated with menopause. However, little information is
known about the influence of isoflavones on brain and especially behavior such
as
anxiety. In this study, the long-term effects of genistein and equol (an
isoflavone
metabolite) were examined for their potential to influence anxiety-related
behav-
iors in male offspring tested as adults in the EPM.
[002781 Pregnant Long-Evans rats were treated via daily subcutaneous injec-
tions from gestational day 14 through 20 and during lactation from postnatal
day 2
through 25 with: 1) control-DMSO vehicle or, 2) genistein @ 3 mg/day or 3)
equol @ 3 mg/day according to the treatment groups. All animals were fed a phy-
toestrogen-low diet tlzroughout these studies (10 ppm isoflavones). There were
no
significant differences in maternal body weights just before birth among the
treat-
ment groups. At birth, body weights were slightly (but not significantly)
higher in
genistein- and equol-treated animals compared to controls. The maternal and
male
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64
offspring genistein and equol levels (@ 25 days post birth) paralleled that of
the
treatments administered.
[00279] At approximately 90 days of age the male offspring were tested by
treatments (control, genistein and equol) in the EPM that was video-taped for
3
minutes and later recorded for the number of entries into- and the percentage
of
total time spent - on the open arms, as previously described. As illustrated
in FIG.
68, equol-treated males displayed significantly greater entries into the open
arms
compared to controls. However, the entries into the open arms were not signifi-
cantly different between the genistein and control groups. Notably, as seen in
FIG. 69, equol-treated males displayed significantly greater time in the open
arms
compared to genistein-treated animals or controls. Also, genistein-treated
males
displayed significantly greater open arm time intervals compared to controls.
Equol time intervals were 2-fold greater than genistein values and genistein
levels
were approximately 9-fold greater than controls.
[00280] These data suggest that: 1) equol is a more potent anti-anxiety agent
compared to genistein and 2) the isoflavone metabolite, equol has long-term
influ-
ences upon anxiety-related behaviors of offspring tested as adults that were
treated
prenatally. The findings of this experiment validate the positive effects of
equol
seen in Example 17 described above for reducing depressive-related behaviors
that
are presumably due to circulating equol levels and not genistein. The results
of
this study have applications to food, nutritional and pharmaceutical
modulation of
anxiety by various routes of administration.
[00281] It is therefore intended that the foregoing detailed description be re-
garded as illustrative rather than limiting, and that it be understood that it
is the
following claims, including all equivalents, that are intended to define the
spirit
and scope of this invention.
