U.S. patent application number 09/900573 was filed with the patent office on 2001-11-29 for composition for and method of preventing or treating breast cancer.
This patent application is currently assigned to Protein Technologies International, Inc.. Invention is credited to Henley, Edna C., Taylor, Richard B..
Application Number | 20010047033 09/900573 |
Document ID | / |
Family ID | 23133790 |
Filed Date | 2001-11-29 |
United States Patent
Application |
20010047033 |
Kind Code |
A1 |
Taylor, Richard B. ; et
al. |
November 29, 2001 |
Composition for and method of preventing or treating breast
cancer
Abstract
The present invention is a composition for preventing,
minimizing, or reversing the development or growth of breast
cancer. The composition contains a combination of a selective
estrogen receptor modulator selected from at least one of
raloxifene, droloxifene, toremifene, 4'-iodotamoxifen, and
idoxifene and at least one isoflavone selected from genistein,
daidzein, biochanin A, formononetin, and their respective naturally
occurring glucosides and glucoside conjugates. The present
invention also provides a method of preventing, minimizing, or
reversing the development or growth of breast cancer in which a
selective estrogen receptor modulator selected from at least one of
raloxifene, droloxifene, toremifene, 4'-iodotamoxifen, and
idoxifene is co-administered with at least one isoflavone selected
from genistein, daidzein, biochanin A, formononetin, and their
naturally occuring glucosides and glucoside conjugates to a woman
having or predisposed to having breast cancer.
Inventors: |
Taylor, Richard B.; (Valley
Park, MO) ; Henley, Edna C.; (Athens, GA) |
Correspondence
Address: |
Richard B. Taylor
Protein Technologies International, Inc.
P.O. Box 88940
St. Louis
MO
63188
US
|
Assignee: |
Protein Technologies International,
Inc.
St. Louis
MO
|
Family ID: |
23133790 |
Appl. No.: |
09/900573 |
Filed: |
July 6, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09900573 |
Jul 6, 2001 |
|
|
|
09294519 |
Apr 20, 1999 |
|
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Current U.S.
Class: |
514/456 ;
514/651 |
Current CPC
Class: |
A61K 31/35 20130101;
A61K 31/35 20130101; A61K 2300/00 20130101; A61K 31/135 20130101;
A61K 31/35 20130101 |
Class at
Publication: |
514/456 ;
514/651 |
International
Class: |
A61K 031/35; A61K
031/138 |
Claims
What is claimed is:
1. A method for preventing, minimizing, or reversing the
development or growth of breast cancer in a woman comprising
co-administering to said woman droloxifene and genistein, said
droloxifene being administered to said woman in an amount effective
to prevent, minimize, or reverse the development or growth of
breast cancer, and said genistein being administered for the
purpose of preventing or minimizing uterine hypertrophy induced by
administration of said droloxifene wherein said genistein is
administered in an amount effective to prevent or minimize uterine
hypertrophy induced by administration of droloxifene.
2. The method of claim 1 wherein co-administration of said
droloxifene and said genistein is concurrent.
3. The method of claim 1 wherein co-administration of said
droloxifene and said genistein is sequential.
4. The method of claim 1 wherein from about 0.5 mg to about 500 mg
of said droloxifene is administered to said woman per day.
5. The method of claim 4 wherein from about 5 mg to about 100 mg of
said droloxifene is administered to said woman per day.
6. The method of claim 1 wherein from about 1 mg to about 1000 mg
of said genistein is administered to said woman per day.
7. The method of claim 6 wherein from about 10 mg to about 200 mg
of said genistein is administered to said woman per day.
8. The method of claim 1 wherein said genistein is administered to
said woman in an amount sufficient to augment prevention,
minimization, or reversal of the development or growth of breast
cancer provided by said droloxifene.
9. The method of claim 1 wherein said genistein is administered to
said woman to prevent or minimize the development of endometrial
cancer.
10. The method of claim 1 wherein said droloxifene and said
genistein are administered in a pharmaceutical preparation.
11. The method of claim 10 wherein said pharmaceutical preparation
is a tablet, capsule, powder, suspension, or solution.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a composition containing a
selective estrogen receptor modulator and at least one isoflavone,
and a method of treating breast cancer while inhibiting selective
estrogen receptor modulator induced uterotrophic effects.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] Tamoxifen (FIG. 1), 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.
[0004] 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.
[0005] Tamoxifen has antiestrogenic effects in breast tissue.
Tamoxifen's antiestrogenic effect in breast tissue is a primary
mechanism by which tamoxifen inhibits the 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.
[0006] Tamoxifen, however, has an estrogenic effect on uterine
tissues when endogenous estrogen levels are low, which occurs in
postmenopausal women and oopherectimized women. Uterine epithelial
cell heights are significantly increased by the estrogenic effect
of tamoxifen in postmenopausal and oopherectimized 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.
[0007] Efforts have been made to develop new selective estrogen
receptor modulators ("SERMS") which act in a mechanism similar to
that of tamoxifen in breast tissue, while avoiding the risks caused
by the estrogenic effects of tamoxifen in uterine tissue. Several
of these SERMS are triphenylethylene tamoxifen analogs. As shown in
FIG. 2, droloxifene is a tamoxifen analog in which a
3'-hydroxyphenyl moiety is substituted in place of a phenyl moiety
of tamoxifen. Droloxifene has a binding affinity for the estrogen
receptor which is ten times that of tamoxifen, has been shown to
have antiestrogenic activity in breast tissue and to be efficacious
in treatment of advanced breast cancer, yet has lower estrogenic
effects in uterus tissue than tamoxifen. Droloxifene, a New
Estrogen Antagonist/Agonist, Prevents Bone Loss in Ovariectomized
Rats, Ke at al., Endocrinology 136:2435-2441 (1995).
[0008] Toremifene, shown in FIG. 2, is a tamoxifen analog having a
4-chloro substituent. Pharmacologically toremifene has quite
similar effects as tamoxifen on breast tissue, acting as potent
antiestrogen. Toremifene also exhibits anti-tumor cytolytic effects
at high doses which are independent of its antiestogenicity,
effects which do not occur with high doses of tamoxifen.
Antiestrogenic Potency of Toremifene and Tamoxifen in
Postmenopausal Women, Homesley et al., Am. J. Clin. Onc.,
16(2):117-122 (1993).
[0009] 4-Iodotamoxifen, shown in FIG. 2, is another tamoxifen
analog, having a 4'-iodophenyl substituent in place of a phenyl
substituent of tamoxifen. lodination of tamoxifen at the 4'-phenyl
postion reduces estrogenic activity, mimicking the high
antiestrogenic activity of the tamoxifen metabolite
4'-hydroxytamoxifen, while giving the compound a longer duration of
action in vivo by blocking formation of the rapidly metabolized
4'-hydroxytamoxifen metabolite. Pyrrolidino-4-iodotamoxifen and
4-Iodotamoxifen, New Analogues of the Antiestrogen Tamoxifen for
the Treatment of Breast Cancer, Chander et al., Cancer Research,
51:5851-5858 (Nov. 1, 1991); Idoxifene: Report of a Phase I Study
in Patients with Metastatic Breast Cancer, Coombes et al, Cancer
Research, 55:1070-1074 (Mar. 1, 1995). 4-Iodotamoxifen has been
shown to have less estrogenic agonist activity in uterine tissue
than tamoxifen, and, therefore, is less likely to cause endometrial
cancer when administered over a long term.
[0010] Idoxifene, also known as pyrrolidino-4-iodotamoxifen, shown
in FIG. 2, is another tamoxifen analog, and is modeled on the
4'-iodotamoxifen analog. Idoxifene has the same general molecular
structure as 4'-iodotamoxifen, except that the N,N-dimethylamino
moiety of 4'-iodotamoxifen is replaced with a pyrrolidino moiety.
Substitution of the pyrrolidino group for the dimethylamino group
reduces possible toxic side effects by inhibiting the
metabolization of the compound by the liver to a desmethyl
metabolite with the concomitant release of potentially toxic
formaldehyde. Idoxifene has a 2.5 to 5 fold higher affinity for ERs
than tamoxifen, and is 1.5-fold more efffective in inhibiting the
growth of MCF-7 breast cancer cells. Idoxifene also has less
uterotrophic estogenic effects than tamoxifen and 4'-iodotamoxifen,
and produced uterotrophic effects comparable to that of tamoxifen
only at a dose which was ten times greater.
Pyrrolidino-4-Iodotamoxifen and 4-Iodotamoxifen, New Analogues of
the Antiestrogen Tamoxifen for the Treatment of Breast Cancer,
Chander et al., Cancer Research, 51:5851-5858 (November 1991);
Idoxifene: Report of a Phase I Study in Patients with Metastatic
Breast Cancer, Coombes et al., Cancer Research, 55:1070-1074 (Mar.
1, 1995).
[0011] Other SERMS which are not tamoxifen analogs have shown
effectiveness in preventing or minimizing the development of breast
cancer. Raloxifene (FIG. 3), a benzothiophene derivative, has shown
potent antiestrogenic inhibition of estradiol binding to the ER and
significantly inhibits estrogen dependent proliferation of MCF-7
cells derived from human mammary tissue. Raloxifene, unlike
tamoxifen and its analogs, exhibits an antiestrogenic effect in
uterine tissue, and provides a nearly complete blockade of
uterotrophic responses to estrogen as well as tamoxifen. Selective
Estrogen Receptor Modulators, Kauffman & Bryant, DN&P, 8(9)
531-539 (November 1995).
[0012] It is desirable to utilize these SERMS to develop new
compositions which may be used to improve the SERMS' prevention or
minimization of the development of breast cancer while reducing
their uterotrophic activity, if any.
SUMMARY OF THE INVENTION
[0013] In one aspect, the present invention is a composition for
preventing or minimizing the development or growth of breast
cancer. The composition comprises a combination of a selective
estrogen receptor modulator selected from at least one of
raloxifene, droloxifene, toremifene, 4-iodotamoxifen, and
idoxifene, and at least one isoflavone selected from genistein
daidzein, biochanin A, formononetin, or their respective naturally
occuring glucosides or glucoside conjugates.
[0014] In another aspect, the present invention is a method for
preventing or minimizing the development or growth of breast cancer
in a human. A selective estrogen receptor modulator and an
isoflavone are co-administered to a human to prevent or minimize
the development or growth of breast cancer. The selective estrogen
receptor is selected from at least one of raloxifene, droloxifene,
toremifene, 4'-iodotamoxifen, and idoxifene. The isoflavone is
selected from at least one of genistein, daidzein, biochanin A,
formononetin, or their naturally occuring glucosides or glucoside
conjugates.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a molecular representation of tamoxifen.
[0016] FIG. 2 is a molecular representation of the selective
estrogen receptor modulators droloxifene, toremifene,
4'iodotamoxifen, and idoxifene.
[0017] FIG. 3 is a molecular representation of the selective
estrogen receptor modulator raloxifene.
[0018] FIG. 4 is a molecular representation of genistein, daidzein,
biochanin A, and formononetin.
[0019] FIG. 5 is a molecular representation of the naturally
occuring glucosides of genistein and daidzein.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0020] 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 selective estrogen
receptor modulators 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 both a single isoflavone or plural
isoflavones when the isoflavone is defined as at least one of a
selected group of isoflavones. "SERM" means a selective estrogen
receptor modulator and its physiologically acceptable salts, other
than tamoxifen, which is a compound which produces estrogen
antagonist effects in one or more desired target tissues (e.g.
breast tissue and uterine tissue), while producing either estrogen
agonist effects or minimal agonism in other non-target tissues.
[0021] The present invention resides in the discovery that the
combination of selected SERMs 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 augment the SERM induced
prevention, minimization, or reversal of the development or growth
of breast cancer, as well as prevent or minimize uterotrophic
effects associated with some SERMs. The SERMs which are useful in
the compositions and methods of the present invention are
droloxifene, toremifene, 4'-iodotamoxifen, idoxifene, and
raloxifene. The isoflavones which are useful in the compositions
and methods of the present invention are genistein, daidzein,
glycitein, biochanin A, formononetin, their naturally occuring
glycosides and their naturally occuring glycoside conjugates, shown
in FIGS. 4 and 5.
[0022] Materials
[0023] The selective estrogen receptor modulator compounds used in
the compositions and methods of the present invention can be
chemically synthesized according to known methods, and include the
salt forms of each of the compounds. Raloxifene,
6-hydroxy-2-(4-hydroxyphenyl)-3-[4-(2--
piperdinoethoxy)benzoyl]benzo[b]thiophene (FIG. 3), and its
physiologically acceptable salts may be produced according to the
methods described in U.S. Pat. Nos. 4,418,068 and 4,133,814, each
of which is incorporated herein by reference. Droloxifene,
E-1-[4'-(2-dimethylaminoet-
hoxy)phenyl]-1-(3'-hydroxyphenyl)-2-phenyl-1-butene (FIG. 2), and
its physiologically acceptable salts may be produced according to
the methods described in U.S. Pat. No. 5,047,431, which is
incorporated herein by reference. Toremifene,
4-chloro-1,2-diphenyl-1-{4-[2-(N,N-dimethylamino)e-
thoxy]-phenyl}-1-butene (FIG. 2), and its physiologically
acceptable salts may be produced by the methods described in U.S.
Pat. No. 4,696,949, which is incorporated herein by reference.
4'-Iodotamoxifen,
E-1-{4-[2-(dimethylamino)ethoxy]phenyl}-1-(4-iodophenyl)-2-phenyl-1-buten-
e (FIG. 2), and its physiologically acceptable salts may be
produced according to combined methods described in Stereoselective
Olefin Formation from the Dehydration of
1-(p-Alkoxyphenyl)-1,2-diphenylbutan-1-- ols: Application to the
Synthesis of Tamoxifen, McCague, J. Chem. Soc. Perkin Trans.,
1:1011-1015 (1987); and Derivatives of Tamoxifen. Dependence of
Antiestrogenicity on the 4-Substituent, McCague et al., J. Med.
Chem., 32(12):2527-2533 (1989), each of which is incorporated
herein by reference. Idoxifene,
E-1-(4-iodophenyl)-1-[4-(2-pyrrolidinoethoxy)phe-
nyl]-2-phenyl-1-butene (FIG. 2), may be produced according to
combined methods described in the references above that provide
methods for producing 4'-iodotamoxifen.
[0024] 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).
[0025] 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 (Chem.
& Ind. (London), p. 201 (1970)), both references of which are
incorporated by reference herein. 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. 685 (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 is incorporated herein by reference. The
isoflavone glucoside daidzin may be synthetically prepared by the
method of Farkas et al. (Ber., Vol. 92, p. 819 (1959)),
incorporated herein by reference. 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.
[0026] 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 is incorporated herein by
reference. The isoflavone glucoside genistin may be synthetically
prepared by the method of Zemplen et al. (Ber., Vol 76B, p. 1110
(1943)), incorporated herein by reference. 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.
[0027] Biochanin A can be synthetically prepared by the method
provided by Baker et al. (Nature 169:706 (1952)), incorporated
herein by reference. Biochanin A can also be separated from red
clover by the method provided by Pope et al. (Chem. & Ind.
(London) p. 1092 (1953)), incorporated herein by reference.
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 of which are
incorporated herein by reference. 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 of which are
incorporated herein by reference.
[0028] 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 compounds can be isolated from the
extract by conventional separation procedures such as reverse phase
high performance liquid chromatography ("HPLC").
[0029] In a preferred embodiment, the isoflavone compounds
genistein, 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 at about
the isoelectric point of soy protein (about pH 4 to pH 5) to
minimize the amount of soy protein extracted by the extractant.
[0030] 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.
[0031] 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.
[0032] 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 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 16
.mu.m 100.ANG. beads available from Eka Nobel, Nobel Industries,
Sweden.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] In a particularly preferred embodiment the soy isoflavone
glucosides and isoflavone glucoside conjugates--6'-O-Mal genistin,
6'-O-Ac genistin, 6'-O-Mal daidzin, 6'-O-Ac daidzin, 6'-O-Mal
glycitin, genistin, daidzin, and glycitin--are converted to their
respective aglucone isoflavone forms--genistein, daidzein, and
glycitein. The 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 isoflavone 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 inhibiting
tyrosine kinase activity.
[0037] 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.degree. C. to
about 121.degree. C., preferably about 25.degree. C. to about
75.degree. 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-.beta.-glucoside bond--preferably a commercially
available beta-glucosidase enzyme, an alpha- or beta-galactosidase
enzyme, a pectinase enzyme, a lactase enzyme, or a glucoamylase
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.degree. C. to
about 75.degree. C., more preferably about 45.degree. C. to about
65.degree. 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.
[0038] 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 substrate for formulation into a
composition for use in the 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 for use in the method of the
present invention contains at least 85% genistein, at least 5%
daidzein, and at least 0.5% glycitein. In yet another preferred
embodiment, each isoflavone is recovered separately in pure
form.
[0039] 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, N.J.
08876, and biochanin A is available from Aldrich Chemical Company,
Inc., 940 West Saint Paul Avenue, Milwaukee, Wis. 53233.
[0040] Methods
[0041] In one aspect the present invention is a method for
preventing or minimizing the development or growth of breast cancer
in a human by co-administering at least one SERM selected from
raloxifene, droloxifene, toremifene, 4-iodotamoxifen, and
idoxifene, and at least one isoflavone selected from genistein,
daidzein, biochanin A, formononetin, their respective glucosides,
and their respective glucoside conjugates. The SERM and isoflavone
may be co-administered prophylactically to prevent the development
of breast cancer in women susceptible of developing breast cancer,
or the SERM and isoflavone may be co-administered to treat breast
cancer by preventing, minimizing, or reversing the growth and
development of the cancer. The SERM may be obtained for use in
accordance with the method the present invention as described
above, or, preferably, may be provided in a composition of the
present invention, as described below. The isoflavone may be
obtained for use in accordance with the method of the present
invention as described above, or, preferably, may be provided in a
composition of the present invention, as described below.
[0042] The SERM and the isoflavone may be co-administered either
concurrently or sequentially within a specified period of time,
preferably daily, on a periodic basis. Most preferably the SERM and
the isoflavone are co-administered concurrently in a composition of
the present invention, as described below, on a periodic basis,
preferably daily. Alternatively, the SERM and the isoflavone are
administered sequentially as separate components. "Sequentially" as
used herein is intended to mean administration of desired amounts
of the SERM 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 the SERM and
isoflavone.
[0043] The SERM is administered in an amount sufficient to prevent
or treat the development or growth of breast cancer in combination
with the isoflavone. The amount of SERM sufficient to prevent or
treat the development or growth of breast cancer in combination
with the isoflavone is dependent on the particular SERM utilized,
the amount and activity of the isoflavone utilized, the size of the
patient to which the SERM is administered, whether the SERM is
administered prophylatically or to treat breast cancer, and if used
in treatment, the extent of the cancer. The amount of SERM
sufficient to prevent the development of breast cancer in a woman
predisposed to breast cancer is preferably at least 0.5 mg per day,
more preferably from about 0.5 mg to about 100 mg per day, and most
preferably from about 5 mg to about 50 mg per day. The amount of
SERM sufficient to treat the development or growth of breast cancer
to prevent, minimize, or reverse the development or growth of the
cancer is preferably at least 0.5 mg per day, more preferably from
0.5 mg to about 500 mg per day, and most preferably from about 40
mg to about 400 mg per day. The SERM may be administered in several
doses per day to achieve the daily amount of the SERM sufficient to
prevent or treat breast cancer, however, it is preferred that the
daily required amount of SERM be administered in one or two
doses.
[0044] The isoflavone is co-administered to the human in an amount
sufficient to prevent or treat the development or growth of breast
cancer in combination with the SERM. The amount of isoflavone
sufficient to prevent or treat the development or growth of breast
cancer in combination with the SERM is dependent on the particular
isoflavone utilized, the amount and activity of the co-administered
SERM, the size of the patient, whether the isoflavone is
administered prophylatically or to treat breast cancer, and if used
in treatment, the extent of the cancer. The amount of isoflavone
sufficient to prevent the development of breast cancer in a woman
predisposed to breast cancer in the present method is preferably at
least 1 mg per day, more preferably from about 10 mg to about 200
mg per day. The amount of isoflavone sufficient to treat the
development or growth of breast cancer to prevent, minimize, or
reverse the development or growth of the cancer is preferably at
least 1 mg per day, more preferably from about 1 mg to about 1000
mg per day, and most preferably from about 50 mg to about 500 mg
per day.
[0045] The isoflavones utilized in the method of the present
invention prevent, minimize, or reverse the growth of breast cancer
by several mechanisms, which in combination with the
anti-estrogenic activity of the SERM in breast tissue, increase the
relative anti-breast cancer activity of each compound. 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
complex inhibits cancer cell growth in much the same manner as
tamoxifen and the SERMs (e.g. daidzein halts cell growth in the G1
phase of the cell cycle, and genistein halts cell growth in the G2
phase of the cell cycle). Second, some of the isoflavones,
particularly genistein and biochanin A, and to a lesser extent
daidzein and formononetin, are tyrosine kinase inhibitors which
inhibit enzymatic tyrosine kinase activity. Tyrosine kinase
activity is necessary for cancerous cells to produce proteins
required for cellular differentiation and growth. Third, the
isoflavones inhibit angiogenesis, thereby preventing 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
further prevent or minimize the development or growth of breast
cancer when co-administered with a SERM effective to prevent or
minimize the growth of breast cancer.
[0046] In a particularly preferred embodiment of the method of the
present invention, the isoflavone is co-administered with the SERM
in an amount sufficient to prevent or minimize SERM induced
uterotrophic effects. Atlhough the SERMs utilized in the present
invention are less uterotrophic than tamoxifen, each of the SERMs
except raloxifene induces uterotrophic effects at relatively high
doses. The isoflavones utilized in the present method have an
antiestrogenic effect in uterine tissues when concenrations of
estrogen or an estrogen agonist SERM are relatively high. One
mechanism by which the isoflavones likely cause an antiestrogenic
effect in uterine tissue in the presence of uterine estrogen
agonist SERMs is by binding to uterine cell ERs and competitively
inhibiting the estrogen agonist SERMs from binding to the ERs.
Unlike uterine tissue estrogen agonist SERMs, the isoflavones do
not cause an estrogenic response upon binding to uterine cell ERs,
therefore, the isoflavones prevent, inhibit, or minimize the
uterotrophic effects caused by uterine endothelial cell ER/SERM
complexes. Preferably the isoflavone is co-administered with the
SERM to prevent or minimize uterotrophic effects in a weight/weight
ratio of isoflavone:SERM of about 0.25:1 to about 100:1, and more
preferably from about 0.5:1 to about 20:1.
[0047] In a particularly preferred embodiment of the method,
co-administration of the isoflavone with a uterine tissue estrogen
agonist SERM in an amount sufficient to prevent or minimize
uterotrophic effects is also effective to prevent or minimize the
development of endometrial cancer when the SERM is used to prevent
or treat breast cancer. As noted above, tamoxifen and uterine
tissue estrogen agonist SERMs cause an increased risk of the
development of endometrial cancer as a result of estrogen-like
activity in uterine tissue and its uterotrophic effects.
Co-administration of the isoflavone together with an uterine tissue
estrogen agonist SERM prevents or minimizes the development of
endometrial cancer by preventing or minimizing SERM induced
uterotrophic effects.
[0048] Compositions
[0049] In another aspect, the present invention is a composition
useful for preventing or minimizing the development or growth of
breast cancer. The composition includes combination of a selective
estrogen receptor modulator selected from at least one of
raloxifene, droloxifene, toremifene, 4-iodotamoxifen, and
idoxifene, and at least one isoflavone selected from genistein,
daidzein, biochanin A, formononetin, their respective naturally
occuring glucosides and glucoside conjugates. These SERM and
isoflavone materials necessary to form compositions in accordance
with the present invention may be obtained as described above. The
composition contains from about 1% to about 99% SERM, by weight of
biologically active ingredients, and from about 1% to about 99%
isoflavone, by weight of biologically active ingredients.
[0050] The SERM 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 co-administered with the
isoflavone. Preferably at least 0.5 mg of the SERM is present in
the composition, more preferably from about 0.5 mg to about 500 mg,
and most preferably from about 5 mg to about 100 mg. Most
preferably, the SERM is present in the composition in an amount
sufficient to prevent, minimize, or reverse the development or
growth of breast cancer by itself.
[0051] Preferably at least 1 mg of the isoflavone is present in the
composition, more preferably from about 1 mg to about 1000 mg, and
most preferably from about 10 mg to about 200 mg. In a preferred
embodiment the isoflavone is present in the composition in an
amount sufficient to augment the composition's SERM induced
prevention or minimization of development or growth of breast
cancer when the composition is administered to a woman. In a more
preferred embodiment, the isoflavone is present in the composition
in an amount sufficient to prevent, minimize, or reverse the
development or growth of breast cancer by itself.
[0052] In another preferred embodiment, the isoflavone is present
in the composition in an amount sufficient to prevent or minimize
the composition's SERM induced uterotrophic effects when the
composition is administered to a woman. The isoflavone should be
present in a ratio of isoflavone:SERM of from about 0.25:1 to about
100:1 by weight, and more preferably from about 0.5:1 to about 50:1
by weight, to be present in the composition in an amount sufficient
to prevent or minimize the composition's SERM induced uterotrophic
effects. In a most preferred embodiment, the isoflavone is present
in the composition in an amount sufficient to augment the
composition's SERM induced prevention or minimization of the
development or growth of breast cancer and to prevent or minimize
the composition's SERM induced uterotrophic effects when the
composition is administered to a woman.
[0053] A composition in accordance with the present invention
containing a SERM and an isoflavone can be prepared by conventional
procedures for blending and mixing compounds. Preferably, the
composition also includes an excipient, most preferably a
pharmacuetical excipient. Compositions containing an excipient and
incorporating the SERM and isoflavone can be prepared by procedures
known in the art. For example, the SERM 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.
[0054] 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.
[0055] The following non-limiting formulations illustrate
pharmaceutical compositions of the present invention.
FORMULATIONS
[0056] The following Formulations 1-4 illustrate pharmaceutical
formulations including a SERM and an isoflavone.
Formulation 1
Gelatin Capsules
[0057] Hard gelatin capsules are prepared using the following
ingredients: SERM 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
[0058] Tablets are prepared using the following ingredients: SERM
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
[0059] Suspensions are prepared using the following ingredients:
SERM 0.5-100 mg/5 ml; Isoflavone 0.1-1000 mg/5 ml; Sodium
carboxymethyl cellulose 50-700 mg/5 ml; Sodium benzoate 0-10 mg/5
ml; Purified water 5 ml; and flavor and color agents as needed.
Formulation 4
Parenteral Solutions
[0060] A parenteral composition is prepared by stirring 1.5% by
weight of active ingredients (SERM 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.
[0061] 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.
* * * * *