Note: Descriptions are shown in the official language in which they were submitted.
CA 02310294 2000-OS-30
COMPOSITION FOR AND METHOD OF PREVENTING OR TREATING
BREAST CANCER
Field of the Invention
The present invention relates to a composition containing tamoxifen and at
least
one isoflavone, and a method of treating breast cancer while inhibiting
tamoxifen induced
uterotrophic effects.
Back ,round of the Invention
Breast cancer is one of the leading causes of cancer mortality among Western
women, and is predicted to become a leading cause of cancer death in Oriental
women in
countries such as Japan in the near future. The American Cancer Society
estimates that 1
in 9 women face a lifetime risk of this disease, which will prove fatal for
about one-
quarter of those afflicted with the disease.
Tamoxifen (Fig. I), a synthetic nonsteroidal selective estrogen receptor
modulator, has been used effectively in the treatment of breast cancer for
over twenty
years. Tamoxifen is one of the most widely prescribed antineoplastic agents in
the
United States and Great Britain, and is one of the initial hormonal treatments
of choice in
both premenopausal and postmenopausal women with estrogen receptor positive
metastatic disease. Furthermore, adjuvant therapy studies show a substantial
reduction of
contralateral primary breast carcinoma in tamoxifen treated women, which
indicates that
tamoxifen may be of use in breast cancer prevention.
Tamoxifen has tissue-specific estrogenic and antiestrogenic effects. Estrogen,
an
ovarian hormone, increases the risk of breast and endometrial cancer by
inducing an
estrogen receptor mediated increase in the frequency of breast and endometrial
cell
division. Cell division is essential in the complex process of genesis of
human cancer
since it per se increases the risk of genetic error - particularly genetic
errors such as
inactivation of tumor suppressor genes.
Tamoxifen has antiestrogenic effects in breast tissue. Tamoxifen's
antiestrogenic
effect in breast tissue is a primary mechanism by which tamoxifen inhibits the
EI193646477US
CA 02310294 2000-OS-30
proliferation of breast cancer cells. Tamoxifen competes with estrogen for
binding to
cytoplasmic estrogen receptors ("ER"), with subsequent inhibition by the
tamoxifen/ER
complex of many of the activities of endogenous estrogen within tumor cells.
Endogenous estrogen binds with ERs to promote cellular activities such as
estrogen/ER-
mediated gene transcription, DNA synthesis, cancer cell growth, and increases
in
autocrine polypeptides such as transforming growth factor-alpha, epidermal
growth
factor, insulin-like growth factor-II, and other growth factors that may be
involved in cell
proliferation. Competitive inhibition of estrogen binding to ERs by tamoxifen
reduces or
prevents such cancer growth inducing cellular activities. As a result of
tamoxifen's
antiestrogenic activity in breast tissue, tamoxifen prevents the transition of
breast cancer
cells from the early G1 phase to the mid-G1 phase of the cell cycle and
exhibits a
cytostatic effect on breast cancer cells. Tamoxifen has been shown to reduce
distant
breast cancer metastasis as well as local-regional recurrence of such cancers
in both node-
negative and node-positive women.
Tamoxifen, however, has an estrogenic effect on uterine tissues when
endogenous
estrogen levels are low, which predominantly occurs in postmenopausal women
and
oopherectimized women. Uterine epithelial cell heights are significantly
increased by the
estrogenic effect of tamoxifen in these women, leading to uterine hypertrophy.
Tamoxifen also causes marked uterine eosinophilia. These effects have been
associated
with endometrial carcinoma, and long term use of tamoxifen is linked to an
increased risk
of endometrial cancer, up to a fivefold excess of risk relative to women not
treated with
tamoxifen therapy. Therefore, application of tamoxifen for long term breast
cancer
prevention and long term treatment of breast cancer has significant associated
risks. It is
desirable to reduce or eliminate these risks.
Summar~of the Invention
In one aspect, the present invention is a composition which is a combination
of
tamoxifen and at least one isoflavone selected from genistein, daidzein,
biochanin A,
formononetin, and their respective naturally occurring glucosides and
glucoside
conjugates. Preferably, the tamoxifen is present in the composition in an
amount
CA 02310294 2000-OS-30
sufficient to prevent, minimize, or reverse the development or growth of
breast cancer in
a woman having or predisposed to breast cancer. Preferably, the isoflavone is
present in
the composition in an amount sufficient to prevent or minimize tamoxifen
induced
uterotrophic effects and/or to prevent minimize, or reverse the development or
growth of
breast cancer in a woman having or predisposed to breast cancer. In a
preferred
embodiment, the isoflavone is present in the composition in an amount
sufficient to
prevent or minimize the risk of development of endometrial cancer.
In a another aspect, the present invention is a method of treating breast
cancer in a
woman. Tamoxifen and at least one isoflavone selected from genistein,
daidzein,
biochanin A, formononetin, and their naturally occurring glucosides and
glucoside
conjugates are co-administered to a woman having breast cancer. The tamoxifen
is
administered to prevent, minimize, or reverse the development or growth of
breast cancer,
and the isoflavone is administered to prevent or minimize tamoxifen induced
uterotrophic
effects and/or to prevent, minimize, or reverse the development or growth of
breast
cancer. In a preferred embodiment, the isoflavone is also administered to
prevent or
minimize the risk of development of endometrial cancer.
In yet another aspect, the present invention is a method of preventing breast
cancer in a woman predisposed to breast cancer. Tamoxifen and at least one
isoflavone
selected from genistein, daidzein, biochanin A, formononetin, and their
respective
naturally occurnng glucosides and glucoside conjugates are co-administered to
a woman
predisposed to breast cancer. Tamoxifen is administered to prevent or minimize
the risk
of development of breast cancer. The isoflavone is administered to prevent or
minimize
tamoxifen induced uterotrophic effects. In a preferred embodiment, the
isoflavone is also
administered to prevent or minimize the risk of development of endometrial
cancer.
Brief Description of the Drawings
Fig.l is a depiction of the molecular structure of tamoxifen
Fig.2 is a depiction of the molecular structures of genistein, daidzein,
glycitein, biochanin
A, and formononetin.
CA 02310294 2000-OS-30
Fig. 3 is a depiction of the molecular structures of specific naturally
occurring glucosides
and glucoside conjugates of genistein, daidzein, and glycitein.
Detailed Descr~tion of the Invention
As used herein, the term "ER" refers to "estrogen receptor". The term "breast
cancer" means any cancer having its origin in breast cells, and includes
metastatic and
local forms of breast cancer (node negative and node positive), as well as ER
positive and
ER negative forms of breast cancer. The term "uterotrophic effect" means the
proliferation of uterine epithelial cells, which frequently is a side effect
of administration
of tamoxifen to women, and which appears to be directly related to development
of
endometrial cancer. As used herein "Mal" represents "malonyl' and "Ac"
represents
"acetyl". The term "minimize", or a derivative thereof, includes a complete or
partial
inhibition of a specified biological effect (which is apparent from the
context in which the
term minimize is used). The term "isoflavone" may mean a single isoflavone or
plural
isoflavones when the "isoflavone" is defined as at least one of a selected
group of
isoflavones. "Tamoxifen" means either tamoxifen or a pharmaceutically
acceptable salt
thereof. "Sequential" or "sequentially" as used herein to describe sequential
administration of tamoxifen and an isoflavone means administration of desired
amount of
tamoxifen and isoflavone individually within a specified periodic period of
time, for
example daily, and is not intended to be limited to immediate consecutive
administration
of tamoxifen and the isoflavone.
The present invention resides in the discovery that the combination of
tamoxifen
with certain isoflavones can be used to treat or prevent breast cancer in a
woman having
or predisposed to breast cancer, and the isoflavones will prevent or minimize
the
development of uterotrophic effects and endometrial cancer, as well as enhance
the
prevention, minimization or reversal of the development or growth of the
breast cancer.
The isoflavones which are useful in the compositions and methods of present
invention
are genistein, daidzein, glycitein, biochanin A, formononetin and their
naturally occurring
glucosides and glucoside conjugates. An isoflavone glucoside refers herein to
an
isoflavone moiety having a carbohydrate monomer covalently bonded thereto, and
an
CA 02310294 2003-07-07
isoflavone glucoside conjugate refers to an isoflavone glucoside having
another molecular
moiety, such as an ester, bonded to the carbohydrate portion of the isoflavone
glucoside.
These compounds are shown in Figs. 2 and 3.
Materials
Tamoxifen (2-[4-(1,2-biphenyl-1-butenyl)phenoxyl-N,N-dimethylethanamine)
(Fig. 1), as used in the methods and compositions of the present invention,
can be
prepared by established procedures. For example, tamoxifen may be prepared by
the
methods described in Belgian patent number 637,389 and British patent number
1,064,629,
both of which may be referred to for further details. Tamoxifen is also
commercially
available, for example from the Aldrich Chemical Company, Inc., 940 West Saint
Paul
Avenue, Milwaukee, Wisconsin, 53233.
The isoflavone compounds used in the compositions and methods of the
present invention are naturally occurring substances which may be found in
plants such as
legumes, clover and the root of the kudzu vine (pueraria root). Common legume
sources
of these isoflavone compounds include soy beans, chick peas and various other
types of
beans and peas. Clover sources of these isoflavone compounds include red
clover and
subterranean clover. Soy beans are a particularly preferred source of the
isoflavone
compounds (except biochanin A which is not present in soy).
The isoflavone compounds may be isolated from the plant sources in which
they naturally occur, or may be synthetically prepared by processes known in
the art. For
example, daidzein may be isolated from red clover as disclosed by Wong (J.
Sci. Food
Agr., Vol. 13, p. 304 (1962)) or may be isolated from the mold Micromonospora
halophytica as provided by Ganguly and Sarre CChem. & Ind. (London), p. 201
(1970)),
both references of which may be referred to for further details. Daidzein may
be
synthetically prepared by the methods provided by Baker et al (J. Chem. Soc.,
p. 274
(1933)), Wesley et al (Ber. Vol. 66, p. 695 (1933)), Mahal et al (J. Chem.
Soc., p. 1769
(1934)), Baker et al (J. Chem. Soc., p. 1852 (1953)), or Farkas (Ber. Vol. 90,
p. 2940
(1957)), each reference of which may be referred to for further details. The
isoflavone
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glucoside daidzin may be synthetically prepared by the method of Farkas et al
(Ber., Vol.
92, p. 819 ( 1959)), which may be referred to for further details. The
daidzein isoflavone
glucoside conjugates 6'-O-Mal daidzin and 6'-O-Ac daidzin can be prepared by a
conventional saponification of daidzin with a malonyl or an acetyl anhydride,
respectively.
Genistein may be synthetically prepared by the methods provided by Baker et
al (J. Chem. Soc., p. 3115 (1928)); Narasimhachari et al (J. Sci. Ind. Res.,
Vol. 12, p. 287
(1953)); Yoder et al, (Proc. Iowa Acad Sci., Vol. 61, p. 271 (1954) and
Zemplen et al
(Acta. Chim. Acad. Sci. Hung., Vol. 19, p. 277 (1959)), each reference of
which may be
referred to for further details. The isoflavone glucoside genistin may be
synthetically
prepared by the method of Zemplen et al (Ber., Vol. 76B, p. 1110 (1943)),
which may be
referred to for further details. The isoflavone glucoside conjugates of
genistein, 6'-O- Mal
genistin and 6'-O-Ac genistin, can be prepared by a conventional
saponification of genistin
with a malonyl or an acetyl anhydride, respectively.
Biochanin A can be synthetically prepared by the method provided by Baker
et al (Nature 169:706 (1952)), which may be referred to for further details.
Biochanin A
can also be separated from red clover by the method provided by Pope et al
CChem. &
Ind. (London) p. 1092 (1953)), which may be referred to for further details.
Formononetin
can be synthetically prepared by the methods disclosed by Wessely et al (Ber.
66:685
(1933) and Kagel et al (Tetrahedron Letters, p. 593 (1962)), both references
which may be
referred to for further details. Formononetin can be isolated from soybean
meal by the
method of Walz (Ann. 489:118 (1931)) or can be isolated from clover species by
the
method of Bradbury et al (J. Chem. Soc. p. 3447 (1951)), both references which
may be
referred to for further details.
It is preferred to extract the isoflavones useful in the compositions and
methods of the present invention from the plant materials in which they
naturally occur.
A preferred method of isolating the isoflavone compounds is to extract the
plant materials
with an alcohol, preferably methanol or ethanol, or an aqueous solution,
preferably an
aqueous alkaline solution, to remove the isoflavones from the plant material.
It is
preferred to comminute the plant material before extracting the isoflavone
compounds to
maximize recovery of the isoflavone compounds from the plant material. The
isoflavone
6
CA 02310294 2000-OS-30
compounds can be isolated from the extract by conventional separation
procedures such
as reverse phase high performance liquid chromatography ("HPLC").
In a preferred embodiment, the isoflavone compounds gginistein, genistin, 6'-O-
Mal genistin, 6'-O-Ac genistin, daidzein, daidzin, 6'-O-Mal daidzin, 6'-O-Ac
daidzin,
glycitein, glycitin, and 6'-O-Mal glycitin are isolated from a soy material,
preferably a
commercially available soy material. Soy materials from which the isoflavone
compounds can be isolated include: soy beans, dehulled soy beans, soy meal,
soy flour,
soy grits, soy flakes (full fat and defatted), soy cotyldeons, soy molasses,
soy protein
concentrate, soy whey, soy whey protein, and soy protein isolate. In one
embodiment,
the isoflavones are extracted from soy beans, dehulled soy beans, soy meal,
soy flour, soy
grits, soy flakes, soy protein concentrate, soy whey protein, or soy protein
isolate,
preferably soy meal, soy flour, soy grits, or soy flakes, with a low molecular
weight
organic extractant, preferably an alcohol, ethyl acetate, acetone, or ether,
and most
preferably aqueous ethyl alcohol or methyl alcohol. Most preferably the
extractant has a
pH of about the isoelectric point of soy protein (about pH 4 to pH 5) to
minimize the
amount of soy protein extracted by the extractant.
The extractant containing the isoflavones is separated from the insoluble soy
materials to form an isoflavone enriched extract. If desired, an isoflavone
enriched
material may be recovered by concentrating the extract to remove the solvent,
thereby
producing a solid isoflavone enriched material.
In a more preferred embodiment the isoflavone compounds are further purified
from other soy materials soluble in the extract by contacting the extract with
a material
which adsorbs the isoflavones in the extract, and eluting the adsorbed
isoflavones out of
the adsorbent material with a solvent which causes the isoflavones to be
differentially
eluted from the adsorbent material.
In a preferred embodiment, the isoflavones are separated from impurities in
the
extract by a conventional reverse phase HPLC separation. After extraction of
the
isoflavones from the soy material and separation of the extract from the
insoluble soy
materials, the extract is filtered to remove insoluble materials that could
plug an HPLC
column. An HPLC column is prepared by packing a conventional commercially
CA 02310294 2000-OS-30
available HPLC column with a particulate adsorbent material which will
releasably bind
the isoflavones and impurities in the extract in a compound specific manner.
The
adsorbent material may be any reverse phase HPLC packing material, however, a
preferred packing material may be chosen by the criteria of load capacity,
separation
effectiveness, and cost. One such preferred packing material is Kromasil C18
l6pm
100. beads available from Eka Nobel, Nobel Industries, Sweden.
The filtered extract is passed through the packed HPLC column until all the
binding sites of the column are fully saturated with isoflavones, which is
detected by the
appearance of isoflavones in the effluent from the column. The HPLC column may
then
be eluted with a solvent to effect the separation. In a preferred embodiment,
the eluent is
a polar solvent such as ethanol, methanol, ethyl acetate, or acetonitrile, and
preferably is
an aqueous alcohol having an alcohol content of between about 30% and about 90
%,
most preferably about 50%, and most preferably the alcohol is ethanol.
The isoflavone compounds and impurities are separately collected from the
column effluent. The isoflavone fractions of the eluent may be identified from
other
eluent fractions in accordance with conventional HPLC and analytical chemistry
techniques. In a preferred embodiment the eluent fractions containing the
aglucone
isoflavones are collected separately since the aglucone isoflavones are
believed to be
particularly active tyrosine kinase inhibitors and anti-angiogenesis agents
which inhibit
the development or progression of breast cancer. Of the aglucone isoflavone
materials,
the fraction of effluent containing daidzein elutes from the column first,
followed by a
glycitein fraction, followed by the more polar genistein.
The isoflavone fractions of the eluent may be collected from the column, and
the
volatile content of the solvent (e.g. alcohol) can be removed by evaporation.
The
isoflavone compounds can be recovered directly if all of the solvent is
removed by
evaporation, or may be recovered by chilling the remaining solvent (e.g.
water) to
crystallize the isoflavones and centrifuging or filtering the remaining
solvent away from
the crystallized isoflavones.
In a particularly preferred embodiment the isoflavone glucosides and
isoflavone
glucoside conjugates -- 6'-O-Mal genistin, 6'-O-Ac genistin, 6'-O-Mal daidzin,
6'-O-Ac
CA 02310294 2000-OS-30
daidzin, 6'-O-Mal glycitin, genistin, daidzin, and glycitin -- are converted
to their
respective ag(ucone isoflavone forms -- genistein, daidzein, and glycitein.
'hhc
conversion of the isoflavone glucoside conjugates and the isoflavone
glucosides to the
aglucone isoflavones can be effected in the substrate from which the
isoflavones are to be
extracted prior to the extraction, or may be effected in the isoflavone
enriched extract
after separation of the extract from the insoluble materials. The aglucone
isollavone
compounds are especially desirable in the compositions and methods of the
present
invention since, as noted above, they are believed to be particularly active
in inhibiting
angiogenesis and tyrosine kinase activity.
The isoflavone glucoside conjugates 6"-O-Mal genistin, 6"-O-Ac genistin, 6"-O-
Mal daidzin, 6"-O-Ac daidzin, and 6"-O-Mal glycitin can be converted to their
respective
glucosides genistin, daidzin, and glycitin by forming an aqueous alkaline
solution of the
substrate containing the isoflavones having a pH of about 6 to about 13,
preferably about
pH 9 to about pH 11, and treating the aqueous alkaline solution at a
temperature of about
2°C to about 121°C, preferably about 25°C to about
75°C, for a period of time sufficient
to effect the conversion, preferably about 30 minutes to about 5 hours, more
preferably
about 30 minutes to about 1.5 hours. The isoflavone glucosides genistin,
daidzin, and
glycitin can be converted to their respective aglucone forms genistein,
daidzein, and
glycitein by contacting the isoflavone glucosides with an enzyme capable of
cleaving a
1,4-13-glucoside bond -- preferably a commercially available beta-glucosidase
enzyme, an
alpha- or beta-galactosidase enzyme, a pectinase enzyme, a lactase enzyme, or
a gluco-
amylase enzyme -- at a pH at which the enzyme is active, typically from about
pH 3 to
about pH 9, and at a temperature of about 25°C to about 75°C,
more preferably about
45°C to about 65°C, for a period of time sufficient to effect
the conversion, typically
about 1 hour to about 24 hours, preferably about 1 hour to about 3 hours.
The aglucone isoflavones can be separated from the substrate using
conventional
separation procedures. For example, the aglucone isoflavones may be extracted
from the
substrate with a low molecular weight alcohol. The aglucone isoflavones may be
separated from the extract by conventional recrystallization processes, or by
HPLC. In a
particularly preferred embodiment, an isoflavone composition isolated from a
soy
CA 02310294 2000-OS-30
substrate for formulation into a composition of the present invention or for
use in a
method of the present invention includes at least 40% genistein, at least 15%
daidzein,
and at least 1% glycitein. In another particularly preferred embodiment of the
invention,
an isoflavone composition isolated from a soy substrate for formulation into a
composition of the present invention or for use in a method of the present
invention
contains at least 85% genistein, at least S% daidzein, and at least 0.5%
glycitein. In yet
another preferred embodiment, each isoflavone is recovered separately in pure
form.
Several of the isoflavone compounds are commercially available, and may be
purchased for formulation into compositions provided in the present invention,
or used in
the methods of the present invention. For example, genistein, daidzein, and
glycitein are
commercially available and may be purchased, for example, from Indofine
Chemical
Company Inc., P.O. Box 473, Somerville, New Jersey 08876, and biochanin A is
available from Aldrich Chemical Company, Inc., 940 West Saint Paul Avenue,
Milwaukee, Wisconsin 53233.
Method
In one aspect, the present invention is a method of treating or preventing
breast
cancer in a woman having or predisposed to having breast cancer by
coadministering to
the woman tamoxifen and at least one isoflavone selected from genistein,
daidzein,
biochanin A, formononetin, and their naturally occurring glucosides and
glucoside
conjugates. The tamoxifen prevents, minimizes, or reverses the development or
growth
of breast cancer. In one embodiment of the invention, the isoflavone prevents
or
minimizes tamoxifen induced uterotrophic effects and endometrial carcinoma. In
another
embodiment of the invention, the isoflavone enhances prevention, minimization,
or
reversal of the development or growth of breast cancer.
A Method of treating breast cancer with tamoxifen and preventing or minimizing
tamoxifen induced uterotrophic effects
One embodiment of the present invention is a method of treating breast cancer
in
which tamoxifen is administered to a woman having breast cancer, and at least
one
l0
CA 02310294 2000-OS-30
isoflavone selected from genistein, daidzein, biochanin A, formononetin, and
their
naturally occurring glucosides and glucoside conjugates is co-administered to
the woman
to prevent or minimize any tamoxifen-induced uterotrophic effects. Tamoxifen
is
administered to the woman to prevent, minimize, or reverse the growth or
development of
breast cancer in the woman. An amount of tamoxifen sufficient to treat breast
cancer in
the method of the invention is at least 5 mg per day, preferably from S mg to
100 mg per
day, more preferably from 10 mg to SO mg per day, and most preferably from 1 S
mg to
45 mg per day. The tamoxifen may be administered in several doses per day to
achieve
the daily amount of tamoxifen sufficient to treat the breast cancer, however,
it is preferred
that the daily required amount of tamoxifen be administered in one or two
doses.
The isoflavone is co-administered to the woman with the tamoxifen to prevent
or
minimize tamoxifen induced uterotrophic effects. The isoflavone can be co-
administered
concurrently or sequentially with the tamoxifen. Most preferably, tamoxifen
and the
isoflavone are co-administered concurrently in a composition of the present
invention,
described below, on a periodic basis, preferably daily. When administered
sequentially
the isoflavone and tamoxifen are administered as separate components on a
periodic
basis, preferably daily, although shorter and longer periods may be used. The
isoflavone
and tamoxifen can each be prepared as described above for sequential
administration, or,
if commercially available, may be purchased from a commercial vendor.
The amount of isoflavone sufficient to prevent or minimize tamoxifen induced
uterotrophic effects when tamoxifen is administered to treat breast cancer
depends on the
amount of tamoxifen administered and the effectiveness of the selected
isoflavone in
competitively inhibiting tamoxifen induced uterotrophic effects. The
isoflavones are
weakly estrogenic substances that have estrogenic or antiestrogenic effects in
certain
tissues depending on the type of tissue and the concentration of endogenous
estrogen or
other compounds having estrogenic activity such as tamoxifen or tamoxifen
metabolites.
The isoflavones used in the present invention have an antiestrogenic effect in
uterine
tissues when concentrations of tamoxifen or estrogen are relatively high, such
as induced
by treatment of a woman with tamoxifen. One mechanism by which the isoflavones
likely cause an antiestrogenic effect in uterine tissue in the presence of
tamoxifen or
CA 02310294 2000-OS-30
tamoxifen metabolites is by binding to uterine cell ERs and competitively
inhibiting
tamoxifen or tamoxifen metabolites from binding to the ERs. Unlike tamoxifen,
the
isoflavones do not cause an estrogenic response upon binding to the uterine
cell ERs,
therefore, the isoflavones prevent, inhibit, or minimize the uterotrophic
effects caused by
uterine endothelial cell tamoxifen/ER or tamoxifen-metabolite/ER complexes.
When tamoxifen is administered to treat breast cancer and the isoflavone is
administered to inhibit tamoxifen induced uterotrophic effects, the isoflavone
is
administered in a weight/weight ratio of isoflavoneaamoxifen of at least
0.25:, more
preferably from about O.S:1 to about 100:1, and most preferably from about 1:1
to about
20:1. Typical daily doses of the isoflavone sufficient to minimize tamoxifen
induced
uterotrophic effects will be at least 1 mg per day, preferably from about 1 mg
to about
1000 mg per day, more preferably from about 10 mg to about S00 mg per day, and
most
preferably from about 30 mg to about 300 mg per day. The isoflavone may be
administered in several doses per day to achieve the daily amount of
isoflavone sufficient
to prevent or minimize tamoxifen induced uterotrophic effects, however, it is
preferred
that the daily required amount of isoflavone be administered in one or two
doses.
In a particularly preferred embodiment of the method, co-administration of the
isoflavone with tamoxifen in an amount sufficient to prevent or minimize
uterotrophic
effects is also effective to prevent or minimize the development of
endometrial cancer.
As noted above, tamoxifen causes an increased risk of development of
endometrial cancer
as a result of tamoxifen's estrogenic activity in uterine tissue and its
uterotrophic effects.
Co-administration of the isoflavone together with tamoxifen, therefore,
preferably
prevents or minimizes the development of endometrial cancer by preventing or
minimizing tamoxifen induced uterotrophic effects.
B Method of ureventin~ or minimizing the risk of developing breast cancer
while
preventing or minimizing uterotrophic effects
Another embodiment of the present invention is a method of preventing or
minimizing the risk of development of breast cancer in which tamoxifen is
administered
to a woman susceptible to developing breast cancer, and at least one
isoflavone selected
l2
CA 02310294 2000-OS-30
from genistein, daidzein, glycitein, biochanin A, formononetin, and their
naturally
occurring glucosides and glucoside conjugates is co-administered to the woman
to
prevent or minimize any tamoxifen-induced uterotrophic effects. Tamoxifen is
administered to the woman in an amount sufficient to prevent or minimize the
risk of
development of breast cancer. The amount of tamoxifen sufficient to prevent or
minimize the risk of development of breast cancer may be less than that
required to treat
breast cancer, and preferably is administered in a smaller amount than that
utilized to
treat breast cancer, particularly since preventative administration is likely
to span a
significant time period. An amount of tamoxifen sufficient to prevent or
minimize the
risk of development of breast cancer is at least 0.5 mg per day, preferably
from about 0.5
mg to about 10 mg per day, more preferably from about 1 mg to about S mg per
day, and
most preferably from about 1.5 mg to about 4.5 mg per day. The tamoxifen may
be
administered in several doses per day to achieve the daily amount of tamoxifen
sufficient
to prevent or minimize the risk of development of breast cancer, however, it
is preferred
that the daily required amount of tamoxifen be administered in one dose.
The isoflavone is co-administered to the woman with the tamoxifen to prevent
or
minimize the tamoxifen induced uterotrophic effects. The isoflavone can be co-
administered concurrently or sequentially with the tamoxifen. When
administered
sequentially the isoflavone and tamoxifen are administered as separate
components on a
periodic basis, preferably daily, although shorter and longer periods may be
used. The
isoflavone and tamoxifen can each be prepared as described above for
sequential
administration, or, if commercially available may be purchased from a
commercial
vendor. When administered concurrently, the isoflavone and tamoxifen may be
combined into a composition of the present invention, as described below,
which is
effective to deliver tamoxifen and the isoflavone concurrently.
The amount of isoflavone sufficient to prevent or minimize tamoxifen induced
uterotrophic effects when the tamoxifen is administered to prevent the
development of
breast cancer depends on the amount of tamoxifen administered and the
effectiveness of
the selected phytoestrogen in competitively inhibiting tamoxifen induced
uterotrophic
effects. The isoflavone is administered in a weight/weight ratio of
isoflavoneaamoxifen
t3
CA 02310294 2000-OS-30
of at least 0.25:1, more preferably from about 0.5:1 to about 100:1, and most
preferably
from about I :1 to about 20:1. Typical daily doses of the isoflavone
sufficient to
minimize tamoxifen induced uterotrophic effects, when the tamoxifen is
administered in a
dosage of between 0.5 mg per day to 10 mg per day, will be at least 0.125 mg
per day,
and more preferably from about 0.25 mg per day to about 1000 mg per day.
In a particularly preferred embodiment, co-administration of the isoflavone
with
tamoxifen to prevent or minimize the risk of development of breast cancer and
to prevent
or minimize uterotrophic effects is also effective to prevent or minimize the
risk of
development of endometrial cancer. The isoflavone-induced prevention or
minimization
of the risk of development of tamoxifen induced endometrial cancer removes the
significant risk associated with long term administration of tamoxifen, and
permits
tamoxifen to be used in a long term breast cancer preventative regimen.
C Method of treatin~Ybreast cancer with tamoxifen and isoflavones
Yet another embodiment of the present invention is a method of treating breast
cancer in which tamoxifen is administered to a woman having breast cancer to
prevent,
minimize, or reverse the growth of the breast cancer, and at least one
isoflavone selected
from genistein, daidzein, biochanin A, formononetin, and their naturally
occurring
glucosides and glucoside conjugates is co-administered to the woman to enhance
the
prevention, minimization, or reversal of the growth of the breast cancer.
Tamoxifen is
administered to the woman to treat the breast cancer in the manner and in the
dosages
described above.
The isoflavone is administered to the woman in an amount sufficient to enhance
the tamoxifen induced prevention, minimization, or reversal of the breast
cancer.
Preferably, the isoflavone is administered to the woman in an amount effective
to
prevent, minimize, or reverse the development or growth of breast cancer by
itself.
The isoflavones utilized in the method of the invention prevent, minimize, or
reverse the growth of breast cancer by several mechanisms. First, the
isoflavones are
anti-estrogenic in breast tissue, and serve to competitively inhibit estrogen
induced
cancerous breast cell division by binding to the ER of the cell, where the
isoflavone/ER
14
CA 02310294 2000-OS-30
complex inhibits cancer cell growth in much the same manner as tamoxifen (e.g.
daidzein
halts cell growth in the G1 phase of the cell cycle, gginistein halts cell
growth in the G2
phase of the cell cycle). Second, some of the isoflavones, particularly
gginistein and
biochanin A, are tyrosine kinase inhibitors which inhibit enzymatic tyrosine
kinase
enzyme activity. Tyrosine kinase activity is necessary for cancerous cells to
produce
proteins required for cellular differentiation and growth. Third, the
isoflavones inhibit
angiogenesis, and thereby inhibit a cancerous cell mass from developing the
network of
blood vessels necessary to support the cell mass, limiting the sustainable
growth of the
cell mass. Fourth, the isoflavones decrease endogenous estrogen levels by
interfering
with pituitary and hypothalmus gland feedback mechanisms which regulate the
release of
gonadotropins such as estradiol. The effect of the combined mechanisms of
action is to
prevent, minimize, or reverse the growth of breast cancer in addition to the
tamioxfen
activity against breast cancer.
The amount of isoflavone sufficient to enhance the tamoxifen induced
prevention,
minimization, or reversal of breast cancer growth may be less than that
required for
prevention or minimization of tamoxifen induced uterotrophic effects since the
isoflavone
is being utilized in cooperation with tamoxifen in the breast tissue, rather
than in
opposition to tamoxifen as in the uterine tissues. The isoflavone, however,
need not be
administered in an amount lesser than that required to prevent or minimize
tamoxifen
induced uterotrophic effects, and is preferably administered in such amounts
to achieve
the dual goals of enhancing the prevention, minimization, or reversal of
breast cancer and
preventing or minimizing tamoxifen induced uterotrophic effects and/or
endometrial
cancer. Preferably the isoflavone is administered in a weight/weight ratio of
isoflavoneaamoxifen of at least 0.1:1, and more preferably from about 0.5:1 to
about
100:1, and most preferably from about 1:1 to about 20:1. Typical daily doses
of the
isoflavone sufficient to enhance tamoxifen's effects against breast cancer
will be at least
0.5 mg per day, preferably from about 0.5 mg per day to about 1000 mg per day,
and
most preferably from about 3 mg per day to about 300 mg per day. The
isoflavone may
be administered in several doses per day to achieve the daily amount of
isoflavones
IS
CA 02310294 2000-OS-30
sufficient to enhance tamoxifen's anti-cancer activity, however, it is
preferred that the
daily required amount of isoflavones be administered in one or two doses.
The isoflavone can be co-administered concurrently or sequentially with the
tamoxifen to treat breast cancer and enhance the prevention, minimization, or
reversal of
the growth or development of the breast cancer with the isoflavone. When
administered
sequentially the isoflavone and tamoxifen are administered as separate
components on a
periodic basis, preferably daily, although shorter and longer periods may be
used. The
isoflavone and tamoxifen can each be prepared as described above for
sequential
administration, or, if commercially available may be purchased from a
commercial
vendor. When administered concurrently, the isoflavone and tamoxifen may be
combined into a composition of the present invention, as described below,
which is
effective to deliver tamoxifen and the isoflavone concurrently.
Composition
The composition of the present invention includes tamoxifen and at least one
of
the isoflavones selected from genistein, daidzein, glycitein, biochanin A,
formononetin,
and their naturally occurring glucosides and glucoside conjugates. The
tamoxifen is
present in the composition in an amount sufficient to prevent, minimize, or
reverse the
development or growth of breast cancer in a woman. When the composition is to
be used
to treat breast cancer, at least 5 mg of tamoxifen are present in the
composition, more
preferably from about S mg to about 100 mg of tamoxifen, even more preferably
from
about 10 mg to about 50 mg of tamoxifen, and most preferably from about 15 mg
to
about 45 mg of tamoxifen. When the composition is to be used to prevent or
minimize
the risk of development of breast cancer in a woman predisposed to breast
cancer, at least
0.5 mg of tamoxifen are present in the composition, more preferably from about
0.5 mg
to about 10 mg of tamoxifen, even more preferably from about 1 mg to about 5
mg of
tamoxifen, and most preferably from about 1.5 mg to about 4.5 mg of tamoxifen.
In one embodiment of the composition of the invention, the isoflavone is
present
in the composition in an amount sufficient to prevent or minimize uterotrophic
effects,
especially uterotrophic effects induced by tamoxifen. To prevent or minimize
CA 02310294 2000-OS-30
uterotrophic effects of tamoxifen in the composition the isoflavone is present
in the
composition in a weight:weight ratio of isoflavoneaamoxifen of at least
0..25:1, and more
preferably from about 0.5:1 to about 100:1, and most preferably from about I
:1 to about
20:1.
When the composition is to be used to treat breast cancer and to prevent or
minimize uterotrophic effects, and the composition contains from about 5 mg to
about
100 mg of tamoxifen, the composition contains at least 0.25 mg of the
isoflavone,
preferably from about 0.25 mg to about 1000 mg of the isoflavone, even more
preferably
from about 10 mg to about 500 mg of the isoflavone, and most preferably from
about 30
mg to about 300 mg of the isoflavone. When the composition is to be used to
prevent the
development of breast cancer and to prevent or minimize uterotrophic effects,
and the
composition contains from about 0.5 mg to about 10 mg of tamoxifen, the
composition
contains at least 0.25 mg of the isoflavone, and more preferably from about
0.25 mg to
about 50 mg of the isoflavone. Most preferably the isoflavone is present in
the
composition in an amount sufficient to prevent or minimize the risk of
development of
endometrial cancer, which is an amount equivalent to that for preventing or
minimizing
uterotrophic effects.
In another embodiment of the invention the isoflavone is present in the
composition in an amount sufficient to enhance the tamoxifen induced
prevention,
minimization, or reversal of the growth of breast cancer. To enhance the
tamoxifen
induced prevention, minimization, or reversal of the growth of breast cancer
the
isoflavone is present in the composition in a weight:weight ratio of
isoflavoneaamoxifen
of at least 0.1:1, more preferably from about 0.5:1 to about 100:1, and most
preferably
from about 1:1 to about 20:1. When tamoxifen is present in the composition
from about
mg to about 100 mg and the isoflavone is to be used to enhance the prevention,
minimization, or reversal of the growth of breast cancer, the composition
contains at least
0.5 mg of the isoflavone, more preferably from about 2.5 mg to about 1000 mg
of the
isoflavone, and most preferably from about 5 mg to about 500 mg of the
isoflavone.
The above compositions preferably also include an excipient, IIlOSL preferably
a
pharmaceutical excipient. Compositions containing an excipient and
incorporating
CA 02310294 2000-OS-30
tamoxifen and the isoflavone can be prepared by procedures known in the art.
For
example, tamoxifen and the isoflavone can be formulated into tablets,
capsules, powders,
suspensions, solutions for parenteral administration including intravenous,
intramuscular,
and subcutaneous administration, and into solutions for application onto
patches for
transdermal application with common and conventional carriers, binders,
diluents, and
excipients.
Inert pharmaceutically acceptable carriers useful to form pharmaceutical
compositions in accordance with the present invention include starch,
mannitol, calcium
sulfate, dicalcium phosphate, magnesium stearate, silicic derivatives, and/or
sugars such
as sucrose, lactose, and glucose. Binding agents include carboxymethyl
cellulose and
other cellulose derivatives, gelatin, natural and synthetic gums including
alginates such as
sodium alginate, polyethylene glycol, waxes and the like. Diluents useful in
the
invention include a suitable oil, saline, sugar solutions such as aqueous
dextrose or
aqueous glucose, and glycols such as polyethylene or polypropylene glycol.
Other
excipients include lubricants such as sodium oleate, sodium acetate, sodium
stearate,
sodium chloride, sodium benzoate, talc, and magnesium stearate, and the like;
disintegrating agents including agar, calcium carbonate, sodium bicarbonate,
starch,
xanthan gum, and the like; and adsorptive carriers such as bentonite and
kaolin.
Coloring and flavoring agents may also be added to the pharmaceutical
compositions.
The following non-limiting formulations illustrate pharmaceutical compositions
of the present invention.
FORMULATIONS
The following Formulations 1-4 illustrate pharmaceutical formulations
including
tamoxifen and an isoflavone.
Formulation 1
Gelatin capsules
~8
CA 02310294 2000-OS-30
r
Hard gelatin capsules are prepared using the following ingredients: Tamoxifen
0.5-100 mg/capsule; Isoflavone 0.1-1000 mg/capsule; Starch, NF 0 - 600
mg/capsule;
Starch flowable powder 0 - 600 mg/ capsule; Silicone fluid 350 centistokes 0 -
20
mg/capsule. The ingredients are mixed, passed through a sieve, and filled into
capsules.
Formulation 2
Tablets
Tablets are prepared using the following ingredients: Tamoxifen 0.5-100
mg/tablet; Isoflavone 0.1-1000 mg/ tablet; Microcrystalline cellulose 20-300
mg/tablet;
Starch 0-50 mg/tablet; Magnesium stearate or stearate acid 0-15 mg/tablet;
Silicon
dioxide, fumed 0-400 mg/tablet; silicon dioxide, colloidal 0-1 mg/tablet, and
lactose 0-
100 mg/tablet. The ingredients are blended and compressed to form tablets.
Formulation 3
Suspensions
Suspensions are prepared using the following ingredients: Tamoxifen 0.5-100
mg/Sml; Isoflavone 0.1-1000 mg/Sml; Sodium carboxymethyl cellulose 50-700
mg/Sml;
Sodium benzoate 0-10 mg/Sml; Purified water 5 ml; and flavor and color agents
as
needed.
Formulation 4
Parenteral solutions
A parenteral composition is prepared by stirring 1.5% by weight of active
ingredients (tamoxifen and isoflavone wt/wt ratio of from 10:1 to 1:10) in 10%
by
volume propylene glycol and water. The solution is made isotonic with sodium
chloride
and sterilized.
The above description is intended to be illustrative of the present invention,
and is
not intended to be limiting. Other embodiments are within the claims.
19
