U.S. patent application number 10/459537 was filed with the patent office on 2003-11-27 for therapy of estrogen-associated disorders.
This patent application is currently assigned to Novogen Research Pty Ltd. Invention is credited to Husband, Alan James, Kelly, Graham Edmund.
Application Number | 20030219499 10/459537 |
Document ID | / |
Family ID | 3806868 |
Filed Date | 2003-11-27 |
United States Patent
Application |
20030219499 |
Kind Code |
A1 |
Kelly, Graham Edmund ; et
al. |
November 27, 2003 |
Therapy of estrogen-associated disorders
Abstract
A method for the treatment, prophylaxis, amelioration or
prevention of disorders associated with abnormally high activity of
steroidal estrogens, which includes administering to a human
subject a composition comprising an isoflavone-containing extract
of clover or chick peas, said extract comprising primarily
biochanin, or a ratio of biochanin to one or more of the
isoflavones from the group formononetin, daidzein and genistein in
the range of from about 2:1 to about 5:1, optionally in association
with one or more pharmaceutically acceptable carriers, excipients,
auxiliaries and/or diluents is described. Also described are
compositions and uses comprising the isoflavone containing
extracts.
Inventors: |
Kelly, Graham Edmund;
(Northbridge, AU) ; Husband, Alan James;
(McMahon's Point, AU) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER
LLP
1300 I STREET, NW
WASHINGTON
DC
20005
US
|
Assignee: |
Novogen Research Pty Ltd
|
Family ID: |
3806868 |
Appl. No.: |
10/459537 |
Filed: |
June 12, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10459537 |
Jun 12, 2003 |
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09647092 |
Dec 5, 2000 |
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6599536 |
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09647092 |
Dec 5, 2000 |
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PCT/AU99/00222 |
Mar 26, 1999 |
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Current U.S.
Class: |
424/725 |
Current CPC
Class: |
A61K 31/35 20130101;
A61P 15/00 20180101; A61K 2300/00 20130101; A61K 31/35 20130101;
A61P 5/32 20180101; A61K 31/35 20130101; A61K 36/48 20130101; A61P
35/00 20180101; A61P 43/00 20180101; A61P 5/30 20180101; A61K 31/35
20130101 |
Class at
Publication: |
424/725 |
International
Class: |
A61K 035/78 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 26, 1998 |
AU |
PP-2607 |
Claims
1. A method for the treatment, prophylaxis, amelioration or
prevention of disorders associated with an abnormally high activity
of steroidal estrogen, which includes administering to a human
subject a composition comprising an isoflavone containing extract
of clover or chick pea, or an isoflavone extract of clover or chick
pea comprising primarily biochanin or biochanin and any one or more
of the isoflavones from the group formononetin, daidzein and
genistein at a ratio from about 2:1 to about 5:1, optionally in
association with one or more pharmaceutically acceptable carriers,
excipients, auxiliaries and/or diluents.
2. A method according to claim 1 wherein the disorder is selected
from uterine fibroids, polycystic ovarian disease, ovarian cysts,
cyclical acne, mastalgia, endometriosis and endometrial
hyperplasia.
3. A method according to claim 1 wherein the isoflavone containing
extract primarily comprising biochanin comprises at least 65%
biochanin by weight, the remainder comprising essentially
formononetin and/or daidzein and/or genistein.
4. A method according to claim 3 comprising 70 to 100%
biochanin.
5. A method according to claim 1 wherein the ratio of biochanin to
one or more of the ioflavones from the group formononetin, daidzein
and genistein is from about 7:3 to about 5:1.
6. A method according to claim 1 wherein the extract of clover or
chick peas is a water/water-miscible organic solvent extract.
7. A method according to claim 1 wherein the clover is red
clover.
8. A method according to claim 1 wherein the isoflavone is in a
glycoside, agylcone, acetyl or malonyl form.
9. A composition comprising an isoflavone extract of clover or
chick pea, said extract comprising biochanin and one or more of the
isoflavones from the group formononetin, daidzein and genistein at
a ratio from about 2:1 to about 5:1, optionally in association with
one or more pharmaceutically acceptable carriers, excipients,
auxiliaries, and/or diluents.
10. A composition according to claim 9 wherein the ratio of
biochanin to one or more of the isoflavones from the group
formononetin, daidzein and genistein is from about 7:3 to about
5:1.
11. A composition according to claim 9 wherein the extract of
clover or chick peas is a water/water-miscible organic solvent
extract.
12. A composition according to claimn 9 wherein the isoflavone is
in a glycoside, agylcone, acetyl or malonyl form.
13. Use of a composition comprising an isoflavone extract of clover
or chick pea, said extract comprising primarily biochanin, or a
ratio of biochanin to one or more of the isoflavones from the group
formononetin, daidzein and genistein in the range of from about 2:1
to about 5:1, optionally in association with one or more
pharmaceutically acceptable carriers, excipients, auxiliaries,
and/or diluents for the manufacture of a medicament for the
treatment, prophylaxis, amelioration or prevention of an disorders
associated with an abnormally high activity of steroidal
estrogen.
14. A use according to claim 13 wherein the disorder is selected
from uterine fibroids, polycystic ovarian disease, ovarian cysts,
cyclical acne, mastalgia, endometriosis and endometrial
hyperplasia.
15. A use according to claim 13 wherein the isoflavone containing
extract primarily comprising biochanin comprises at least 65%
biochanin by weight, the remainder comprising isoflavones from the
group formononetin, daidzein and genistein.
16. A use according to claim 14 comprising 70 to 100%
biochanin.
17. A use according to claim 13 wherein the ratio of biochanin to
one or more of the isoflavones from the group formononetin,
daidzein and genistein is in the range of from about 7:3 to about
5:1.
18. A use according to claim 13 wherein the isoflavone is in a
glycoside, agylcone, acetyl or malonyl form.
Description
[0001] This invention relates to methods and compositions for the
treatment, prophylaxis, amelioration or prevention of disorders
associated with abnormally high activity of steroidal estrogens,
using a plant extract enriched for estrogenic isoflavones
preferably comprising predominantly biochanin or a high ratio of
biochanin to a mixture of other estrogenic isoflavones comprising
formononetin, daidzein and genistein. The targeted conditions
specifically include uterine fibroids, polycystic ovarian disease,
ovarian cysts, mastalgia, cyclical acne, endometriosis and
endometrial hyperplasia.
[0002] The aforementioned conditions usually are considered as a
group because of a number of common characteristics. They occur
almost exclusively in pre-menopausal women and therefore are
thought to be associated with high blood levels of steroidal sex
hormones, and they are associated with benign, hyperplastic and
proliferative changes in the tissues of the female reproductive
tract (with the exception of cyclical acne).
[0003] The prominent role of steroidal estrogen in these conditions
is suggested by the observations firstly that these conditions and
symptoms occur almost predominantly in pre-menopausal women and
usually disappear with the onset of menopause, secondly, they often
get worse during the mid-phase of the menstrual cycle when cyclical
estrogen levels are highest, they usually are exacerbated by the
use of steroidal estrogen therapy, and lastly they usually respond
to therapies such as GnrH agonists, oral contraception, or
oophorectomy, all of which have the effect of reducing steroidal
estrogen production in the body.
[0004] Uterine fibroids are a particularly common problem with more
than 30% of women developing fibroids by the time they reach
menopause. Fibroids are benign adenomas originating in the uterine
wall and composed of fibrous tissue. For some women, the fibroids
can be small and essentially asymptomatic. For other women, the
fibroids can be of such a size to cause symptoms such as severe
menstrual bleeding and uterine cramping (with fibroids that disrupt
the endometrium) or constipation and urinary frequency (with large,
space-occupying fibroids that grow out into the pelvic cavity).
Estrogen therapy is known to aggravate fibroids and the symptoms
associated with fibroids [Grodstein et al. "Postmenopausal hormone
use and colecystectomy in a large prospective study." Obstetrics
and Gynaecology: 1994: 83(1), 5-11; Akkad et al. "Abnormal uterine
bleeding on HRT: The importance of intrauterine structural
abnormalities." Obstetrics and Gynaecology, 1995. 86(3): 330-334;
Sener et al. "The effects of HRT on uterine fibroids in
postmenopausal women." Fertility and Sterility, 1996. 65(2):
354-357]. The usual treatment for fibroids is surgical removal
(hysterectomy or hysterotomy).
[0005] Endometrial hyperplasia is thought to affect between 5-15%
of pre-menopausal women. It involves an abnormal thickening of the
endometrium that is not completely shed at the time of
menstruation. The symptoms can include painful and heavy
menstruation, and painful sexual intercourse. Treatment usually
consists of surgery (curettage or hysterectomy).
[0006] Endometriosis affects about 5% of pre-menopausal women. This
condition is due to the appearance of endometrium in the peritoneal
cavity. Patches of endometrium can grow on the serosal surface of
the ovaries, uterus, bladder, large intestine or the peritoneum.
These patches respond to normal hormonal changes over the menstrual
cycle in parallel to that of the endometrium in the uterus and can
bleed, swell and cause severe pelvic discomfort and pain. Standard
therapy of endometriosis is surgical ablation of the abnormal
tissue, although the recurrence rate is high, requiring ongoing
surgical treatment [Namnoum et al. "Incidence of symptom recurrence
after hysterectomy for endometriosis." Fertility and Sterility,
1995. 64(5): 898-902]. Endometriosis also is aggravated by estrogen
therapy [Goh et al. "Postmenopausal endometrioma and HRT" Australia
New Zealand of Obstetrics and Gynaecology, 1992. 32(4):
384-385].
[0007] Ovarian cysts are thought to affect up to about 20% of
pre-menopausal women. The pathology is that of multiple, incomplete
follicles within the body of the ovary. A variant of this condition
is known as polycystic ovarian disease which is characterised by
excessive androgen production from the follicles stimulated by
abnormally high insulin levels. Excess androgens are often
converted to steroidal estrogen. The usual symptoms of polycystic
ovarian disease are hirsutism and acne. The normal treatment for
these conditions are GnrH agonists that function by down-regulating
the release of gonadotrophins (FSH and LH) from the hypothalamus,
thereby inhibiting and further ovulation.
[0008] Mastalgia is also known as cyclical mastalgia or fibrocystic
breast disease. It is characeterised by the retention of fluid in
cysts within the fibrous tissue of the breast. It normally is
associated with swelling, pain and tenderness, with symptoms
usually worsening about the middle of the menstrual cycle when
estradiol levels peak in the blood. No effective therapy for this
condition is known.
[0009] Cyclical acne normally is restricted to post-adolescent
women. It is associated with severe acne over the face and upper
torso and normally the acne worsens on a cyclical basis in parallel
with the menstrual cycle. The normal therapy for this condition is
oral contraception in order to regulate ovulation and estrogen
production.
[0010] In general, the management of these aforementioned
conditions is unsatisfactory. Surgery is the most common method of
treatment, and apart from the dramatic and intrusive nature of this
approach, an inevitable outcome often is sterility as a result of
removal of ovaries and/or uterus. The use of GnrH agonists also is
not without adverse consequences as it invariably leads to
premature menopause, with its attendant increased risks of
osteoporosis and heart disease.
[0011] The underlying causes of or risk factors for the
aforementioned conditions are unknown. Genetic risk factors are not
reported, neither are lifestyle risk factors. There is no reliable
epidemiological data reported that links the incidence of any one
of these conditions or the group of conditions as a whole to
specific communities or racial groups, or to lifestyle factors such
as diet. However, it generally is recognised that the conditions or
symptoms are associated with excess estrogen stimulation of tissues
of the female reproductive tract and that it would be prudent to
avoid any situation likely to aggravate estrogenic activity.
[0012] Plant estrogens including estrogenic isoflavones recently
have come to medical attention because they mimic the
effect/activity of steroidal estrogens and their biologically
active analogues by binding to and activating estrogen receptors on
animals (including human) cells. Plant isoflavones such as
formononetin, biochanin, daidzein and genistein are known to be
estrogenic in vitro, acting as agonists for the human estrogen
receptor. Their ability to function as estrogens in the body is
well understood and the epidemiological link between diets high in
these estrogenic isoflavones and low incidences of certain
estrogen-deficiency states also is well documented. This has led to
considerable interest in the use of dietary estrogens such as
isoflavones to provide a supplementary estrogenic activity in
menopausal women, providing relief from estrogen-deficiency
symptoms including hot flushes, mood swings, osteoporosis,
hypertension, and hypercholesterolaemia.
[0013] Given the current understanding about the estrogenic action
of isoflavones, and the adverse consequences of estrogen therapy on
the aforementioned pre-menopausal conditions and symptoms,
estrogenic isoflavones would appear to be contra-indicated for
subjects suffering from those conditions or symptoms. While the
incidences of these conditions are adequately reported in women in
Western countries where the diet is low in dietary isoflavones,
there is no known data on the incidences in communities that
typically ingest high levels of estrogenic isoflavones. It would be
reasonable, however, to assume that it would particularly
contra-indicated for pre-menopausal women at risk of the
aforementioned conditions and symptoms to extensively consume
foodstuffs such as clover that is the richest source of estrogenic
isoflavones in nature. Therefore, the applicants were surprised to
find that isoflavone-containing extracts of clover or chickpeas
were particularly beneficial in the treatment of estrogen-sensitive
conditions.
[0014] Clovers (Trifolium spp.) are one of the richest sources of
estrogenic isoflavones in the plant kingdom, with some cultivars
containing up to 5% of their dry weight as estrogenic isoflavones.
The use of subterranean clovers in agriculture is associated with a
condition in sheep known as `clover disease`. This disease is due
to excessive ingestion of estrogenic isoflavones resulting in
hyperplastic changes in the lower reproductive tract. The linings
of the uterus, cervix and vagina of sheep undergo dramatic
proliferative changes and the ovaries show multiple cyst formation,
all of which produces temporary and then permanent infertility. The
pathology of these changes in sheep closely parallels that in
humans with conditions such as endometrial hyperplasia. The
likelihood that estrogenic isoflavones might exacerbate such
conditions in women, particularly in those women with little prior
exposure to isoflavones, is therefore is a real concern.
[0015] Clovers such as red clover have enjoyed some therapeutic use
by herbal practitioners over the centuries for various human
ailments such as asthma and eczema and have not been noted as
having any association with increased incidence of reproductive
disorders. It should be noted that the traditional herbal use of
red clover employs the flower and this part of the plant contains
very little isoflavone. Also, red clover occurs in multiple
cultivars and many cultivars have very low isoflavone levels. Thus
it is not possible to ascribe any putative therapeutic benefit from
herbal medicinal use to their isoflavone content, and nor is it
possible to ascribe any apparent lack of adverse side effects to
the safety of isoflavones.
[0016] In spite of this background, the applicant surprisingly has
found that clover extracts preferably those enriched for certain
estrogenic isoflavones may be used in the prevention and treatment
of conditions of the female reproductive tract associated with
excessive estrogen activity. Even more surprising is the finding
that a particular ratio of different types of estrogenic
isoflavones in the extract is primarily responsible for the
demonstrated therapeutic effects. Preferably the extract comprises
biochanin as the principal isoflavone. In the context of this
invention, it has been found that the preferred ratio of biochanin
to the other three main estrogenic isoflavones embraces the various
naturally-occurring forms of isoflavones including their aglycone,
glycoside, acetyl or malonyl forms.
[0017] In accordance with a first aspect of this invention there is
provided a method for the treatment, prophylaxis, amelioration or
prevention of disorders associated with an abnormally high activity
of steroidal estrogen, which includes administering to a human
subject a composition comprising an isoflavone containing extract
of clover or chick pea, or an isoflavone extract of clover or chick
pea comprising primarily biochanin or biochanin and any one or more
of the isoflavones from the group formononetin, daidzein and
genistein at a ratio from about 2:1 to about 5:1, optionally in
association with one or more pharmaceutically acceptable carriers,
excipients, auxiliaries and/or diluents.
[0018] Preferably the disorder is selected from uterine fibroids,
polycystic ovarian disease, ovarian cysts, cyclical acne,
mastalgia, endometriosis and endometrial hyperplasia.
[0019] By reference to an extract comprising "primarily biochanin"
is meant a biochanin content of at least 65% by weight isoflavone
content, such as 70 to 100%, more preferably 75 to 90% biochanin,
the remainder of isoflavone content comprising essentially
formononetin, daidzein and genistein.
[0020] The high ratio of biochanin to the other three estrogenic
isoflavones in the range from about 2:1 to about 5:1 is responsible
for the optimal unexpected therapeutic effects according to this
invention. Outside of this range of ratios, the therapeutic
benefits effectively are not observed, or if observed are
considerably less effective. The ratio of biochanin to the other
three isoflavones may, for example, be from about 7:3 to about
5:1.
[0021] In a further aspect of this invention there is provided a
composition comprising an isoflavone-rich extract from clover or
chick pea, said extract comprising a ratio of biochanin to
formononetin, daidzein and genistein at a ratio from about 2:1 to
about 5:1, optionally in association with one or more
pharmaceutically acceptable carriers, excipients, auxiliaries,
and/or diluents.
[0022] In a further aspect of the invention there is provided use
of a composition comprising an isoflavone extract of clover or
chick pea, said extract comprising primarily biochanin, or a ratio
of biochanin to formonentin, daidzein and genistein at a ratio of
from about 2:1 to about 5:1, optionally in association with one or
more pharmaceutically acceptable carriers, excipients, auxiliaries,
and/or diluents for the manufacture of a medicament for the
treatment, prophylaxis, amelioration or prevention of disorders
associated with an abnormally high activity of steroidal
estrogen.
[0023] Scientific interest in isoflavones up until now has focused
on daidzein and genistein. This is in part because of the belief
that these two isoflavones are the most prominent in the human
diet. But mainly it is because of the general belief that
formononetin and biochanin-are unlikely to have any biological
significance in their own right. Formononetin and biochanin are
methylated versions of daidzein and genistein respectively and it
is thought that humans have the capacity to convert formononetin to
daidzein and biochanin to genistein. In sheep, the only animal
species in which isoflavone metabolism has been extensively
studied, this conversion is virtually 100% effective. Sheep consume
predominantly formononetin and biochanin as a result of eating
isoflavone-rich clovers, with virtually all formononetin being
demethylated to daidzein and all biochanin being demethylated to
genistein. Reports of analyses of blood or urine of humans
ingesting diets rich in isoflavones has failed to demonstrate any
formononetin and biochanin as being present, leading to the general
assumption that the same situation applies in humans. That is, even
if formononetin and biochanin are present in the diet, there is a
general assumption that they are converted to daidzein and
genistein and that it is these two isoflavones that hold the true
source of biological significance to humans. The dominant use of
soya as a source of dietary isoflavones by the health food and
pharmaceutical industries is testament to this, with soya
containing predominantly daidzein and genistein (95-99%) and only
trace amounts of biochanin and formononetin.
[0024] The inventors were therefore very surprised to find that an
extract of red clover or chick peas, preferably enriched for or
comprising biochanin as the principal isoflavone, provided a
therapeutic effect quite distinct from that seen with the same dose
of isoflavones containing predominantly daidzein and genistein.
This suggests that biochanin provides a distinctive and
individualistic biological effect.
[0025] It has been recognized for some time that the four
estrogenic isoflavones have an individual biological profile. That
is, genistein is approximately ten fold more estrogenic than
daidzein, and genistein has potent anti-cancer actions while
daidzein has virtually none. The finding that biochanin might
therefore also have a distinctive biological profile is not
surprising, but that it should be able to maintain this distinction
when its presence in the human body has not previously been
demonstrated is surprising.
[0026] This finding was even more surprising given what generally
is known about the action of isoflavones at the cellular level and
the manner in which they function as estrogens. Compounds can exert
an estrogenic effect in the body in one of two ways, either
indirectly, by stimulating the brain to produce gonadotropins that
in turn cause the body to make more estradiol, or directly, by
attaching to and activating the human estrogen receptor (hER). The
isoflavones are thought to act directly in the same way as the
steroidal estrogen, estradiol, and some evidence exists to support
that belief. Recent studies, however, point to isoflavones having
an important difference to that of estradiol in the way they act as
estrogens. Two distinct types of hER now have been identified,
known as alpha-ER and beta-ER. Whereas estradiol binds with similar
strengths to both ER types and appears to be able to stimulate both
receptor types equally, the isoflavonoids are quite different. They
show significantly greater agonism to the beta-ER than to the
alpha-ER. The significance of this phenomenon lies in the fact that
alpha-ER dominates on tissues of the female reproductive tract
(vagina, endometrium, breast, ovary), while beta-ER dominates on
most other tissues (brain, bone, endothelium etc.). The fact that
isoflavones have weak affinity for alpha-ER is confirmed by
clinical studies and epidemiological observations that diets rich
in estrogenic isoflavones have little or no stimulatory effect on
female reproductive tissue in post-menopausal women. But the
experience with `Clover Disease` in sheep teaches us that this most
likely is a dose-related effect--if sufficient isoflavones are
ingested, then it is possible to achieve stimulation of the
alpha-ER, producing proliferation of the reproductive tissues.
Importantly, however, at normal dietary dosages, estrogenic
isoflavones appear to have little or no impact on tissues
expressing mainly alpha-ER.
[0027] Without wishing to be bound by any particular theory of
action, there are multiple possible mechanisms by which biochanin
may be providing a therapeutic benefit in reducing the impact of
steroidal estrogens on the reproductive tissues. One likely
explanation is the way in which biochanin interacts with the
alpha-ER, but this again is surprising given that there is no known
difference in the way in which any of the four estrogenic
isoflavones behave as ligands with this receptor.
[0028] It has been theorised that one of the biological effects of
isoflavones is their ability to `competitively inhibit` the action
of steroidal estrogens such as estradiol. By this process, the
isoflavonoid, being a weaker estrogen, once bound to the hER and
occupying the receptor site, prevents the more estrogenic estradiol
reaching the receptor. Isoflavones are weak estrogens, having an
estrogenic potency between about 1:250 to 1:2,000 that of
estradiol, and while they have considerably lower affinity for the
hER compared to estradiol, the substantially greater numbers of
isoflavones in the blood relative to estradiol means that a
significant proportion of hER will be occupied by isoflavones. This
theory holds that even though the isoflavonoid produces a weak
estrogenic response, the fact that it has prevented a more potent
response by estradiol means that the net effect has been one of a
reduced estrogenic effect. This theory could be used to explain the
ability of isoflavones to reduce the impact of estradiol on the
female reproductive tissues during the pre-menopausal years when
estradiol levels are at their highest.
[0029] Biochanin has about the same estrogenic potency as daidzein
and there is some evidence that it has lower binding affinity for
the hER compared to daidzein. Therefore even if
competitive-inhibition is a contributing factor the clinical
observation by the inventors then it is even more surprising that
an extract principally comprising biochanin should be more
efficacious than an extract principally comprising daidzein and
genistein.
[0030] Preferably the extract of clover is prepared by a
water/water-miscible organic solvent extract of clover. The ratio
of water to water-miscible organic solvent is generally in the
range of 1:10 to 10:1 and may for example comprise equal
proportions of water and solvent or from 1% to 30% (v/v)
water-miscible organic solvent. Any water-miscible organic solvent
or a mixture of such solvents may be used. The water-miscible
organic solvent is preferably a C2-10, more preferably C14
water-miscible organic solvent (such as methanol, ethanol,
propanol, propylene glycol, erythrite, butanol, butanediol,
acetonitrile, ethyleneglycol, ethylacetate, glycidol, glycerol
dihydroxyacetone or acetone). The extract in this regard is
prepared by exposing the plant material to the water/water-miscible
solvent mix. Optionally the mixture may include an enzyme, which
cleaves isoflavone glycosides to the aglucone form. The mixture may
be vigorously agitated so as to form an emulsion. The temperature
of the mix may range, for example, from an ambient temperature to
boiling temperature. Exposure time may be between one hour to
several weeks. One convenient extraction period is twenty-four
hours at 90 C. The extract is separated from undissolved plant
material and the water-miscible organic solvent removed, such as by
distillation, rotary evaporation or other standard procedures for
solvent removal. The resultant extract containing water-soluble and
non-water soluble components may be dried to give an isoflavone
containing extract, which may be formulated with one or more
pharmaceutically acceptable carriers, excipients and/or
auxiliaries. The extract following distillation contains a small
amount of oil which includes isoflavones in their aglucone form
(referred to herein as isoflavones). This isoflavone-enriched oil,
which may be dried, for example in the presence of silica, may be
formulated with one or more carriers, excipients and/or auxiliaries
to give an isoflavone containing extract. Alternatively,
isoflavones may be further concentrated by addition to the oil of a
non-water soluble organic solvent such as hexane, heptane or octane
having high solubility for oils but low solubility for isoflavones.
The oil readily partitions into the organic solvent, and an
enriched isoflavone containing extract falls out of solution. The
recovered extract may be dried, for example in an oven at 50 C to
about 120 C, and formulated with one or more pharmaceutically
acceptable carriers, excipients and/or auxiliaries.
[0031] The clover (Trifolium spp.) may be red clover (T. pratense),
subterranean clover (T. subterranean), white clover (T. repens), or
any clover related species, or chick pea variety. Preferably the
clover is red clover (T. pratense) or subterranean clover (T.
subterranean).
[0032] The compositions according to the present invention may
include one or more pharmaceutically acceptable carriers. Carriers
are selected so as to be acceptable in the sense of being
ingredients in the composition and must not be deleterious to the
patient. The carriers may be solid or a liquid, or both, and may be
formulated with the extract as a unit-dose, for example a tablet,
which may contain from 0.5% to 80% by weight of extract or up to
100% by weight to extract. Compositions may be prepared by any of
the well known techniques of pharmacy, for example admixing the
extract, optionally including excipients, diluents (for example
water) and auxiliaries as are well known in the pharmaceutical
field.
[0033] The compositions according to the invention may include one
or more agents, such as vitamins (for example, Vitamin A, Vitamin B
group, Vitamin C, Vitamin D, Vitamin E and Vitamin K), and minerals
(for example, magnesium, iron, zinc, calcium and manganese in the
form of pharmaceutically acceptable salts).
[0034] The compositions of the invention include those suitable for
oral, rectal, optical, buccal (for example sublingual), parental
(for example subcutaneous, intramuscular, intradermal and
intravenous) and transdermal administration. The most suitable
route in any given case will depend on the nature and severity of
the condition being treated and the state of the patient.
[0035] Compositions suitable for oral administration may be
presented in discrete units, such as capsules, cachets, lozenges,
or tablets, 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. Such compositions may be prepared by any
suitable method of pharmacy, which includes the step of bringing
into association the active compound, and one or more suitable
carriers (which may contain one or more accessory ingredients as
noted above). In general the compositions of the invention are
prepared by uniformly and intimately admixing the extract with a
liquid or finely divided solid carrier, or both, and then, if
necessary, shaping the resulting mixture. For example, a tablet may
be prepared by comprising or moulding a powder or granules
containing the extract, optionally with one or more accessory
ingredients. Compressed tables may be prepared by compressing in a
suitable machine, the extracts in the form of a powder or granules
optionally mixed with a binder, lubricant, inert diluents, and/or
surface active/dispersing agent(s). Moulded tablets may be made by
moulding, in a suitable machine, the powdered compound moistened
with an inert liquid binder.
[0036] Suitable carriers may be fillers, such as sugars, for
example lactose, saccharose, mannitol or sorbitol, cellulose
preparations and/or calcium phosphates, for example tricalcium
phosphate or calcium hydrogen phosphate, and also binders, such as
starch pastes using, for example, corn, wheat, rice or potato
starch, gelatin, tragacanth, methylceullose and/or
polyvinylpyrrolidone, and, if desired, disintegrators, such as the
above-mentioned starches, also carboxymethyl starch, cross linked
polyvinyl pyrrolidone, agar or alginic acid or a salt thereof, such
as sodium alginate. Excipients may be flow conditioners and
lubricants, for example silicic acid, talc, stearic acid or salts
thereof, such as magnesium or calcium stearate, and/or polyethylene
glycol. Dragee cores are provided with suitable, optionally
enteric, coatings, there being used, inter alia, concentrated sugar
solutions which may comprise gum arabic, talc,
polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide,
or coating solutions in suitable organic solvents or solvent
mixtures, or, for the preparation of enteric coatings, solutions of
suitable cellulose preparations, such as acetylcellulose phthalate
or hydroxypropylmethylcellulose phthalate. Dyes or pigments may be
added to the tablets or dragee coatings, for example for
identification purposes or to indicate different doses of active
ingredients.
[0037] Other orally administrable pharmaceutical compositions are
dry-filled capsules made, for example, of gelatin, and soft, sealed
capsules made of gelatin and a plasticiser, such as glycerol or
sorbitol. The dry-filled capsules may comprise the extracts in the
form of granules, for example in admixture with fillers, such as
lactose, binders, such as starches, and/or glicants, such as talc
or magnesium stearate, and, where appropriate, stabilisers. In soft
capsules, the extract is preferably dissolved or suspended in
suitable liquids, such as fatty oils, paraffin oil or liquid
polyethylene glycols, to which stabilisers may also be added.
[0038] Formulations suitable for buccal (sublingual) administration
include lozenges comprising the extracts in a flavoured base,
usually sucrose and acacia or tragacanth; and pastilles comprising
the compound in an inert base such as gelatin and glycerin or
sucrose and acacia.
[0039] Compositions of the present invention suitable for
parenteral administration conveniently comprise sterile aqueous
preparations of the extracts, which preparations are preferably
isotonic with the blood of the intended recipient. These
preparations are preferably administered intravenously, although
administration may also be effected by means of subcutaneous,
intramuscular, or intradermal injection. Suitable compositions
include water soluble extracts and also suspensions of the active
ingredient, such as corresponding oily injection suspensions, there
being used suitable lipophilic solvents or vehicles, such as fatty
oils, for example sesame oil, or synthetic fatty acid esters, for
example ethyl oleate or triglycerides, or aqueous injection
suspensions comprising viscosity-increasing substances, for example
sodium carboxymethylcellulose, sorbitol and/or dextran, and, where
appropriate, also stabilisers. As an example compositions may
conveniently be prepared by admixing the extracts with water or a
glycine buffer and rendering the resulting solution sterile and
isotonic with the blood. Injectable formulations according to the
invention may contain from 0.1% to 60% w/v of the extract and may,
for example, be administered at a rate of 0.1 ml/minute/kg.
[0040] Formulations suitable for rectal administration are
preferably presented as unit dose suppositories. These may be
prepared by admixing the extracts with one or more conventional
solid carriers, for example cocoa butter, and then shaping the
resulting mixture.
[0041] Compositions suitable for topical administration to the skin
preferably take the form of an ointment, cream, lotion, paste, gel,
spray, aerosol, or oil. Carriers which may be used include
petroleum jelly, lanoline, polyethylene glycols, alcohols, and a
combination of two or more thereof.
[0042] The extract is generally present at a concentration of from
0.1% to 30% weight/weight, for example from 0.5% to 10%
weight/weight. The isoflavone ratio in the composition is
preferably in the range of biochanin:formononetin from 2:1 to 8:1.
Small or trace amounts of daidzein and biochanin may be
present.
[0043] Compositions suitable for transdermal administration may be
presented as discrete patches adapted to remain in intimate contact
with the epidermis of the recipient for a prolonged period of time.
Such patches may contain the extracts in an optionally buffered
aqueous solution.
[0044] Compositions suitable for transdermal administration may
also be delivered by iontophoresis (see, for example,
Pharmaceutical Research 3 (6), 318 (1986)) and typically take the
form of an optionally buffered aqueous solution of the extracts.
Such compositions may, for example, contain citrate or bis/tris
buffer (pH 6) or ethanol/water, with for example 0.05% to 30% w/w
extract.
[0045] Compositions may be prepared in a manner, and in a
form/amount as is conventionally practised. See for example,
Goodman & Gillman, The Pharmalogical Basis of Therapeutics (7th
Edition, 1985) and Remington's Pharmaceutical Science (Mack
Publishing Company, 10th Edition), both of which are incorporated
herein by reference. Compositions may contain, for example, from
0.1 mg to 2 g extract, such as 0.1 mg to 200 mg.
[0046] The extracts may be in the form of a powder, a slurry, in
aqueous solution (for example containing a small amount of oil),
particulate form, or dissolved in an organic solvent (such as
methanol, ethanol, ethylacetate or dimethyl sulphoxide).
[0047] What constitutes an effective amount of the compositions of
the present invention will depend upon a number of factors, such as
specific mode of administration, the condition being treated, the
condition of the patient and the judgement of the health care
giver. Examples of dosages of extracts are about 0.1 mg to about
200 mg per day, such as in the order of 1.5 mg/kg (body
weight)/day.
[0048] Preferably the extract comprises biochanin as the principal
isoflavone. In the context of this invention, it has been found
that the preferred ratio of biochanin to the other three main
estrogenic isoflavones embraces the various naturally-occurring
forms of isoflavones including their aglycone, glycoside, acetyl or
malonyl forms.
[0049] This invention will now be described with reference to the
following non-limiting examples.
EXAMPLE 1
[0050] A cultivar of red clover specially selected for its high
biochanin content (85% biochanin, 10% formononetin, 2.5% daidzein
and 2.5% genistein) is harvested and snap-frozen within 4 hours by
exposure to liquid nitrogen. The material can be stored in this
form for up to several years. For extraction, the frozen material
is crushed to a fine powder, thawed and placed in a fine gauze bag,
which is immersed in a solution of 60% ethanol in water. Extraction
is carried out at 90.degree. C. for 24 hours. The supernatant is
separated from the undissolved plant material, and the solvent
removed by distillation. The aqueous phase containing isoflavones
in an aglucone form is extracted with an organic solvent (either
petroleum ether or hexane or acetyl acetate) to remove oils and
other polar compounds. The solvent then is removed by distillation
and the aqueous phase taken to near-dryness by rotary evaporation.
The tar-like residue then is oven-dried. The final powder typically
contains 40-50% isoflavones in the ratio of biochanin:
formononetin, daidzein and genistein of 5:1.
[0051] The ratio of biochanin to formononetin, daidzein and
genistein may be adjusted from about 2:1 to 5:1. At this ratio the
unexpected therapeutic effects of the invention are observed. The
biochanin to formononetin, daidzein and genistein can be easily
adjusted by HPLC fractionation of the Example 1 material,
extracting a red clover cultivar comprising the appropriate ratio
of components (ie. from 2:1-5:1 biochanin to formononetin, daidzein
and genistein), or by adjusting the concentration of the extracting
organic solvent (less organic solvent less extracted
biochanin).
EXAMPLE 2
[0052] The plasma and urinary profiles of isoflavones after acute
and chronic administration of 22 mg of isoflavones (genistein 0.5
g, daidzein 0.5 mg, biochanin 26 g, and formononetin 14 mg) in
fourteen subjects was analysed. The composition corresponds to the
Promensil product (Registered Trade Mark, Novogen Research Pty Ltd,
Australia). Venous plasma and urine were collected at intervals for
24 h after which subjects commenced taking two tablets each day for
two weeks and repeated plasma samples and urine were collected for
a 48 h period after the last does. Plasma and urine isoflavones
were assayed by HPLC.
[0053] After acute dosing all four isoflavones appeared rapidly in
plasma and reached peak levels around 6 h. Although daidzein and
genistein were present in much higher concentrations than their
methylated precursors, indicating a rapid demethylation of these
compounds, formononetin and biochanin remained detectable in both
plasma and urine at all times. This was a quite unexpected
finding.
EXAMPLE 3
[0054] Pharmaceutical compositions are prepared from the extracts
according to the examples above.
[0055] 1. The following composition is prepared in the form of a
tablet:
[0056] 40-60 mg of extract from Example 1
[0057] 340 mg of a standard tablet inert carrier
[0058] This composition is tableted to provide a 380-400 mg
tablet.
[0059] 2. The following composition is prepared in the form of a
capsule:
[0060] 40-60 mg of extract of Example 1 or 2
[0061] 190 mg of a standard pharmaceutical inert carrier
[0062] all contained in a non-toxic gelatin capsule.
[0063] The carriers referred to above include cellulose
(microcrystalline), calcium hydrogen phosphate, soy polysacchardie,
magnesium stearate and silica-colloidal (anhydrous).
EXAMPLE 4
[0064] A 38-year old pre-menopausal woman with uterine fibroids
suffered from severe and irregular menstrual bleeding. Ultrasound
examination had confirmed the presence of a number of fibroids that
were protruding through the endometrium. She had been taking a soy
supplement delivering about 45 mg of both genistein and daidzein
daily for six months with no apparent change in her symptoms.
Within three months of starting treatment with red clover extract
according to Example 3, her bleeding and menstrual pain became much
less severe and her menstruation returned essentially to normal. An
ultrasound confirmed that the fibroids had significantly reduced in
size.
EXAMPLE 5
[0065] A 36-year old pre-menopausal woman had suffered
endometriosis for over 5 years with progressive worsening. A
laparoscopic examination had confirmed the presence of extensive
endometriosis lesions over her ovaries and serosal surface of the
uterus. For 1 year she had tried a dietary change including eating
tofu and soymilk on a regular basis, both foods known to contain
high levels of daidzein and genistein. The condition continued to
worsen and finally she was scheduled for surgery for laser burning
of the islets of endometriosis tissue. She took a red clover
extract according to Example (3) for 3 months prior to surgery. At
the time of surgery her symptoms (pelvic pain) had reduced
considerably, but it was decided to still proceed with the surgery.
Laparoscopy failed to reveal any evidence of endometriosis lesions.
She remains symptom-free 9 months later with ongoing daily
treatment with the red clover extract.
EXAMPLE 6
[0066] A 44-year old woman had suffered ovarian cysts for 8 years,
producing dysmennorhoea and low-grade pain. Various treatments
including hormonal treatments had failed to improve the condition.
After taking the red clover extract according to Example (3) for 3
months, her menstrual period returned to normal frequency and level
of bleeding and the pain subsided. An ultrasound examination
confirmed the absence of ovarian cysts on either ovary. She remains
well 6 months later with no evidence of return of the cysts.
EXAMPLE 7
[0067] A 29-year old pre-menopausal woman had suffered endometrial
hyperplasia for 9 years. The condition caused symptoms of heavy,
frequent menstruation and menstrual cramps and pain. She had been
treated periodically by curettage which had provided temporary
relief only. After taking the red clover extract according to
Example (3) for 4 months, her menstruation returned to normal
frequency, length, duration and level of bleeding.
EXAMPLE 8
[0068] A 19-year old pre-menopausal woman suffering from severe
acne and cyclical mastalgia was given the red clover extract
according to Example (3) for a period of two menstrual cycles. Her
pain score diary showed significant improvement for breast pain and
tenderness, and a noticable improvement was seen in her acne.
EXAMPLE 9
[0069] A young woman, Mrs M, with recurrent endometriosis, and
suffered severe pain and irregular bleeding. Following daily
treatment according to Example 4, her monthly pain and irregular
bleeding were minimal, and prognosis for conception was improved.
Improvements was seen shortly after treatment began.
[0070] Throughout this specification, unless the context requires
otherwise, the word "comprise", or variations such as "comprises"
or "comprising" or the term "includes" or variations thereof, will
be understood to imply the inclusion of a stated element or integer
or group of elements or integers but not the exclusion of any other
element or integer or group of elements or integers. In this
regard, in construing the claim scope, an embodiment where one or
more features is added to any of claims is to be regarded as within
the scope of the invention given that the essential features of the
invention as claimed are included in such an embodiment.
REFERENCES
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"Postmenopausal hormone use and colecystectomy in a large
prospective study" Obstetrics and Gynaecology: 1994: 83(1),
5-11.
[0072] 2. Akkad, A. A., Habiba, M. A., Ismail, N., Abrams, K., and
al-Azzawi, F. "Abnormal uterine bleeding on HRT: The importance of
intrauterine structural abnormalities" Obstetrics and Gynaecology,
1995. 86(3): 330-334.
[0073] 3. Sener, A. G., Seckin, N. C., Ozmen, S., Gokinen, O., Dou,
N. and Ekici, E. "The effects of HRT on uterine fibroids in
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[0074] 4. Namnoum, A. G., Hickman, T. N., Goodman, S. B., Gehlbach,
D. L., and Rock, J. A. "Incidence of symptoms recurrence after
hysterectomy for endometriosis" Fertility and Sterility, 1995.
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[0075] 5. Goh, J. T. and Hall, B. B. "Postmenopausal endometrioma
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[0076] 6. Sanchez-Guerrero, J., Liang, M. H., Karlson, E. W.,
Hunter, D. J. and Colditz, G. A. "Postmenopausal estrogen therapy
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[0077] 7. Troissi. R. J., Speizer, F. E., Willett, W. C.,
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