U.S. patent application number 12/221478 was filed with the patent office on 2009-03-19 for anti-neoplastic compositions comprising extracts of black cohosh.
This patent application is currently assigned to The Trustees of Columbia University in the City of New York. Invention is credited to Linda Saxe Einbond, I. Bernard Weinstein.
Application Number | 20090075919 12/221478 |
Document ID | / |
Family ID | 33567253 |
Filed Date | 2009-03-19 |
United States Patent
Application |
20090075919 |
Kind Code |
A1 |
Einbond; Linda Saxe ; et
al. |
March 19, 2009 |
Anti-neoplastic compositions comprising extracts of black
cohosh
Abstract
The present invention provides a composition for use in treating
or preventing neoplasia, comprising an effective actein. The
present invention also provides a composition for use in treating
or preventing neoplasia, comprising an effective anti-neoplastic
amount of an ethyl acetate extract of black cohosh. The present
invention further provides a combination of anti-neoplastic agents,
comprising an effective anti-neoplastic amount of an ethyl acetate
extract of black cohosh and an effective anti-neoplastic amount of
at least one additional chemopreventive or chemotherapeutic agent.
Methods for treating and preventing neoplasia are also
provided.
Inventors: |
Einbond; Linda Saxe;
(Crestwood, NY) ; Weinstein; I. Bernard; (New
York, NY) |
Correspondence
Address: |
BRYAN CAVE LLP
1290 Avenue of the Americas
New York
NY
10104
US
|
Assignee: |
The Trustees of Columbia University
in the City of New York
|
Family ID: |
33567253 |
Appl. No.: |
12/221478 |
Filed: |
August 4, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10746960 |
Dec 23, 2003 |
7407675 |
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12221478 |
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60437159 |
Dec 27, 2002 |
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Current U.S.
Class: |
514/33 ;
536/18.1 |
Current CPC
Class: |
A61K 31/704 20130101;
A61P 35/00 20180101; A61K 36/71 20130101; A61K 31/704 20130101;
A61K 36/71 20130101; A61K 45/06 20130101; A61P 35/02 20180101; A61K
2300/00 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/33 ;
536/18.1 |
International
Class: |
A61K 31/704 20060101
A61K031/704; C07H 15/24 20060101 C07H015/24; A61P 35/00 20060101
A61P035/00 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was made with government support under NIH
Grant No. 3P50 AT 00090-02S2. As such, the United States Government
has certain rights in this invention.
Claims
1. A composition for use in treating or preventing neoplasia,
comprising an effective anti-neoplastic amount of an ethyl acetate
extract of black cohosh.
2. The composition of claim 1, wherein the ethyl acetate extract
comprises at least one triterpene glycoside compound.
3. The composition of claim 2, wherein the triterpene glycoside
compound is selected from the group consisting of actein,
cimifugoside, cimigenol glycoside, cimiracemoside A, and
23-epi-26-deoxyactein.
4. The composition of claim 3, wherein the triterpene glycoside
compound is actein.
5. The composition of claim 4, wherein the effective
anti-neoplastic amount of actein is between about 0.5 .mu.g/ml and
about 40.0 .mu.g/ml.
6. The composition of claim 5, wherein the effective
anti-neoplastic amount of actein is between about 1.0 .mu.g/ml and
about 3.0 .mu.g/ml.
7. The composition of claim 1, wherein the ethyl acetate extract
comprises at least one aglycone.
8. The composition of claim 7, wherein the at least one aglycone is
cimigenol.
9. A composition of anti-neoplastic agents, comprising an effective
anti-neoplastic amount of an ethyl acetate extract of black cohosh
and an effective anti-neoplastic amount of at least one additional
chemopreventive or chemotherapeutic agent.
10. The composition of claim 9, which is a synergistic
combination.
11. The composition of claim 9, wherein the anti-neoplastic agents
are combined in a single formulation.
12. The composition of claim 9, wherein a separate, individual
formulation of the ethyl acetate extract of black cohosh is
combined with a separate, individual formulation of the at least
one additional chemopreventive or chemotherapeutic agent.
13. The composition of claim 9, wherein the ethyl acetate extract
comprises a triterpene glycoside compound.
14. The composition of claim 13, wherein the triterpene glycoside
compound is selected from the group consisting of actein,
cimifugoside, cimigenol glycoside, cimiracemoside A, and
23-epi-26-deoxyactein.
15. The composition of claim 14, wherein the triterpene glycoside
compound is actein.
16. The composition of claim 9, wherein the ethyl acetate extract
comprises at least one aglycone.
17. The composition of claim 16, wherein the at least one aglycone
is cimigenol.
18. The composition of claim 9, wherein the at least one additional
chemopreventive or chemotherapeutic agent is selected from the
group consisting of adriamycin, cisplatin, docetaxel, doxorubicin,
5-fluorouracil, herceptin, paclitaxel, tamoxifen, and
vinblastine.
19. The composition of claim 18, wherein the at least one
additional chemopreventive or chemotherapeutic agent is
paclitaxel.
20. The composition of claim 9, wherein the ethyl acetate extract
of black cohosh comprises actein and the at least one additional
chemopreventive or chemotherapeutic agent is paclitaxel.
21. The composition of claim 20, wherein the effective
anti-neoplastic amount of actein is between about 0.5 .mu.g/ml and
about 40.0 .mu.g/ml, and the effective anti-neoplastic amount of
paclitaxel is between about 0.5 nM and about 5.0 nM.
22. A composition for use in treating or preventing neoplasia,
comprising an effective anti-neoplastic amount of actein.
23. The composition of claim 22, wherein the neoplasia is a
carcinoma, a lymphocytic leukemia, a myeloid leukemia, a malignant
lymphoma, a malignant melanoma, a myeloproliferative disease, a
sarcoma, a brain tumor, a childhood tumor, or a mixed type of
neoplasia.
24. The composition of claim 22, further comprising an effective
anti-neoplastic amount of at least one additional chemopreventive
or chemotherapeutic agent.
25. A composition for use in treating or preventing disorders
caused by or related to the abnormality of at least one factor
selected from the group consisting of cyclin D1, cdk4, Her2,
I.kappa.B, I.kappa..kappa.B, NF-.kappa.B, p21, p27, PPAR.gamma.,
and ppRb, wherein the composition comprises an effective amount of
actein.
26. A pharmaceutical composition, comprising the composition of
claim 1, 9, 22, or 24 and a pharmaceutically acceptable
carrier.
27. A method for treating or preventing neoplasia in a subject,
comprising administering to the subject an amount of an ethyl
acetate extract of black cohosh effective to treat or prevent the
neoplasia.
28. The method of claim 27, wherein the neoplasia is a carcinoma, a
lymphocytic leukemia, a myeloid leukemia, a malignant lymphoma, a
malignant melanoma, a myeloproliferative disease, a sarcoma, a
brain tumor, a childhood tumor, or a mixed type of neoplasia.
29. The method of claim 28, wherein the carcinoma is breast cancer,
colon cancer, lung cancer, ovarian cancer, prostate cancer, bladder
cancer, uterine cancer, or skin cancer.
30. The method of claim 27, wherein the ethyl acetate extract
comprises at least one triterpene glycoside compound.
31. The method of claim 30, wherein the triterpene glycoside
compound is actein.
32. The method of claim 31, wherein the effective amount of actein
is between about 0.5 .mu.g/ml and about 40.0 .mu.g/ml.
33. The method of claim 27, wherein the ethyl acetate extract
comprises at least one aglycone.
34. The method of claim 33, wherein the at least one aglycone is
cimigenol.
35. A method for treating or preventing neoplasia in a subject,
comprising administering to the subject an amount of an ethyl
acetate extract of black cohosh effective to treat or prevent the
neoplasia, in combination with an amount of at least one additional
chemopreventive or chemotherapeutic agent effective to treat or
prevent the neoplasia.
36. The method of claim 35, wherein a synergistic anti-neoplastic
effect results.
37. The method of claim 35, wherein administration is
concurrent.
38. The method of claim 35, wherein administration is
sequential.
39. The method of claim 35, wherein administration is
alternate.
40. The method of claim 35, wherein the ethyl acetate extract
comprises actein.
41. The method of claim 35, wherein the at least one additional
chemopreventive or chemotherapeutic agent is selected from the
group consisting of cisplatin, docetaxel, doxorubicin,
5-fluorouracil, herceptin, paclitaxel, tamoxifen, and
vinblastine.
42. The method of claim 41, wherein the at least one additional
chemopreventive or chemotherapeutic agent is paclitaxel.
43. The method of claim 35, wherein the ethyl acetate extract of
black cohosh comprises actein and the at least one additional
chemopreventive or chemotherapeutic agent is paclitaxel.
44. The method of claim 33, wherein the effective amount of actein
is between about 0.5 .mu.g/ml and about 40.0 .mu.g/ml, and the
effective amount of paclitaxel is between about 0.5 nM and about
5.0 nM.
45. A method for treating or preventing neoplasia in a subject,
comprising administering to the subject an amount of actein
effective to treat or prevent the neoplasia.
46. The method of claim 45, wherein the neoplasia is a carcinoma, a
lymphocytic leukemia, a myeloid leukemia, a malignant lymphoma, a
malignant melanoma, a myeloproliferative disease, a sarcoma, a
brain tumor, a childhood tumor, or a mixed type of neoplasia.
47. The method of claim 46, wherein the carcinoma is breast cancer,
colon cancer, lung cancer, ovarian cancer, prostate cancer, bladder
cancer, uterine cancer, or skin cancer.
48. The method of claim 45, further comprising administering to the
subject an amount of at least one additional chemopreventive or
chemotherapeutic agent effective to treat or prevent the
neoplasia.
49. The method of claim 48, wherein the at least one additional
chemopreventive or chemotherapeutic agent is selected from the
group consisting of cisplatin, docetaxel, doxorubicin,
5-fluorouracil, herceptin, paclitaxel, tamoxifen, and
vinblastine.
50. A method for treating or preventing disorders in a subject
caused by or related to the abnormality of at least one factor
selected from the group consisting of cyclin D1, cdk4, Her2,
I.kappa.B, I.kappa..kappa.B, NF-.kappa.B, p21, p27, PPAR.gamma.,
and ppRb, comprising administering to the subject an amount of
actein effective to treat or prevent the disorder.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 60/437,159, filed on Dec. 27, 2002, and
entitled "ANTICANCER COMPOSITIONS OF EXTRACTS OF BLACK COHOSH", the
contents of which are hereby incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0003] Black cohosh, Actaea racemosa L. (Cimicifuga racemosa), a
perennial in the buttercup family (Ranunculaceae), is frequently
used to treat gynecological and other conditions. In particular,
the roots and rhizomes of black cohosh have been used to treat a
variety of disorders, including inflammatory conditions, diarrhea,
dysmenorrhea, and rheumatism; they have also been used to stimulate
menstrual flow and to suppress coughs (Foster, S., Black cohosh:
Cimicifuga racemosa. A literature review. HerbalGram, 45:35-49,
1999).
[0004] Additionally, black cohosh has been used as a natural
alternative to hormone-replacement therapy. In fact, American women
are increasingly turning to black cohosh as a "more natural"
alternative to estrogen, in the belief that it has the benefits,
without the risks, of estrogen-replacement therapy. To date, a
standardized black cohosh extract (Remifemin), developed in
Germany, has been studied, both in animals and in short-term
clinical trials of menopausal women. These studies suggest that the
extract alleviates a variety of menopausal symptoms, particularly
hot flashes (Lehmann-Willenbrock and Riedel, Clinical and
endocrinological examinations concerning therapy of climacteric
symptoms following hysterectomy with remaining ovaries. Zent. Bl.
Gynakol., 110:611-18, 1988; Stoll, W., Phytotherapy influences
atrophic vaginal epithelium: double-blind study--cimicifuga vs.
estrogenic substances. Therapeuticum, 1:23-31, 1987). Although most
studies report that black cohosh is free of significant
side-effects, these studies have not been carried out for a length
of time sufficient to ensure the safety of black cohosh with
respect to uterine function and/or the induction or stimulation of
breast cancer growth. Since the population using black cohosh
(i.e., middle-aged females in developed countries) is at a higher
risk for breast cancer, research is needed to clarify whether black
cohosh extracts stimulate or inhibit breast cancer cells. Such
studies could also identify new approaches to breast cancer
prevention and treatment.
[0005] The components of the black-cohosh rhizome have been
examined in several studies. It is known that the rhizome contains
triterpene glycosides, aromatic acids, cinnaminic acid esters,
sugars, tannins, and long-chain fatty acids (Zheng et al., CimiPure
(Cimicifuga racemosa): a standardized black cohosh extract with
novel triterpene glycoside for menopausal women. In Phytochem.
Phytopharm., Shahidi and Ho, eds. (Champaign, Ill.: AOCS Press,
2000) pp. 360-70). However, little is known about the mechanisms by
which these compounds are metabolized in vivo.
[0006] Crude extracts of black cohosh, and several components
present in black cohosh, have been shown to exhibit biological
activity. Fukinolic acid (2-E-caffeoylfukiic acid) exhibited weak
estrogenic activity on MCF7 cells (Kruse et al., Fukic and piscidic
acid esters from the rhizome of Cimicifuga racemosa and the in
vitro estrogenic activity of fukinolic acid. Planta. Med.,
65:763-64, 1999); it also inhibited the activity of neutrophil
elastase, which is involved on the inflammatory process (Loser et
al., Inhibition of neutrophil elastase activity by cinnamic acid
derivatives from Cimicifuga racemosa. Planta. Med., 66:751-53,
2000). Bioactivity-guided fractionation of the methanolic extract
resulted in the isolation of nine antioxidant compounds. Of these,
methyl caffeate was the most active in reducing menadione-induced
DNA damage in cultured S30 breast cancer cells (Burdette et al.,
Black cohosh (Cimicifuga racemosa L.) protects against
menadione-induced DNA damage through scavenging of reactive oxygen
species: bioassay-directed isolation and characterization of active
principles. J. Agric. Food Chem., 50:7022-28, 2002). None of the
compounds was cytotoxic to S30 cells (Burdette et al., Black cohosh
(Cimicifuga racemosa L.) protects against menadione-induced DNA
damage through scavenging of reactive oxygen species:
bioassay-directed isolation and characterization of active
principles. J. Agric. Food Chem., 50:7022-28, 2002).
[0007] Extracts and components purified from black cohosh have also
been shown to exhibit anti-cancer activity, in vitro and in vivo.
Extracts of black cohosh (ethanol extract, 0.1% v/v) inhibited the
growth of serum-stimulated T-47D breast cancer cells (Dixon-Shanies
and Shaikh, Groxyth inhibition of human breast cancer cells by
herbs and phytoestrogens. Oncol. Rep., 6:1383-87, 1999), and, at
doses starting at 2.5 .mu.g/ml, inhibited the proliferation of the
mammary carcinoma cell line, 435 (Nesselhut et al., Studies on
mammary carcinoma cells regarding the proliferation potential of
herbal medication with estrogen-like effects. Archives of
Gynecology and Obstetrics, 254:817-18, 1993). Furthermore,
isopropanolic extracts of black cohosh inhibited estrogen-induced
proliferation of MCF7 cells, and enhanced the inhibitory effect of
tamoxifen (Bodinet and Freudenstein, Influence of Cimicifuga
racemosa on the proliferation of estrogen receptor-positive human
breast cancer cells. Breast Cancer Research and Treatment, 76:1-10,
2002).
[0008] More recently, it has been shown that cycloartane glycosides
isolated from black cohosh inhibit the growth of human oral
squamous cell carcinoma cells (Watanabe et al., Cycloartane
glycosides from the rhizomes of Cimicifuga racemosa and their
cytotoxic activities. Chem. Pharm. Bull., 50:121-25, 2002).
Additionally, recent studies by Sakurai et al. have indicated that
triterpene glycosides and aglycones--the most active of which is
cimigenol--inhibit Epstein-Barr virus early antigen activation
(induced by 12-O-tetradecanoylphorbol-13-acetate) in Raji cells
(Sakurai et al., Antitumor agents 220. Antitumor-promoting effects
of cimigenol and related compounds on Epstein-Barr virus activation
and two-stage mouse skin carcinogenesis. Bioorg. Med. Chem.
11:1137-40, 2003). Cimigenol has also been shown to inhibit mouse
skin tumor promotion using DMBA as an initiator and TPA as a
promoter.
[0009] All of the foregoing studies, however, have been limited in
scope, and have not addressed issues of specificity and mechanism
of action.
SUMMARY OF THE INVENTION
[0010] The invention disclosed herein generally relates to the
effects of extracts of black cohosh on the growth and progression
of the cell cycle, and on the expression of proteins involved in
cell-cycle control in cancer-cell lines. More particularly, the
present invention relates to the effects of actein and
triterpene-glycoside extracts of black cohosh on neoplastic
cells--when used alone or in combination with a chemopreventive or
chemotherapeutic agent.
[0011] Accordingly, in one aspect, the present invention provides a
composition for use in treating or preventing neoplasia, comprising
an effective anti-neoplastic amount of an ethyl acetate extract of
black cohosh.
[0012] In another aspect, the present invention provides a
combination of anti-neoplastic agents, comprising an effective
anti-neoplastic amount of an ethyl acetate extract of black cohosh
and an effective anti-neoplastic amount of at least one additional
chemopreventive or chemotherapeutic agent. In one embodiment of the
invention, the combination is a synergistic combination.
[0013] In a further aspect, the present invention provides a
composition for use in treating or preventing neoplasia, comprising
an effective anti-neoplastic amount of actein. In one embodiment,
the composition further comprises an effective anti-neoplastic
amount of at least one additional chemopreventive or
chemotherapeutic agent.
[0014] In yet another aspect, the present invention provides a
method for treating or preventing neoplasia in a subject, by
administering to the subject an amount of an ethyl acetate extract
of black cohosh effective to treat or prevent the neoplasia.
[0015] In still another aspect, the present invention provides a
method for treating or preventing neoplasia in a subject, by
administering to the subject an amount of an ethyl acetate extract
of black cohosh effective to treat or prevent the neoplasia, in
combination with an amount of at least one additional
chemopreventive or chemotherapeutic agent effective to treat or
prevent the neoplasia. In one embodiment of the invention, a
synergistic anti-neoplastic effect results.
[0016] Furthermore, the present invention provides a method for
treating or preventing neoplasia in a subject, comprising
administering to the subject an amount of actein effective to treat
or prevent the neoplasia. In one embodiment, the method further
comprises administering to the subject an amount of at least one
additional chemopreventive or chemotherapeutic agent effective to
treat or prevent the neoplasia.
[0017] Additional aspects of the present invention will be apparent
in view of the description that follows.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 is an illustration of the methods of the invention
which were used to fractionate black cohosh.
[0019] FIGS. 2A and 2B illustrate the effect of black cohosh
extracts on the growth of MCF7 cells. FIG. 2A shows the effect of
the ethyl acetate extract when MCF7 cells were treated with the
indicated concentrations of the ethyl acetate fraction for
increasing times. FIG. 2B shows the effect of actein when MCF7
cells were treated with the indicated concentrations of actein for
increasing times. In each case, the number of viable cells was
determined using a Coulter Counter, and the control contained 0.08%
DMSO. Bars=SD
[0020] FIG. 3 shows the structures of the triterpene glycoside
compounds of the invention.
[0021] FIG. 4 depicts the effect of actein and the effect of the
ethyl acetate fraction of black cohosh on MCF7 cell-cycle
distribution at 48 h. MCF7 cells were treated with 0, 30, and 60
.mu.g/ml of the ethyl acetate extract, or actein, and then analyzed
at 48 h by DNA flow cytometry. The values indicate the percentage
of cells in the indicated phases of the cell cycle.
[0022] FIG. 5 illustrates the effect of actein on the G1 phase of
the cell cycle in MCF7 cells. MCF7 cells were treated with 10 (14.8
.mu.M), 20, or 40 .mu.g/ml actein, and then analyzed at 24 and 48 h
by DNA flow cytometry. The values indicate the percentage of cells
in the G1 phase of the cell cycle.
[0023] FIGS. 6A-6F show Western-blot analyses of MCF7 cells treated
with actein. The cells were treated with 0, 20, or 40 .mu.g/ml
actein. 20 .mu.g/ml actein is equivalent to 29.6 .mu.M actein.
After 3, 10, and 24 h, extracts were analyzed by Western blotting
with antibodies to: cyclin D1 (FIG. 6A); ppRb (FIG. 6B); cdk4 (FIG.
6C); p21.sup.cip1 (FIG. 6D); EGFR (FIG. 6E); and phospho-EGFR (FIG.
6F). An antibody for .beta.-actin was used as a loading
control.
[0024] FIG. 7 shows the effects of actein alone, and in combination
with paclitaxel, on cell proliferation in MDA-MB-453 (Her2
overexpressing) human breast cancer cells. MDA-MB-453 cells were
treated with all combinations of 3 concentrations of actein and 3
concentrations of paclitaxel, and the solvent control, for 96 h.
The number of viable cells was determined using a Coulter Counter.
Similar results were obtained in two additional studies. The
control contained 0.044% DMSO. bars=SD
[0025] FIG. 8 illustrates a Western-blot analysis of extracts
obtained from MDA-MB-453 cells treated with actein. The cells were
treated with 0, 20, or 40 .mu.g/ml of actein. After 3 and 24 h,
extracts were prepared and analyzed by Western blotting with an
antibody to Her2 or an antibody to phospho-Her2 (p-Her2). An
antibody to .beta.-actin was used as a loading control. The
staining intensities of the visualized blots were quantified using
NIH image software. For each protein, the relative band intensities
were determined by comparing treated samples with untreated
controls. These values were then normalized (fold), using
.beta.-actin as an internal control.
[0026] FIG. 9 presents a reporter promoter analysis of extracts
obtained from MDA-MB-453 cells treated with actein. Using
lipofectin, triplicate samples of MDA-MB-453 breast cancer cells
were co-transfected with DNA of the indicated reporter plasmid,
using .beta.-gal DNA as an internal control. The cells were then
treated with actein at 0, 20, and 40 .mu.g/ml, in quadruplicate.
Luciferase and .beta.-gal activities were determined, as previously
described (Masuda et al., Effects of epigallocatechin-3-gallate on
growth, epidermal growth factor receptor signaling pathways, gene
expression, and chemosensitivity in human head and neck squamous
cell carcinoma cell lines. Clinical Cancer Research, 7:4220-29,
2001). Luciferase activities were normalized to .beta.-gal
activities. left panel=cyclin D1; right panel=nuclear factor kappa
B (NF-.kappa.B); bars=SD
[0027] FIG. 10 sets forth the effects of the aglycone cimigenol and
the triterpene glycosides cimigenol glycoside, and actein, purified
from black cohosh, on cell proliferation in MDA-MB-453 cells.
MDA-MB-453 cells were exposed to increasing concentrations of the
indicated purified components for 96 h, and the number of viable
cells was determined using a Coulter Counter.
[0028] FIG. 11 shows the effects of butanol fractions from black
cohosh on cell proliferation in MCF7 cells. MCF7 cells were exposed
to increasing concentrations of the indicated purified components,
for 26 or 96 h, and the number of viable cells was determined using
a Coulter Counter.
[0029] FIG. 12 demonstrates the effects of the components ferulic
and isoferulic acid, purified from black cohosh, on cell
proliferation in MCF7 cells. MCF7 cells were exposed to increasing
concentrations of the indicated purified components for 96 hrs, and
the number of viable cells was determined using a Coulter
Counter.
[0030] FIG. 13 illustrates the effects of actein on cyclin D1 mRNA
in MCF7 cells (RT-PCR). MCF7 cells were treated with DMSO or actein
for 3, 10, or 24 h. RNA was isolated and analyzed by RT-PCR, using
primers for cyclin D1 and actin (control). The staining intensities
of the visualized blots were quantified using NIH image software.
The relative band intensities were determined by comparing treated
samples with untreated controls. These values were then normalized
(fold), using .beta.-actin as an internal control.
[0031] FIG. 14 demonstrates the effects of actein on cyclin D1 mRNA
in MDA-MB-453 cells (RT-PCR). MDA-MB-453 cells were treated with
DMSO or actein for 3, 10, or 24 h. RNA was isolated and analyzed by
RT-PCR, using primers for cyclin D1 and actin (control). The
staining intensities of the visualized blots were quantified using
NIH image software. The relative band intensities were determined
by comparing treated samples with untreated controls. These values
were then normalized (fold), using .beta.-actin as an internal
control.
[0032] FIG. 15 illustrates MCF7 cells treated with 0, 20, or 40
.mu.g/ml actein. 20 .mu.g/ml actein is equivalent to 29.6 .mu.M.
After 3, 10, and 24 h, extracts were analyzed by Western blotting
with an antibody to p21. An antibody for .beta.-actin was used as a
loading control.
[0033] FIG. 16 shows the effects of actein on p21 mRNA in
MDA-MB-453 cells (RT-PCR). MDA-MB-453 cells were treated with DMSO
or actein for 3, 10, or 24 h. RNA was isolated and analyzed by
RT-PCR, using primers for cyclin D1 and actin (control). The
staining intensities of the visualized blots were quantified using
NIH image software. The relative band intensities were determined
by comparing treated samples with untreated controls. These values
were then normalized (fold), using .beta.-actin as an internal
control.
[0034] FIG. 17 illustrates MDA-MB-453 cells that were treated with
0, 20, or 40 .mu.g/ml actein. 20 .mu.g/ml actein is equivalent to
29.6 .mu.M. After 3, 10, and 24 h, extracts were analyzed by
Western blotting, with an antibody to ik.beta.. An antibody for
.beta.-actin was used as a loading control.
[0035] FIG. 18 illustrates MDA-MB-453 cells that were treated with
0, 20, or 40 .mu.g/ml actein (20 .mu.g/ml actein is equivalent to
29.6 .mu.M). After 3 and 24 h, extracts were analyzed by Western
blotting, with an antibody to PPAR.gamma.. An antibody for
.beta.-actin was used as a loading control.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The Examples below provide the first detailed examination of
the effects on human breast cancer cells of extracts and purified
compounds present in black cohosh. In these studies, the roots and
rhizomes of black cohosh were extracted with MeOH/H.sub.2O, and
fractionated by solvent-solvent partitioning to yield three
fractions: hexane, ethyl acetate (EtOAc), and H.sub.2O. The EtOAc
fraction exhibited the greatest growth-inhibitory activity. This
fraction inhibited growth of both the ER.sup.+MCF7 and
ER.sup.-/Her2+ MDA-MB-453 human breast cancer cell lines, with
IC.sub.50 values of about 18 .mu.g/ml and 10 .mu.g/ml,
respectively. The normal human mammary epithelial cell line,
MCF10F, was much less sensitive to growth inhibition by this
extract (with an IC.sub.50 value of 46 .mu.g/ml). It is possible
that the greater sensitivity of the malignant cells may reflect, in
part, the difference in growth rates of the malignant and
non-malignant cells.
[0037] The inventors tested the effects of crude extracts, methanol
and ethanol, as well as ethanol extracts provided by Pure World,
native and plus expedient: the IC.sub.50 values for these extracts
after 96 hours of treatment were: methanol: 100 .mu.g/ml; ethanol:
>200 .mu.g/ml; Pure World native: 175 .mu.g/ml; and Pure World
plus expedient: 195 .mu.g/ml. To partition the phytochemicals
according to polarity, the water portion was also partitioned
sequentially with hexane and n-butanol (n-BuOH). The n-BuOH
fraction was tested for its effect on the growth of MDA-MB-453
breast cancer cells. The IC50 value after 96 hours of treatment
was: 40 .mu.g/ml.
[0038] The inventors also examined the effects of the EtOAc
fraction of black cohosh on SW480 human colon cancer cells. The
IC.sub.50 values after 48 hours of incubation using the MTT assay
were: SW480: 42 .mu.g/ml; MCF7: 38 .mu.g/ml (Luo et al., PM-3, a
benzo-g-pyran derivative isolated from propolis, inhibits growth of
MCF-7 human breast cancer cells. Anticancer Res 21: 1665-1672,
2001).
[0039] The inventors further demonstrated that the EtOAc fraction
of black cohosh induced cell-cycle arrest in MCF7 human breast
cancer cells at G1 at 30 .mu.g/ml, and at G2/M at 60 .mu.g/ml. The
triterpene glycoside fraction that was obtained by polyamide column
chromatography, and the specific triterpene glycosides (actein,
23-epi-26-deoxyactein, and cimiracemoside A), inhibited growth of
MCF7 human breast cancer cells and induced cell-cycle arrest at G1.
At 60 .mu.g/ml, actein induced a less-pronounced G1 arrest.
Therefore, it is likely that, at high concentrations, actein and
related compounds affect proteins that regulate later phases in the
cell cycle.
[0040] Because the triterpene glycosides induced cell-cycle arrest
at G1, the inventors decided to ascertain the effect of the most
potent compound, actein, on cell-cycle proteins that control G1
cell-cycle progression. As discussed below, actein decreased the
level of cyclin D1, cdk4, and the hyperphosphorylated form of pRb,
and increased the level of the cdk inhibitory protein,
p21.sup.cip1, in MCF7 cells--changes that may contribute to the
arrest in G1. The inventors also found that actein reduced the
level of cyclin D1 mRNA within 3 h of treatment, and significantly
reduced the level at 24 h, suggesting an effect at the level of
transcription. The level of the EGFR was not altered after
treatment with actein; nor was there a consistent effect on the
level of the phosphorylated form of the EGFR (p-EGFR), which
reflects its state of activation. Thus, the EGFR did not appear to
be a direct target for actein. This result is in contrast to the
effect of another plant-derived compound--a fiavonol,
epigallocatechin-gallate--which is the active component in green
tea (Masuda et al., Effects of epigallocatechin-3-gallate on
growth, epidermal growth factor receptor signaling pathways, gene
expression, and chemosensitivity in human head and neck squamous
cell carcinoma cell lines. Clinical Cancer Research, 7:4220-29,
2001). Previous studies have also indicated that micromolar
concentrations of the aglycone compounds, cyanidin and delphinidin,
inhibited activation of the EGFR and cell proliferation in the
human vulva carcinoma cell line, A431, whereas the corresponding
glycosides had a minimal effect (Meiers et al., The anthocyanidins
cyanidin and delphinidin are potent inhibitors of the epidermal
growth-factor receptor. J. Agric. Food Chem., 49:958-62, 2001). The
inventors tested the effects of the aglycone cimigenol on the
growth of human breast cancer cells. Cimigenol was less active than
cimigenol glycoside.
[0041] Triterpene molecules are structurally related to steroids,
and have been present in the plant kingdom for millions of years.
Some may have evolved to become ligands for receptors on animal
cells (Sporn and Suh, Chemoprevention of cancer. Carcinogenesis,
21:525-30, 2000). However, the mode of action triterpene glycosides
is not well understood. Studies by Haridas et al. (Avicins:
triterpenoid saponins from Acacia victoriae (Bentham) induce
apoptosis by mitochondrial perturbation. Proc. Natl. Acad. Sci.
USA, 98:5821-26, 2001) indicate that avicins--triterpenoid saponins
from the plant Acacia victoriae (Bentham)--are potent inhibitors of
the transcription factor, nuclear factor kappa B (NF-.kappa.B), and
act by inhibiting its translocation to the nucleus and its capacity
to bind DNA--perhaps by altering sulfhydryl groups critical for
NF-.kappa.B activation. Betulinic acid, a pentacyclic triterpene
present in the bark of white birch trees, is a selective inhibitor
of human melanoma (Pisha et al., Discovery of betulinic acid as a
selective inhibitor of human melanoma that functions by induction
of apoptosis. Nat. Med, 1: 1046-51, 1995). It induces apoptosis in
neuroectodermal tumors by a direct effect on mitochondria (Fulda
and Debatin, Betulinic acid induces apoptosis through a direct
effect on mitochondria in neuroectodermal tumors. Med. Pediatr.
Oncol., 35:616-18, 2000).
[0042] Suh et al. (Novel triterpenoids suppress inducible nitric
oxide synthase (iNOS) and inducible cyclooxygenase (COX-2) in mouse
macrophages. Cancer Res., 58:717-23, 1998) have generated a series
of derivatives of the triterpenes, oleanic and ursolic acids, that
are highly potent in suppressing the expression of inducible nitric
oxide synthase and cyclooxygenase-2 in primary mouse macrophages.
Indeed, the derivative, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic
acid (CDDO), is 1000 times more potent than oleanic acid in this
cell system (Spom and Suh, Chemoprevention of cancer.
Carcinogenesis, 21:525-30, 2000). Suh et al. also found that CDDO
displays potent differentiating, anti-proliferative, and
anti-inflammatory activities (Suh et al., A novel synthetic
oleanane triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic
acid, with potent differentiating, antiproliferative, and
anti-inflammatory activity. Cancer Res., 59:336-41, 1999).
[0043] CDDO further induces apoptosis by a caspase-8-dependent
mechanism (Ito et al., The novel triterpenoid CDDO induces
apoptosis and differentiation of human osteosarcoma cells by a
caspase-8 dependent mechanism. Mol. Pharmacol., 5:1094-99, 2001;
Pedersen et al., The triterpenoid CDDO induces apoptosis in
refractory CLL B cells. Blood, 8:2965-72, 2002), and inhibits
NF-.kappa.B-mediated gene expression, following translocation of
the activated form to the nucleus (Stadheim et al., The novel
triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)
potently enhances apoptosis induced by tumor necrosis factor in
human leukemia cells. J. Biol. Chem., 19:16448-55,2002). It is a
ligand for the peroxisome proliferator activated receptor-.gamma.
(PPAR-.gamma.) (Wang et al., A synthetic triterpenoid,
2-cyano-2,12-dioxooleana-1,9-dien-28-oic acid (CDDO), is a ligand
for the peroxisome proliferator-activated receptor gamma. Mol.
Endocrinol., 14:1550-56, 2000), but the specific cellular target of
CDDO and related compounds, for mediating the above biologic
effects, is not known.
[0044] The triterpene glycoside, actein, and the fraction of black
cohosh enriched for triterpene glycosides (which are selective for
human breast cancer versus normal mammary epithelial cells),
synergize with several classes of chemotherapy agents. For example,
the inventors have demonstrated that actein has synergy with the
taxane, paclitaxel; the antimetabolite, 5-fluorouracil (5-FU); the
Her2 antibody, herceptin; the anthracycline antibiotic,
doxorubicin; and the platinum analog, cisplatin. Additionally, the
inventors have shown that black cohosh extracts have synergy with
paclitaxel and doxorubicin. Because it is easier to prepare
enriched extracts, the extracts of black cohosh might represent the
preferred sources to be used in combination with such
chemotherapeutic agents.
[0045] In view of the foregoing, the present invention provides
methods for treating and preventing neoplasia in a subject. The
subject is preferably a mammal (e.g., humans, domestic animals, and
commercial animals, including cows, dogs, monkeys, mice, pigs, and
rats). More preferably, the subject is a human.
[0046] As used herein, "neoplasia" refers to the uncontrolled and
progressive multiplication of cells under conditions that would not
elicit, or would otherwise cause cessation of, the multiplication
of normal or non-neoplastic cells. Neoplasia results in the
formation of a neoplasm, which is any new and abnormal growth,
particularly a new growth of tissue, in which the growth is
uncontrolled and progressive. Malignant neoplasms are distinguished
from benign in that the former show a greater degree of anaplasia,
or loss of differentiation and orientation of cells, and have the
properties of invasion and metastasis. Thus, neoplasia includes
"cancer", which refers herein to a proliferation of cells having
the unique trait of loss of normal controls, resulting in
unregulated growth, lack of differentiation, local tissue invasion,
and metastasis (Beers and Berkow, eds., The Merck Manual of
Diagnosis and Therapy, 17.sup.th ed. (Whitehouse Station, N.J.:
Merck Research Laboratories, 1999) 973-74, 976, 986, 988, 991).
[0047] Neoplasias which may be treated and/or prevented by the
methods of the present invention include, without limitation,
carcinomas, particularly those of the bladder, breast, cervix,
colon, head, kidney, lung, neck, ovary, prostate, and stomach;
lymphocytic leukemias, particularly acute lymphoblastic leukemia
and chronic lymphocytic leukemia; myeloid leukemias, particularly
acute monocytic leukemia, acute promyelocytic leukemia, and chronic
myelocytic leukemia; malignant lymphomas, particularly Burkitt's
lymphoma and Non-Hodgkin's lymphoma; malignant melanomas;
myeloproliferative diseases; sarcomas, particularly Ewing's
sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma, peripheral
neuroepithelioma, and synovial sarcoma; and mixed types of
neoplasias, particularly carcinosarcoma and Hodgkin's disease.
Preferably, the methods of the present invention are used to treat
or prevent breast cancer, colon cancer, leukemia, lung cancer,
malignant melanoma, ovarian cancer, or prostate cancer. More
preferably, the cancer is breast cancer.
[0048] The method of the present invention comprises administering
to the subject an ethyl acetate extract of black cohosh or a
composition comprising actein. It is well known that black cohosh
is a medicinal plant from the genus Cimicifuga (or Actea) and the
species racemosa. The ethyl acetate extract of black cohosh is a
partially-purified extract, enriched for triterprene glycosides. It
is a safe and effective extract, with few side effects. The ethyl
acetate extract of black cohosh may be prepared in any suitable
manner that maintains or enriches the triterpene glycosides
component in the extract. By way of example, the method of
extraction may comprise a first extraction of the rhizome of black
cohosh, with an aqueous solution of a lower alkyl alcohol, followed
by partitioning of the aqueous alcohol layer with a lower alkyl
acetate. A preferred lower alkyl alcohol is methanol, and a
preferred lower alkyl acetate is ethyl acetate. The resultant
extract comprises triterpene glycoside compounds and cinnamic acid
esters. Of interest to the invention are the triterpene glycosides,
which can be separated from the cinnamic acid esters by
purification of the ethyl acetate extract. Such triterpene
glycosides include, without limitation, actein, cimifugoside,
cimigenol glycoside, cimiracemoside A, 23-epi-26-deoxyactein and
the aglycone cimigenol. Preferably, the triterpene glycoside
compound is actein.
[0049] The individual triterpenoid components in the ethyl acetate
extract of black cohosh can be individually separated by
purification. The ethyl acetate extract may be maintained in any
form, provided that the activity of the triterpene glycosides, and
of each component therein, is maintained. Activity of the
triterpene glycosides may be assayed by reference to the Examples
presented below. Furthermore, actein and related triterprene
glycosides may be modified to increase their activity, while
retaining their selectivity for neoplastic cells.
[0050] In accordance with the method of the present invention, the
ethyl acetate extract of black cohosh may be administered to the
subject in an anti-neoplastic amount, which is an amount that is
effective to treat or prevent neoplasia in the subject. As used
herein, "anti-neoplastic" includes the ability to inhibit or
prevent the development or spread of a neoplasm, and the ability to
limit, suspend, terminate, or otherwise control the development,
maturation, and proliferation of cells in a neoplasm. As further
used herein, an amount of the ethyl acetate extract of black cohosh
that is "effective to treat or prevent the neoplasia" is an amount
that is effective to ameliorate or minimize the clinical impairment
or symptoms of the neoplasia, or to inhibit their development. For
example, the clinical impairment or symptoms of the neoplasia may
be ameliorated or minimized by diminishing any pain or discomfort
suffered by the subject; by extending the survival of the subject
beyond that which would otherwise be expected in the absence of
such treatment; by inhibiting or preventing the development or
spread of the neoplasm; or by limiting, suspending, terminating, or
otherwise controlling the development, maturation, and
proliferation of cells in the neoplasm.
[0051] Exemplary doses of actein, administered intraperitoneally,
may be between about 0.5 .mu.g/ml and about 40.0 .mu.g/ml, and
preferably, between about 1 .mu.g/ml and about 3.0 .mu.g/ml.
However, the amount of actein effective to treat or prevent
neoplasia or other disorders in a subject will vary depending on
the particular factors of each case, including the target molecule,
the type of neoplasia, the stage of neoplasia, the subject's
weight, the severity of the subject's condition, and the method of
administration. These amounts can be readily determined by the
skilled artisan, based upon known procedures, including analysis of
titration curves established in vivo, dose-response experiments
analogous to those provided in the Examples, and methods and assays
disclosed herein.
[0052] The ethyl acetate extract of black cohosh or the actein
composition may be administered to a human or animal subject by
known procedures, including, without limitation, oral
administration, parenteral administration, transdermal
administration, and by way of catheter. Preferably, the ethyl
acetate extract of black cohosh or the actein composition is
administered parenterally, by epifascial, intracapsular,
intracranial, intracutaneous, intrathecal, intramuscular,
intraorbital, intraperitoneal, intraspinal, intrasternal,
intravascular, intravenous, parenchymatous, subcutaneous, or
sublingual injection.
[0053] For oral administration, a formulation comprising the ethyl
acetate extract of black cohosh or the actein composition may be
presented as capsules, tablets, powders, granules, or as a
suspension. The formulation may have conventional additives, such
as lactose, mannitol, corn starch, or potato starch. The
formulation also may be presented with binders, such as crystalline
cellulose, cellulose derivatives, acacia, corn starch, and
gelatins. Additionally, the formulation may be presented with
disintegrators, such as corn starch, potato starch, and sodium
carboxymethylcellulose. The formulation also may be presented with
dibasic calcium phosphate anhydrous or sodium starch glycolate.
Finally, the formulation may be presented with lubricants, such as
talc and magnesium stearate.
[0054] For parenteral administration (i.e., administration by
injection through a route other than the alimentary canal), the
ethyl acetate extract of black cohosh or the actein composition may
be combined with a sterile aqueous solution that is preferably
isotonic with the blood of the subject. Such a formulation may be
prepared by dissolving a solid active ingredient in water
containing physiologically-compatible substances, such as sodium
chloride, glycine, and the like, and having a buffered pH
compatible with physiological conditions, so as to produce an
aqueous solution, then rendering said solution sterile. The
formulation may be presented in unit or multi-dose containers, such
as sealed ampoules or vials. The formulation may be delivered by
any mode of injection, including, without limitation, epifascial,
intracapsular, intracranial, intracutaneous, intrathecal,
intramuscular, intraorbital, intraperitoneal, intraspinal,
intrasternal, intravascular, intravenous, parenchymatous,
subcutaneous, and sublingual.
[0055] For transdermal administration, the ethyl acetate extract of
black cohosh or the actein composition may be combined with skin
penetration enhancers, such as propylene glycol, polyethylene
glycol, isopropanol, ethanol, oleic acid, N-methylpyrrolidone, and
the like, which increase the permeability of the skin to the ethyl
acetate extract of black cohosh, and permit the ethyl acetate
extract of black cohosh to penetrate through the skin and into the
bloodstream. The ethyl acetate extract of black cohosh, or the
actein composition, /enhancer composition also may be further
combined with a polymeric substance, such as ethylcellulose,
hydroxypropyl cellulose, ethylene/vinylacetate, polyvinyl
pyrrolidone, and the like, to provide the composition in gel form,
which may be dissolved in a solvent, such as methylene chloride,
evaporated to the desired viscosity, and then applied to backing
material to provide a patch.
[0056] In accordance with the method of the present invention, the
ethyl acetate extract of black cohosh or the actein composition
also may be administered to a subject by way of a pharmaceutical
composition for use in treating or preventing neoplasia. The
pharmaceutical composition of the present invention comprises an
effective anti-neoplastic amount of the ethyl acetate extract of
black cohosh or an effective amount of the actein composition and a
pharmaceutically-acceptable carrier. The
pharmaceutically-acceptable carrier must be "acceptable" in the
sense of being compatible with the other ingredients of the
composition, and not deleterious to the recipient thereof. The
pharmaceutically-acceptable carrier employed herein is selected
from various organic or inorganic materials that are used as
materials for pharmaceutical formulations, and which may be
incorporated as analgesic agents, buffers, binders, disintegrants,
diluents, emulsifiers, excipients, extenders, glidants,
solubilizers, stabilizers, suspending agents, tonicity agents,
vehicles, and viscosity-increasing agents. If necessary,
pharmaceutical additives, such as antioxidants, aromatics,
colorants, flavor-improving agents, preservatives, and sweeteners,
may also be added. Examples of acceptable pharmaceutical carriers
include carboxymethyl cellulose, crystalline cellulose, glycerin,
gum arabic, lactose, magnesium stearate, methyl cellulose, powders,
saline, sodium alginate, sucrose, starch, talc, and water, among
others.
[0057] In the pharmaceutical composition of the present invention,
the ethyl acetate extract of black cohosh is provided in an
effective anti-neoplastic amount. For example, where the ethyl
acetate extract comprises actein, the actein may be present in an
amount between about 0.5 .mu.g/ml and about 40.0 .mu.g/ml.
Preferably, the actein is present in an amount between about 1.0
.mu.g/ml and about 3.0 .mu.g/ml.
[0058] The pharmaceutical composition of the present invention may
be prepared by methods well-known in the pharmaceutical arts.
Actein may be obtained from plant extracts or by chemical
synthesis. For example, the ethyl acetate extract of black cohosh
may be brought into association with a carrier or diluent, as a
suspension or solution. Optionally, one or more accessory
ingredients (e.g., buffers, flavoring agents, surface active
agents, and the like) also may be added. The choice of carrier will
depend upon the route of administration.
[0059] Since multiple genetic and epigenetic targets are altered in
the process of carcinogenesis, combination chemoprevention and
chemotherapy are generally optimal. Accordingly, the present
invention further provides a method for treating or preventing
neoplasia in a subject, by administering to the subject an amount
of an ethyl acetate extract of black cohosh or the actein
composition, as described above, in combination with an amount of
at least one additional chemopreventive or chemotherapeutic agent
effective to treat or prevent the neoplasia. As used herein, the
term "effective" also covers the dosages at which the
chemopreventive or chemotherapeutic agent by itself does not have
any significant effect on neoplasia but may significantly promote
or enhance the anti-neoplastic effects of the ethyl acetate extract
of black cohosh, and vice-versa.
[0060] Examples of additional chemopreventive or chemotherapeutic
agents for use in the method of the present invention include,
without limitation cisplatin, docetaxel, doxorubicin,
5-fluorouracil (5-FU), herceptin, paclitaxel, tamoxifen, and
vinblastine, and any fragments, analogues, and derivatives thereof.
In a preferred embodiment, the chemopreventive or chemotherapeutic
agent is paclitaxel. Ethyl acetate extracts of black cohosh, and
additional chemopreventive or chemotherapeutic agents, are referred
to herein as "anti-neoplastic agents."
[0061] By way of example, the term "paclitaxel" includes a natural
or synthetic functional variant of paclitaxel which has paclitaxel
biological activity, as well as a fragment of paclitaxel having
paclitaxel biological activity.
[0062] As used herein, the term "paclitaxel biological activity"
refers to paclitaxel activity which interferes with cellular
mitosis by affecting microtubule formation and/or action, thereby
producing antimitotic and anti-neoplastic effects. Methods of
preparing paclitaxel and its analogues and derivatives are
well-known in the art, and are described, for example, in U.S. Pat.
Nos. 5,569,729; 5,565,478; 5,530,020; 5,527,924; 5,484,809;
5,475,120; 5,440,057; and 5,296,506. Paclitaxel and its analogues
and derivatives are also available commercially. For example,
synthetic paclitaxel can be obtained from Bristol-Myers Squibb
Company, Oncology Division (Princeton, N.J.), under the registered
trademark Taxol.TM.. Moreover, paclitaxel may be synthesized in
accordance with known organic chemistry procedures that are readily
understood by one skilled in the art. Taxol for injection may be
obtained in a single-dose vial, having a concentration of 30 mg/5
ml (6 mg/ml per 5 ml) (Physicians' Desk Reference, 54.sup.th ed.
(Montvale, N.J.: Medical Economics Company, Inc., 2000) 307,
682).
[0063] Paclitaxel and its analogues and derivatives have been used
successfully to treat leukemias and tumors. In particular,
paclitaxel is useful in the treatment of breast, lung, and ovarian
cancers. Since paclitaxel is frequently utilized in the treatment
of human cancers, a strategy to enhance its utility in the clinical
setting, by combining its administration with that of an ethyl
acetate extract of black cohosh, may be of great benefit to many
subjects suffering from malignant neoplasias, particularly advanced
cancers.
[0064] In the method of the present invention, administration of an
ethyl acetate extract of black cohosh "in combination with" one or
more additional chemopreventive or chemotherapeutic agents refers
to co-administration of the anti-neoplastic agents.
Co-administration may occur concurrently, sequentially, or
alternately. Concurrent co-administration refers to administration
of the anti-neoplastic agents at essentially the same time. For
concurrent co-administration, the courses of treatment with the
ethyl acetate extract of black cohosh, and with the one or more
additional chemopreventive or chemotherapeutic agents, may be run
simultaneously. For example, a single, combined formulation,
containing both an amount of the ethyl acetate extract of black
cohosh and an amount of the additional chemopreventive or
chemotherapeutic agent, in physical association with one another,
may be administered to a subject. By way of example, the single,
combined formulation may consist of a liquid mixture, containing
amounts of both anti-neoplastic agents, which may be injected into
a subject, or an oral formulation, containing amounts of both
anti-neoplastic agents, which may be orally administered to a
subject.
[0065] It is also within the confines of the present invention that
an amount of the ethyl acetate extract of black cohosh, and an
amount of the one or more additional chemopreventive or
chemotherapeutic agents, may be administered concurrently to a
subject, in separate, individual formulations. Accordingly, the
method of the present invention is not limited to concurrent
co-administration of the anti-neoplastic agents in physical
association with one another.
[0066] In the method of the present invention, the ethyl acetate
extract of black cohosh, and the one or more additional
chemopreventive or chemotherapeutic agents, also may be
co-administered to a subject in separate, individual formulations
that are spaced out over a period of time, so as to obtain the
maximum efficacy of the combination. Administration of each drug
may range in duration from a brief, rapid administration to a
continuous perfusion. When spaced out over a period of time,
co-administration of the anti-neoplastic agents may be alternate or
sequential. For alternate co-administration, partial courses of
treatment with the ethyl acetate extract of black cohosh may be
alternated with partial courses of treatment with the one or more
additional chemopreventive or chemotherapeutic agents, until a full
treatment of each drug has been administered. For sequential
co-administration, one of the anti-neoplastic agents is separately
administered, followed by the other. For example, a full course of
treatment with the ethyl acetate extract of black cohosh may be
completed, and then may be followed by a full course of treatment
with the one or more additional chemopreventive or chemotherapeutic
agents. Alternatively, for sequential co-administration, a full
course of treatment with the one or more additional chemopreventive
or chemotherapeutic agents may be completed, then followed by a
full course of treatment with the ethyl acetate extract of black
cohosh.
[0067] The anti-neoplastic agents of the present invention (i.e.,
the ethyl acetate extract of black cohosh or the actein composition
and the one or more additional chemopreventive or chemotherapeutic
agents, either in a single, combined formulation, or in separate,
individual formulations) may be administered to a human or animal
subject by known procedures, including, but not limited to, oral
administration, parenteral administration, and transdermal
administration, as described above. Preferably, the anti-neoplastic
agents of the present invention are administered orally or
intravenously. For oral administration, the formulations of the
ethyl acetate extract of black cohosh or the actein composition and
the one or more additional chemopreventive or chemotherapeutic
agents (whether individual or combined) may be presented as
capsules, tablets, powders, granules, as a suspension, or in any
other form described herein. For parenteral administration, the
formulations of the ethyl acetate extract of black cohosh or the
actein composition and the one or more additional chemopreventive
or chemotherapeutic agents (whether individual or combined) may be
combined with a sterile aqueous solution which is preferably
isotonic with the blood of the subject. Such formulations may be
prepared in accordance with methods described herein. For
transdermal administration, the formulations of the ethyl acetate
extract of black cohosh or the actein composition and the one or
more additional chemopreventive or chemotherapeutic agents (whether
individual or combined) may be combined with skin penetration
enhancers, such as propylene glycol, polyethylene glycol,
isopropanol, ethanol, oleic acid, N-methylpyrrolidone, and the
like, and prepared in accordance with methods described herein.
[0068] Additionally, in accordance with the method of the present
invention, the ethyl acetate extract of black cohosh or the actein
composition and the one or more additional chemopreventive or
chemotherapeutic agents are administered to a subject in amounts
effective to treat or prevent neoplasia and other disorders in the
subject. As discussed above, exemplary doses of actein may range
from about 0.5 .mu.g/ml to about 40.0 .mu.g/ml; exemplary doses of
paclitaxel, for example, may range from 0.5 nM to about 5.0 nM.
However, the amounts of the ethyl acetate extract of black cohosh,
or the actein composition, and the one or more additional
chemopreventive or chemotherapeutic agents, that are effective to
treat or prevent neoplasia in a subject will vary depending on the
particular factors of each case, including the type and stage of
disorders (e.g. neoplasia), the subject's weight, the severity of
the subject's condition, and the method of administration. These
amounts can be readily determined by the skilled artisan, based
upon known procedures, including analysis of titration curves
established in vivo, dose-response experiments analogous to those
provided in the Examples, and methods and assays disclosed
herein.
[0069] In one embodiment of the present invention, an ethyl acetate
extract of black cohosh or the actein composition is administered
to a subject in combination with at least one additional
chemopreventive or chemotherapeutic agent, such that a synergistic
anti-neoplastic effect is produced. As used herein, a "synergistic
anti-neoplastic effect" refers to a greater-than-additive
anti-neoplastic effect which is produced by a combination of two
drugs, and which exceeds that which would otherwise result from
individual administration of either drug alone.
[0070] In the method of the present invention, combination therapy
using an ethyl acetate extract of black cohosh or the actein
composition and at least one additional anti-neoplastic agent
preferably results in an anti-neoplastic effect that is greater
than additive, as determined by any of the measures of synergy
known in the art. One measure of synergy between two drugs is the
fractional inhibitory concentration (FIC) (Hall et al., The
fractional inhibitory concentration (FIC) index as a measure of
synergy. J. Antimicrob. Chemother., 11(5):427-33, 1983). This
fractional value is determined by expressing the IC.sub.50 of a
drug acting in combination, as a function of the IC.sub.50 of the
drug acting alone. For two interacting drugs, the sum of the FIC
value for each drug represents the measure of synergistic
interaction. Where the FIC is less than 1, there is synergy between
the two drugs. An FIC value of 1 indicates an additive effect. The
smaller the FIC value, the greater the synergistic interaction.
[0071] Another measurement of synergy is the combination index (CI)
method of Chou and Talalay (Quantitative analysis of dose-effect
relationships: the combined effects of multiple drugs or enzyme
inhibitors. Adv. Enzyme Regul., 22:27-55, 1984), which is based on
the median-effect principle. This method calculates the degree of
synergy, additivity, or antagonism between two drugs at various
levels of cytotoxicity. Where the CI value is less than 1, there is
synergy between the two drugs. Where the CI value is 1, there is an
additive effect, but no synergistic effect. CI values greater than
1 indicate antagonism. The smaller the CI value, the greater the
synergistic effect.
[0072] As the inventors have demonstrated herein, administration of
an ethyl acetate extract of black cohosh, in combination with at
least one additional chemopreventive or chemotherapeutic agent,
frequently results (unexpectedly) in a synergistic anti-neoplastic
effect, by providing greater efficacy than would result from use of
either of the anti-neoplastic agents alone. In these cases, the
ethyl acetate extract of black cohosh enhances the effects of the
additional chemopreventive or chemotherapeutic agent; therefore,
lower doses of one or both of the anti-neoplastic agents may be
used in treating and preventing neoplasias, resulting in increased
chemotherapeutic/chemopreventive efficacy, and decreased
side-effects.
[0073] By way of example, the ethyl acetate fraction of black
cohosh (2 .mu.g/ml) may be combined with doxorubicin (0.2 .mu.g/ml;
0.34 .mu.M) or paclitaxel (4 nM) for a synergistic effect.
Furthermore, actein (2 .mu.g/ml; 3.0 .mu.M) may be combined with
5-FU (0.002 .mu.g/ml, 0.015 .mu.M) for a synergistic effect; actein
(0.2 or 2 .mu.g/ml) may be combined with herceptin (8 .mu.g/ml; 54
nM) for a synergistic effect; actein (1 .mu.g/ml) may be combined
with paclitaxel (1 nM) for a synergistic effect; actein (2
.mu.g/ml; 3.0 .mu.M) may be combined with doxorubicin (0.2
.mu.g/ml; 0.34 .mu.M) for a synergistic effect; actein (2 .mu.g/ml;
2.8 .mu.M) may be combined with tamoxifen (2 .mu.g/ml; 5.4 .mu.M)
for a synergistic effect; actein (2 .mu.g/ml; 3.0 .mu.M) may be
combined with cisplatin (2 .mu.g/ml; 6.7 .mu.M) for a synergistic
effect; and actein (2 .mu.g/ml; 3.0 .mu.M) may be combined with
vinblastine (4 .mu.g/ml; 4.4 .mu.M) for an additive effect. In a
preferred embodiment of the present invention, actein (e.g., about
0.5 .mu.g/ml to about 5.0 .mu.g/ml) is administered to a subject in
combination with paclitaxel (e.g., about 0.5 nM to about 5.0
nM).
[0074] As shown herein, administration of the ethyl acetate extract
of black cohosh (particularly the extract containing one or more
triterpene glycosides, such as actein, cimifugoside, cimigenol
glycoside, cimiracemoside A, and 23-epi-26-deoxyactein), or the
actein composition, in combination with one or more additional
chemopreventive or chemotherapeutic agents (particularly the
anti-neoplastic agents, cisplatin, docetaxel, doxorubicin,
5-fluorouracil, herceptin, paclitaxel, tamoxifen, and vinblastine),
may unexpectedly result in a synergistic anti-neoplastic effect by
providing greater efficacy than would result from use of either of
the anti-neoplastic agents alone. Accordingly, it is also within
the confines of the present invention that a formulation of the
ethyl acetate extract of black cohosh or the actein composition and
a formulation of the one or more additional chemopreventive or
chemotherapeutic agents (whether individual or combined) may be
further associated with a pharmaceutically-acceptable carrier,
thereby comprising a combination of anti-neoplastic agents. In one
embodiment of the invention, the combination of anti-neoplastic
agents is a synergistic combination. As used herein, a "synergistic
combination" of anti-neoplastic agents refers to a combination of
anti-neoplastic agents that achieves a greater anti-neoplastic
effect than would otherwise result if the anti-neoplastic agents
were administered individually.
[0075] The formulations of the combination of the present invention
may be prepared by methods well-known in the pharmaceutical arts
and described herein. Exemplary acceptable pharmaceutical carriers
have been discussed above. An additional carrier, Cremophor.TM.,
may be useful, as it is a common vehicle for Taxol.
[0076] In the combination of the present invention, the relative
proportions of the ethyl acetate extract of black cohosh (including
the triterpene glycoside compounds) or the actein composition and
the one or more chemopreventive or chemotherapeutic agents will
depend on the specific application of the combination. Thus, while
certain proportions may be beneficial in treating one type of
tumor, entirely different proportions may be beneficial in treating
other tumors. Such a determination can be made by a person skilled
in the art, in accordance with methods known in the art and
described in the Examples provided below. Some preferred
combinations, containing at least one triterpene glycoside compound
in the ethyl acetate extract of black cohosh, and at least one
additional chemopreventive or chemotherapeutic agent, may be
formulated such that the amount of the triterpene glycoside is
selected synergistically to enhance the effect of the
chemopreventive or chemotherapeutic agents, while alleviating
unwanted side effects attributable to such agents. Exemplary
combinations comprising the ethyl acetate extract of black cohosh,
and at least one additional chemopreventive or chemotherapeutic
agent, are described above. In a preferred embodiment of the
present invention, the combination comprises actein (e.g., about
0.5 .mu.g/ml to about 5.0 .mu.g/ml) and paclitaxel (e.g., about 0.5
nM to about 5.0 nM).
[0077] In the combination of anti-neoplastic agents of the present
invention, the ethyl acetate extract of black cohosh, or the actein
composition, and the one or more additional chemopreventive or
chemotherapeutic agents, may be combined in a single formulation,
such that the extract is in physical association with the agent.
This single, combined formulation may consist of a liquid mixture,
containing amounts of both the extract and the agent, which may be
injected into a subject, or an oral formulation, containing amounts
of both the extract and the agent, which may be orally administered
to a subject.
[0078] Alternatively, in the combination of the present invention,
a separate, individual formulation of the extract may be combined
with a separate, individual formulation of the agent. For example,
an amount of the extract may be packaged in a vial or unit dose,
and an amount of the agent may be packaged in a separate vial or
unit dose. A combination of the extract and the agent then may be
produced by mixing the contents of the separate vials or unit doses
in vitro. Additionally, a synergistic combination of the extract
and the agent may be produced in vivo by co-administering to a
subject the contents of the separate vials or unit doses, according
to the methods described above. Accordingly, the combination of the
present invention is not limited to a combination in which amounts
of the extract and the agent are in physical association with one
another in a single formulation.
[0079] It is also within the confines of the present invention for
the ethyl acetate extract of black cohosh, or the actein
composition, and the one or more additional chemopreventive or
chemotherapeutic agents, to be co-administered in combination with
radiation therapy or an anti-angiogenic compound (either natural or
synthetic). Examples of anti-angiogenic compounds with which the
anti-neoplastic agents may be combined include, without limitation,
angiostatin, thalidomide, and thrombospondin.
[0080] The combination of anti-neoplastic agents of the present
invention comprises an effective anti-neoplastic amount of the
ethyl acetate extract of black cohosh and an effective
anti-neoplastic amount of the one or more additional
chemopreventive or chemotherapeutic agents. As used herein, an
"effective anti-neoplastic amount" of the extract or the agent is
an amount of the extract or the agent that is effective to
ameliorate or minimize the clinical impairment or symptoms of
neoplasia in a subject, in either a single or multiple dose.
[0081] The present invention is described in the following
Examples, which are set forth to aid in the understanding of the
invention, and should not be construed to limit in any way the
scope of the invention as defined in the claims which follow
thereafter.
EXAMPLES
Example 1
Chemicals and Reagents
[0082] Polyamide resin SC6<0.07 mm was purchased from Alltech
Associates, Inc. (Deerfield, Ill.). RP.sub.18CC silica gel (40
.mu.M) was obtained from J. T. Baker (Phillipsburg, N.J.), and the
RP.sub.18F.sub.254 plate (1-mm layer thickness) was obtained from
EM Science (Darmstadt, Germany). Actein, 27-deoxyactein
(23-epi-26-deoxyactein) (Zheng et al., CimiPure (Cimicifuga
racemosa): a standardized black cohosh extract with novel
triterpene glycoside for menopausal women. In Phytochem.
Phytopharm., Shahidi and Ho, eds. (Champaign, Ill.: AOCS Press,
2000) pp. 360-70), cimifugoside, and cimiracemoside A were obtained
from ChromaDex (Laguna Hills, Calif.), and 27-deoxyactein was also
obtained from Herbstandard (Chesterfield, Mo.). Tamoxifen,
5-fluorouracil (5-FU), doxorubicin, cisplatin, and paclitaxel were
purchased from Sigma (St. Louis, Mo.). Herceptin was obtained from
Genentech (CA). Cimigenol and cimigenol glycoside were obtained
from Dr. WC Ye (Department of Phytochemistry, China Pharmaceutical
University, Nanjing 210009, China).
[0083] Black cohosh extracts and purified components were dissolved
in dimethylsulfoxide (DMSO) (Sigma Chemical Co.). Water (H.sub.2O)
was distilled and deionized. All solvents and reagents were reagent
grade.
Example 2
Plant Material
[0084] Black cohosh roots and rhizomes (GFP) were obtained from
PureWorld Botanicals (South Hackensack, N.J.; lot number
9-2677).
Example 3
Separation of the Ethyl Acetate Extract
[0085] As shown in FIG. 1, black cohosh roots and rhizomes were
extracted with 80% methanol (MeOH)/H.sub.2O, and partitioned with
n-hexane. Two layers were obtained: a water layer and an n-hexane
layer. N-hexane was used to extract the non-polar phytochemicals,
respectively, with yields of 0.05% hexane, 0.73% ethyl acetate, and
1.69% water. The water layer was partitioned with ethyl acetate,
and two fractions were obtained: a water layer and an ethyl acetate
layer. Ethyl acetate was used to extract the mid-polar and polar
phytochemicals. The ethyl acetate layer was dried and evaporated to
yield an ethyl acetate extract. The triterpene glycosides and
cinnamic acid esters were separated from the ethyl acetate extract
by polyamide chromatography (Kruse et al., Fukic and piscidic acid
esters from the rhizome of Cimicifuga racemosa and the in vitro
estrogenic activity of fukinolic acid. Planta. Med., 65:763-64,
1999).
Example 4
Cell Cultures
[0086] MDA-MB-453 human breast cancer cells (HER2 overexpressing,
ER negative), MCF7 cells (ER positive, HER2 low), MDA-MB-231 cells
(ER negative, HER2 low), MCF10F cells (normal mammary epithelial
cells), and SW480 colon cancer cells were obtained from ATCC
(Manassas, Va.). BT474 clone Sc-1 cells (ER positive, Her2
overexpressing) were the kind gift of Dr. S. Friedman (Incyte
Pharmaceuticals). Cells were grown in Dulbecco's Modified Eagle
medium (DMEM) (Gibco BRL Life Technologies, Inc., Rockville, Md.)
containing 10% (v/v) fetal bovine serum (FBS) (Gibco BRL), at
37.degree. C. and 5% CO.sub.2. The medium was supplemented with
bovine insulin (0.01 mg/ml) for the growth of BT474 cells.
Example 5
Cell-Growth Assays
[0087] Cell cultures were treated with increasing concentrations of
extracts and/or purified compounds for increasing times and
cytoxicity (for SW480 cells) measured using the MTT
{3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H tetrazolilum bromide}
(Dojindo, Tokyo, Japan) method (Luo et al., PM-3, a benzo-g-pyran
derivative isolated from propolis, inhibits growth of MCF-7 human
breast cancer cells. Anticancer Res 21: 1665-1672, 2001) and
inhibition of cell proliferation by performing cell counts using a
Coulter Counter (Lim et al., 1999, supra). For the cell count
assay, breast cancer cells were seeded, in triplicate, at
2.times.10.sup.4 cells per well, in 24- or 96-well plates. Two or 3
days later, the medium was replaced with fresh medium--with or
without black cohosh extracts or purified compounds--and the number
of attached viable cells was counted at increasing times (or, to
determine IC.sub.50 values, at 48 or 96 h), using a Coulter
Counter, model Z.sub.F (Coulter Electronics Inc., Hialeah, Fla.)
(Lim et al., Sulindac derivatives inhibit growth and induce
apoptosis in human prostate cancer cell lines. Biochem. Pharmacol.,
58:1097-107, 1999).
[0088] For the MTT assay, cells were seeded at 3.times.10.sup.3
cells per well in 96-well plates; 24 hours later the medium was
replaced with fresh medium containing black cohosh extracts or
components and assayed with MTT reagents at 48 hours.
[0089] To determine the combination index (CI) for potential
combination therapies, the inventors treated the breast cancer
cells with all combinations of 3 concentrations of the black cohosh
component and 3 concentrations of the chemotherapy agent, using a
solvent control. Surviving cells were counted using the Coulter
Counter (Masuda et al., Effects of epigallocatechin-3-gallate on
growth, epidermal growth factor receptor signaling pathways, gene
expression, and chemosensitivity in human head and neck squamous
cell carcinoma cell lines. Clinical Cancer Research, 7:4220-29,
2001). Data that were obtained were analyzed for possible
synergistic effects using previously-described methods (e.g., the
median-effect plot method of Chou and Talalay (Quantitative
analysis of dose-effect relationships: the combined effects of
multiple drugs or enzyme inhibitors. Adv. Enzyme Regul., 22:27-55,
1984)). The CIs were calculated using the index-isobologram method
(Soriano et al., Synergistic effects of new chemopreventive agents
and conventional cytotoxic agents against human lung cancer cell
lines. Cancer Res., 59:61, 78-84, 1999) based on the median-effect
principle of Chou and Talalay, 1984, sulpra.
Example 6
Statistical Analysis
[0090] The inventors were interested in determining the effects of
combinations of actein and paclitaxel concentrations on MDA-MB-453
cells. In calculating statistical significance, a Two-Way Analysis
of Variance (ANOVA) was performed to test whether the effects of
paclitaxel and actein concentrations were independent, or were
related, or "interacted" with each other (alpha=0.05; significant
difference=p<0.05; very significant difference=p<0.01). If
the F-test showed that the interaction was significant, the Least
Significant Difference method (LSD) was then used for multiple
comparisons, to clarify the significance between the different
combinations of paclitaxel and actein concentrations.
Example 7
Cell-Cycle Analyses
[0091] To obtain exponential cultures of breast cancer cells,
3.times.10.sup.5 cells were plated onto 10-cm dishes, and grown for
2-3 days; the medium was then replaced with fresh medium containing
black cohosh extracts or purified components alone and in
combination with chemotherapy agents. Synchrony: To synchronize the
cells, 3.times.10.sup.5 cells were plated onto 10 cm dishes and
grown for 2 days in DMEM supplemented with 10% fetal bovine serum.
The medium was then replaced with DMEM containing 0.25% FBS (Imoto
et al., 1997) and black cohosh extracts or purified components.
[0092] After incubation for 1-3 days, the supernatant was
collected, and the cells were trypsinized, collected, and washed
with phosphate buffered saline (PBS) containing 5% FBS. Cell
pellets were re-suspended in 1 ml of PBS plus 5% FBS. Thereafter, 5
ml of 70% ethanol were added drop-wise, while vortexing the tube,
and the mixture was stored at 4.degree. C. Cells were centrifuged,
washed with PBS plus 5% FBS, and re-suspended in 400 .mu.l of
propidium iodide (0.1 mg/ml) (Sigma Chemical Co.). 400 .mu.l (2
mg/ml) of RNase (Sigma Chemical Co.) were added, and the cells were
incubated in the dark at room temperature for 30 min. The
suspension was filtered through a 41-.mu.M spectra/mesh filter
(Spectrum Medical Industries, Calif.), and analyzed with a
FACScalibur instrument (Becton Dickinson, Franklin Lakes, N.J.)
equipped with Cell Quest software (Becton Dickinson). The
percentage of cells in different cell-cycle phases was then
calculated (Lim et al., Sulindac derivatives inhibit growth and
induce apoptosis in human prostate cancer cell lines. Biochem.
Pharmacol., 58:1097-107, 1999; Luo et al., PM-3, a benzo-g-pyran
derivative isolated from propolis, inhibits growth of MCF7 human
breast cancer cells. Anticancer Research, 21:1665-72, 2001; Soh et
al., Cyclic GMP mediates apoptosis induced by sulindac derivatives
via activation of c-Jun NH2-terminal kinase 1. Clin. Cancer Res.,
10:4136-41, 2000).
Example 8
Western-Blot Analysis
[0093] Cells were treated for increasing times with approximately
the IC.sub.50 concentration, or twice the IC.sub.50 concentration,
of actein. The cells were harvested, washed with PBS, and sonicated
in extraction buffer according to the procedure of Han et al. (Han
et al., Stable overexpression of cyclin D1 in a human mammary
epithelial cell line prolongs the S-phase and inhibits growth.
Oncogene, 10:953-61, 1995). The lysates were subjected to
electrophoresis on a 10% or 12.5% SDS-polyacrylamide gel, and then
transferred to a polyvinylidene difluoride (PVDF) membrane. The
membrane was blocked with milk protein, and incubated with a
solution containing the primary antibody against the following:
cyclin D1 (Upstate Biotechnology, Lake Placid, N.Y.), p21.sup.cip1
(Oncogene Research Products, Darmstadt, Germany), ppRb (ser 780,
Medical and Biological Laboratories, Nagoya, Japan), cdk4 (Upstate
Biotechnology, Lake Placid, N.Y.), EGFR (clone-74, Transduction
Laboratories, Lexington, Ky.), p-EGFR (phospho (Y1173)-EGFR) (Cell
Signaling, Beverly, Mass.), actin (Sigma, St. Louis, Mo.),
Her-2/neu (Cell Signaling, Beverly, Mass.), or
phospho-(Y1248)-Her-2/neu (Cell Signaling, Beverly, Mass.)),
I.kappa.B (Sant Cruz Biotechnolgy, Santa Cruz, Calif.),
I.kappa..kappa.B (Sigma, St. Louis, Mo.) and PPAR.gamma. (Santa
Cruz biotechnology, Santa Cruz, Calif.) (Masuda et al.,
Epigallocatechin-3-gallate inhibits activation of HER-2/neu and
downstream signaling pathways in human head and neck and breast
carcinoma cells. Clin. Cancer Res., 9: 3486-91, 2003). The membrane
was washed, and incubated with horseradish peroxidase conjugated
secondary antibody.
[0094] Protein bands were visualized with the ECL-enhanced
chemiluminescence system, according to the manufacturer's
directions (Amersham Pharmacia Biotech) (Sgambato et al.,
Overexpression of p27 (Kip1) inhibits the growth of both normal and
transformed human mammary epithelial cells. Cancer Research,
58:3448-54, 1998). The staining intensities of the visualized blots
were quantified using NIH image software. For each protein, the
relative band intensities were determined by comparing treated
samples with untreated controls. These values were then normalized,
using .beta.-actin as an internal control.
Example 9
Thin-Layer Chromatography Analysis
[0095] Extracts were tested for triterpene glycosides and cinnamic
acid esters using silica gel 60 F.sub.254 plates (0.25-mm layer
thickness) and RP.sub.18 F.sub.254 plates (1-mm layer thickness)
from EM Science (Darmstadt, Germany). The solvent system for the
silica gel thin-layer chromatography (TLC) was chloroform-MeOH
(9:1); the solvent system for the RP18 plates was MeOH--H2O (9:1).
After development, the compounds were visualized under UV, and
visualized by spraying with vanillin in 10% (v/v) H.sub.2SO.sub.4
in ethanol (EtOH).
Example 10
Polyamide Chromatography
[0096] Polyamide SC6 resin (1.5 gm), pre-conditioned with MeOH (15
min) and H.sub.2O (10 min), was packed under pressure in a 12-ml
syringe (approximately 3.3 cm in height, with a column volume of
4.5 ml); the syringe was then rinsed with water. The black cohosh
ethyl acetate extract (100 mg) was dissolved in 1 ml of
H.sub.2O/MeOH (1:1), and adsorbed to the polyamide column for 20
min before elution. The column was then eluted sequentially, twice,
with 6 ml of H.sub.2O/MeOH (50:50), H.sub.2O/MeOH (75:25), MeOH,
EtOH, and EtOH+0.1% TFA, to yield 10 fractions (Kruse et al., Fukic
and piscidic acid esters from the rhizome of Cimicifuga racemosa
and the in vitro estrogenic activity of fukinolic acid. Planta.
Med., 65:763-64, 1999).
Example 11
Cyclin D1 Reporter Assay
[0097] The cyclin D1 promoter luciferase reporter plasmid,
1745CD1LUC, was prepared by Dr. R. Pestell (Albert Einstein Cancer
Center, New York, N.Y.). The method used for transient transfection
reporter assays was previously described (Soh et al., Novel roles
of specific isoforms of protein kinase C in activation of the c-fos
serum response element. Molecular and Cellular Biology, 19:1313-24,
1999). Using lipofectin, triplicate samples of MDA-MB-453 breast
cancer cells (1.times.10.sup.5 cells in 35-mm plates) were
co-transfected using DNA of the indicated reporter plasmid (1
.mu.g) and the .beta.-gal plasmid as an internal control (10 .mu.g
of the pCMV-b-gal plasmid) in opti-MEM 1 medium (Life Technologies,
Inc.). After 24 h, the medium was replaced with serum-free medium
containing the indicated concentrations of actein. After 24 h,
cells were harvested, and luciferase activity was determined with
the luciferase assay system (Promega Corp. Madison, Wis.);
.beta.-gal activities were determined with the .beta.-gal enzyme
assay system (Promega). Luciferase activities were normalized to
.beta.-gal activities, to correct for differences in transfection
efficiency.
Example 12
Bioactivity-Guided Fractionation
[0098] Open chromatography techniques were used to fractionate the
extracts further. The stationary phases used included Diaion HP-20,
Sephadex, normal and reversed-phase silica, and polyamide.
[0099] 1. Alcoholic black cohosh powder extract was redissolved in
MeOH/H.sub.2O, and evaporated to dryness, leaving the water
portion.
[0100] 2. To partition the phytochemicals according to polarity,
the water portion was partitioned sequentially with hexane and
n-butanol (n-BuOH). The three resulting fractions--hexane, n-BuOH,
and water--were evaporated to dryness, and tested for their effects
on the growth of MCF7 breast cancer cells. The n-BuOH extract
showed high activity (FIG. 11).
[0101] 3. The n-BuOH extract was further separated using Diaion
HP-20 as a stationary phase, and eluting sequentially with
MeOH/H.sub.2O (1:1), MeOH, and acetone. By thin-layer
chromatography, the MeOH/H.sub.2O (1:1) contained mostly
UV-absorbing compounds (aromatic acid derivatives), while the MeOH
contained mostly triterpenoids (FIG. 11).
[0102] 4. Further separation of the MeOH/H.sub.2O fraction, over
silica gel, RP.sub.18, and polyamide columns, yielded isoferulic
acid, ferulic acid, and caffeic acid. Preliminary experiments
indicated that isoferulic, the more potent, and ferulic acids were
active in suppressing the growth of MCF7 human breast cancer cells
(FIG. 12).
[0103] Summarized below are results obtained by the inventors in
connection with the experiments described in Examples 1-12:
Effects of Extracts of Black Cohosh on the Growth of Human Breast
Cancer Cells
[0104] Black cohosh roots and rhizomes were extracted with
MeOH/H.sub.2O, and fractionated by solvent-solvent partitioning to
yield three fractions: hexane, ethyl acetate (EtOAc), and H.sub.2O
(FIG. 1). These fractions were assayed for growth inhibition on
human breast cancer cell lines. By TLC, it was determined that
triterpene glycosides are present at the highest level in the EtOAc
extract; low levels were detected in the hexane and water
extracts.
[0105] The effects of increasing amounts of the three black cohosh
fractions on the growth of the (ER+) human breast cancer cell line,
MCF7, were determined after exposure of the cells for 96 h. The
results, expressed as IC.sub.50 values (i.e., the concentration
that causes approximately 50% inhibition of growth), are set forth
in Table 1. The results indicate that the EtOAc extract was the
most active fraction.
[0106] The inventors tested the effects of crude extracts, methanol
and ethanol, as well as ethanol extracts provided by Pure World,
native and plus expedient: the IC.sub.50 values for these extracts
after 96 hours of treatment of MDA-MB-453 cells were: methanol: 100
.mu.g/ml; ethanol: >200 .mu.g/ml; PW native 175 .mu.g/ml: and PW
expedient: 195 .mu.g/ml.
[0107] To partition the phytochemicals according to polarity, the
water portion was also partitioned sequentially with hexane and
n-butanol (n-BuOH). The n-BuOH fraction was tested for its effect
on the growth of MDA-MB-453 breast cancer cells. The IC50 value
after 96 hours of treatment was: 40 .mu.g/ml.
[0108] The inventors also examined the effects of the EtOAc
fraction of black cohosh on SW480 human colon cancer cells. The
IC50 values after 48 hours of incubation using the MTT assay were:
SW480: 42 .mu.g/ml; MCF7: 38 .mu.g/ml (Luo et al., PM-3, a
benzo-g-pyran derivative isolated from propolis, inhibits growth of
MCF-7 human breast cancer cells. Anticancer. Res. 21: 1665-1672,
2001).
TABLE-US-00001 TABLE 1 Effects of black cohosh extracts on MCF7
cells. IC.sub.50 Values (.mu.g/ml) Black Cohosh Extracts H.sub.2O
extract 150 ethyl acetate extract 18 hexane extract 28 Purified
Components Actein 14 (21 .mu.M) 23-epi-26-deoxyactein 21 (32 .mu.M)
Cimifugoside 22 (36 .mu.M) cimiracemoside A 41 (61 .mu.M)
[0109] The effects of two concentrations of the EtOAc fraction on
the growth of MCF7 cells were examined at increasing times.
Exposure to 20 .mu.g/ml of the EtOAc fraction led to partial
inhibition of cell proliferation as early as 24 h after addition;
40 .mu.g/ml resulted in complete inhibition and cell death after 72
h (FIG. 2A), while 60 .mu.g/ml resulted in cell death at 24 h.
[0110] Two major signaling pathways in breast cancer cells are the
ER-mediated signaling pathway (exemplified in the
estrogen-dependent human breast cancer cell line, MCF7) and the
HER2-mediated signaling pathway (exemplified in the
estrogen-independent human breast cancer cell line, MDA-MB-453,
which overexpresses HER2 (erb2, c-neu), a membrane-associated
tyrosine kinase receptor (p185 HER2)). Clinical studies indicate
that a reciprocal relationship often occurs in the expression of
the two pathways in primary human breast cancers (Tsutsui et al.,
Prognostic value of c-erbB2 expression in breast cancer. J. Surg.
Oncol., 79:216-33, 2002). It was important, therefore, for the
inventors to determine if black cohosh extracts have different
effects on the two cell types. Accordingly, the following three
breast cancer cell lines were tested: MCF7 (ER positive, HER2 low),
MDA-MB-231 (ER negative, HER2 low), and MDA-MB-453 (HER2
overexpressing, ER negative).
[0111] Treatment with the EtOAc fraction for 48 h inhibited the
growth of all three cell lines, with IC.sub.50 values in the range
of 20-40 .mu.g/ml (Table 2). The Her2 overexpressing cells were the
most sensitive. It is of interest that the normal human mammary
epithelial cell line, MCF10F, was considerably less sensitive, with
an IC.sub.50 value of 85 .mu.g/ml.
TABLE-US-00002 TABLE 2 Effects of black cohosh extracts on breast
cancer cells. Cells Receptors Expressed IC.sub.50 (.mu.g/ml)
MDA-MB-453 ER-/HER2+ 18 MCF7 ER+/HER2- 35 MDA-MB-231 ER+/HER2- 39
MCF10F Normal Mammary 85 Epithelial Cells (ER-)
[0112] Observed over a 48-h period, the approximate doubling times
for the malignant cells were 36 h for MDA-MB-453, 32 h for MCF7,
and 30 h for MDA-MB-231; the approximate doubling time for the
non-malignant MCF10F cells was 48 h. It is possible that the
greater sensitivity of the malignant cells may reflect, in part,
the difference in growth rates. The IC.sub.50 values were less when
the cells were treated for 96 h: 18 .mu.g/ml for MCF7 cells, 10
.mu.g/ml for MDA-MB-453 cells, and 46 .mu.g/ml for MCF10F cells.
Based upon these results, it can be concluded that the EtOAc
fraction of black cohosh does not act specifically through the ER
or the Her2 receptors.
Characterization of the Active Components in the Ethyl Acetate
Extract
[0113] As the ethyl acetate extract of black cohosh contains many
components, it was important for the inventors to identify the
specific active compounds and their modes of action.
[0114] To separate the triterpene glycosides from the aromatic
acids and esters, the ethyl acetate extract was fractionated on a
polyamide SC6 column (Kruse et al., Fukic and piscidic acid esters
from the rhizome of Cimicifuga racemosa and the in vitro estrogenic
activity of fukinolic acid. Planta. Med., 65:763-64, 1999). The
first four fractions (water/methanol--50:50; 75:25), which are
enriched for triterpene glycosides, suppressed the growth of MCF7
cells. Incubation with fraction 1 (5.7 .mu.g/ml) resulted in 25%
cell death; incubation with fraction 2 (23 .mu.g/ml) resulted in
67% cell death; and incubation with fraction 3 (30 .mu.g/ml)
resulted in 73% cell death. In view of these results, it appears
that the triterpene glycosides are among the active components in
the ethyl acetate extract.
Effects of Triterpene Glycoside Fraction and Pure Components on
Cell Proliferation
[0115] To ascertain the nature of the triterpene glycosides of
black cohosh, the purified triterpene glycosides (set forth in FIG.
3) were tested for growth inhibition on MCF7 cells (FIG. 2B and
Table 1). Actein, which has an hydroxyl group on the C-26 position
of 23-epi-26-deoxyactein (Chen et al., Isolation, structure
elucidation, and absolute configuration of 26-deoxyactein from
Cimicifuga racemosa and clarification of nomenclature associated
with 27-deoxyactein. J. Nat. Prod., 65:601-05, 2000)), had an IC50
of 21 .mu.M; it was approximately 1.5-fold more potent than
23-epi-26-deoxyactein or cimifugoside, and approximately 3 times
more potent than cimiracemoside A, in inhibiting the growth of MCF7
cells (table 1). The substitution of an hydroxyl on the aglycone
moiety can significantly alter this inhibitory activity.
[0116] The effects of two concentrations of actein on the
proliferation of MCF7 cells were examined at increasing times.
Treatment with actein (15 .mu.g/ml) resulted in partial inhibition
of growth, within 24 h after addition of the compound, while
treatment with actein at 30 .mu.g/ml resulted in complete
inhibition of growth (FIG. 2B). In additional studies, it was found
that MCF7 cells were approximately three times more sensitive to
growth inhibition by actein than the MCF10F normal mammary
epithelial cells; the respective IC.sub.50 values were 14 .mu.g/ml
vs. 42 .mu.g/ml, when measured at 96 h of exposure. The mean of the
MCF7 cells that were alive (38.0%.+-.3.0) after 96 h of treatment
with actein (20 .mu.g/ml) was significantly less than the mean of
the MCF10F cells that were alive (63.8%.+-.1.4) after 96 h of
treatment with actein (20 .mu.g/ml) (p<0.01). As was the case
for the EtOAc fraction, the MDA-MB-453 cells were the most
sensitive to treatment with actein--with an IC.sub.50 value of
approximately 8 .mu.g/ml at 96 h.
Effects of the EtOAc Extract and Purified Components of Black
Cohosh on Cell-Cycle Kinetics
[0117] The ability of an extract or purified compound to affect
specific phases of the cell cycle may provide clues to its
mechanism of action (Weinstein, I. B., Disorders of cell circuitry
during multistage carcinogenesis: the role of homeostasis.
Carcinogenesis, 5:857-64, 2000). To determine the effects of black
cohosh on the cell cycle, MCF7 cells were treated with 30 and 60
.mu.g/ml of the EtOAc fraction of black cohosh, or 30 and 60
.mu.g/ml of actein, for 48 h. The cells were then stained with
propidium iodide, and analyzed by DNA flow cytometry (FIG. 4).
After exposure to 30 .mu.g/ml of the EtOAc fraction, there was an
increase of cells in G1 (from 70% to 82%) when compared to the DMSO
solvent control, and a concomitant decrease of cells in S (9% to
3%) and G2/M (19% to 12%). After treatment with 60 .mu.g/ml of the
EtOAc fraction, there was a decrease of cells in G1 (68% to 58%)
and an increase of cells in G2/M (21% to 31%).
[0118] The above results indicate that the extract contains more
than one component, with the more active or abundant component
inducing G1 arrest, and the less active component inducing G2/M
arrest, and/or that individual components in the extract exert
different effects at different concentrations. To distinguish
between these possibilities, cells were treated with the purified
compound, actein, at 30 and 60 .mu.g/ml. Exposure to actein at 30
.mu.g/ml also resulted in an increase of cells in G1 (70% to 82%)
and a decrease of cells in G2/M (19% to 12%). After exposure to 60
.mu.g/ml of actein, there was also an increase of cells in G1 (68%
to 77%), and a decrease of cells in G2/M (21% to 18%). Thus, with
60 .mu.g/ml of actein, the inventors did not observe the increase
in G2/M cells that was seen with 60 .mu.g/ml of the EtOAc extract
(FIG. 4).
[0119] To examine in greater detail the effects of actein on
cell-cycle progression, MCF7 cells were treated with 0, 10, 20, or
40 .mu.g/ml of actein, and analyzed at 0, 24, and 48 h by DNA flow
cytometry. FIG. 5 summarizes the results obtained with respect to
the percent of cells in G1. When cells were treated with 10
.mu.g/ml of actein, the percentage of cells in G1 increased from
64% at time zero to 75% at 24 h, and to 77% at 48 h. With 20
.mu.g/ml of actein, the respective values were 64%, 77%, and 87%;
with 40 .mu.g/ml of actein, the respective values were 64%, 74%,
and 79%. These increases in the G1 population were associated with
decreases in both the S and G2/M populations of cells. Indeed, the
maximal increase in the G1 population occurred at about 20 .mu.g/ml
actein. Therefore, it is possible that, at high concentrations,
actein and related compounds affect proteins that regulate later
phases in the cell cycle. The triterpene glycoside fraction of
black cohosh (polyamide eluate, fraction 3), 23-epi-26-deoxyactein,
and cimiracemoside A also induced cell-cycle arrest at G1, when
tested at about 40 .mu.g/ml.
[0120] Treatment with the EtOAc fraction at 30 .mu.g/ml induced a
small amount of apoptosis for 48 h (1.3%); at 60 .mu.g/ml, there
was a further increase in apoptosis (3.2%), as determined by the
sub G1 fraction (FIG. 4). When the cells were exposed to 20
.mu.g/ml actein for 48 h, approximately 1.4% of the population
displayed apoptosis; at 72 h, this value was 3.6%, when assessed by
the size of the sub G1 peak.
Effects of Actein on the Expression of Specific Proteins Involved
in Cell-Cycle Control and Apoptosis
[0121] Since actein induces cell-cycle arrest at G1, the inventors
examined the effect of actein on proteins which control the
progression of the cell cycle. Cyclin D1 was of particular
interest, since it plays a critical role in mediating the
transition from G1 to S, is overexpressed in approximately 50-60%
of primary human breast carcinomas (Joe et al., Cyclin D1
overexpression is more prevalent in non-Caucasian breast cancer.
Anticancer Res., 21:3535-39, 2001), and is overexpressed in several
human breast cancer cell lines (Han et al., Effects of sulindac and
its metabolites on growth and apoptosis in human mammary epithelial
and breast carcinoma cell lines. Breast Cancer Res. Treat.,
48:195-203, 1998). Therefore, the inventors monitored possible
changes in cellular levels of cyclin D1 by Western-blot analysis of
extracts obtained from control and actein-treated cells.
[0122] Treatment of MCF7 cells with 40 .mu.g/ml of actein for 3 or
10 h resulted in a partial decrease, and treatment for 24 h caused
a marked decrease, in the cellular level of cyclin D1, when
compared to comparable time points in the control (untreated)
cells. Indeed, after treatment with 40 .mu.g/ml for 24 h, there was
almost a complete loss of this protein (FIG. 6A). The MCF10F normal
mammary epithelial cells did not express an appreciable level of
cyclin D1. Thus, the inventors could not assess the effect of
actein on cyclin D1 in these cells.
[0123] Cyclin D1 binds to and activates the cyclin dependent
kinases, cdk4 and cdk6; the resulting complexes phosphorylate and
inactivate pRb (retinoblastoma protein), thereby preventing pRb
from inhibiting the transcription factor, E2F, and allowing the
cells to progress from G1 to S (Weinstein, I. B., Disorders of cell
circuitry during multistage carcinogenesis: the role of
homeostasis. Carcinogenesis, 5:857-64, 2000). The inventors
examined the effect of actein on the cellular level of the
inactivated, hyperphosphorylated form of Rb (designated ppRb).
After treatment with actein, the intensities of the ppRb bands
relative to the .beta.-actin bands were: 1.51 (3 h, 20 .mu.g/ml),
1.59 (3 h, 40 .mu.g/ml), 0.61 (10 h, 20 .mu.g/ml), 0.64 (10 h, 40
.mu.g/ml), 0.80 (24 h, 20 .mu.g/ml), and 0.43 (24 h, 40 .mu.g/ml).
The inventors found that there was a increase in the level of ppRb
at 3 hours and a decrease at 10 hours after treating MCF7 cells
with 20 or 40 .mu.g/ml actein; there was a marked decrease at 48
hours after exposure to 40 .mu.g/ml actein (FIG. 6B). The inventors
also observed a decrease in the level of cdk4 at 10 hours after
treatment with 20 or 40 .mu.g/ml actein and a pronounced decrease
at 24 hours after exposure to 40 .mu.g/ml actein (FIG. 6C).
[0124] The cdk inhibitory protein p21.sup.cip1 negatively regulates
the activity of the cyclin D1/cdk4 complex. Therefore, the
inventors examined the effect of actein on this protein. After
exposure to actein, the intensities of the p21.sup.cip1 bands
relative to the .beta.-actin bands were: 1.47 (3 h, 20 .mu.g/ml),
1.17 (3 h, 40 .mu.g/ml), 1.75 (10 h, 20 .mu.g/ml), 1.37 (10 h, 40
.mu.g/ml), 0.94 (24 h, 20 .mu.g/ml), and 0.78 (24 h, 40 .mu.g/ml).
Thus treatment of MCF7 cells with 20 or 40 .mu.g/ml of actein
induced an increase in p21.sup.cip1 within 3 hours and this
increase persisted at 10 hours. The increase was more pronounced
after treatment with 20 .mu.g/ml. However, this increase was not
seen with the 20 or 40 .mu.g/ml dose at. 24 hours (FIG. 6D).
[0125] In view of the foregoing, the ability of actein to arrest
cells in G1 (FIG. 5) may be due to the decreased expression of
cyclin D1 and cdk4, and the increased expression of
p21.sup.cip1--both of which result in a decrease in the level of
the hyperphosphorylated form of pRb.
[0126] The level of the epidermal growth factor receptor (EGFR),
which is overexpressed in various cancers (Masuda et al., Effects
of epigallocatechin-3-gallate on growth, epidermal growth factor
receptor signaling pathways, gene expression, and chemosensitivity
in human head and neck squamous cell carcinoma cell lines. Clinical
Cancer Research, 7:4220-29, 2001), was not significantly affected
by treatment with actein (FIG. 6E). There was also not a consistent
effect of actein on the phosphorylated and activated form of EGFR
(p-EGFR). However, the inventors did observe a significant decrease
with the 40 .mu.g/ml dose at 24 h (FIG. 6F).
The Effects of Actein and the Ethyl Acetate Extract of Black
Cohosh--Alone and in Combination with Chemotherapy Agents--On the
Proliferation of Human Breast Cancer Cells
[0127] It was essential for the inventors to explore the effects of
actein (the structure of which is set forth in FIG. 3) and extracts
from black cohosh on Her2 overexpressing breast cancer cells, such
as MDA-MB-453 cells, because these cells appeared to be more
sensitive to inhibition by the black cohosh components, and because
Her2 overexpressing breast cancers have a poorer clinical
prognosis. To determine the interaction of black cohosh with
chemotherapeutic drugs, actein was combined with several different
classes of drugs. Among the chemotherapy drugs tested were the
taxane, paclitaxel (Taxol); the selective estrogen receptor
modulator (SERM), tamoxifen; the anthracycline antibiotic,
doxorubicin; the anti-Her2 monoclonal antibody, herceptin (rhumab
Her2); the antimetabolite, 5-fluorouracil; the platinum analog,
cisplatin; and the vinca alkaloid, vinblastine. The SERM,
tamoxifen, was tested on ER+ MCF7 cells; the Her2 antibody and the
remainder of the agents were tested on MDA-MB-453 cells. The
combinations of actein with herceptin and the EtOAc extract with
doxorubicin were also tested on BT474 human breast cancer cells,
which form xenografts in athymic mice.
[0128] The results for the combination of actein and Taxol are
shown in FIG. 7. IC.sub.50 values obtained from the graphs were
used to calculate the combination index (CI) (Table 3). The
inventors found that actein (2 .mu.g/ml) potentiates the effect of
Taxol at concentrations of 1 and 4 .mu.M. These concentrations are
reported to be attainable in the blood after treatment with
Taxol.
TABLE-US-00003 TABLE 3 Combination index values for the combination
of actein and paclitaxel on MDA-MB-453 cells. Actein (.mu.g/mL)
Taxol (nM) 0.1 1 10 0.25 2.10 -- 1.70 -- 1.00 +/- 1 1.15 - 0.75 ++
0.05 +++ 4 1.10 +/- 0.70 ++ 0.00 +++ Symbols: CI -- >1.3
antagonism - 1.1-1.3 moderate antagonism +/- 0.9-1.1 additive
effect + 0.8-0.9 slight synergism ++ 0.6-0.8 moderate synergism +++
<0.6 synergism IC.sub.50 values determined from the graphs in
FIG. 7 were used to obtain combination index values: CI =
{IC.sub.50 (actein + paclitaxel)/IC.sub.50 (actein alone)} +
{IC.sub.50 (paclitaxel + actein)/IC.sub.50 (actein alone)}.
Results of Statistical Analyses
[0129] In the two-way ANOVA analysis, the F-test showed very
significant differences (p values approaching to zero) among the
paclitaxel concentrations, among the actein concentrations, and
among the combinations of actein and paclitaxel concentrations
(i.e., there were very significant interactions between the actein
and paclitaxel concentrations).
[0130] The LSD t-test indicated that, under the fixed paclitaxel
concentrations, 0 and 0.25 nM, there were very significant
differences (p<0.01) among the four different actein
concentrations. Under the paclitaxel concentration, 1 nM, there
were very significant differences between the actein
concentrations, 0 and 1 .mu.g/ml, and between the actein
concentrations, 0.1 and 1 .mu.g/ml. The addition of 0.1 .mu.g/ml
actein to 1 nM paclitaxel did not produce a significant effect;
however, the addition of 1 .mu.g/ml did.
TABLE-US-00004 TABLE 4 2-way ANOVA. 1 2 3 average DMSO 236742
255918 216444 236368 1 actein .1 169636 175420 179339 174798.3 2
actein 1 147900 148206 148502 148202.7 3 actein 10 110402 105501
104339 106747.3 4 tax.25 189954 205308 199987 198416.3 1 actein .1
tax .25 165780 168440 164526 166248.7 2 actein 1 tax .25 145603
159404 152503.5 3 actein 1 tax .25 107455 98838 107202 104498.3 4
tax1 125602 119850 110420 118624 1 actein .1 tax 1 126449 125399
125924 2 actein 1 tax 1 105302 100944 92668 99638 3 actein 10 tax 1
76402 72726 76398 75175.33 4 tax4 42388 35462 33332 37060.67 1
actein .1 tax 4 40902 35448 35962 37437.33 2 actein 1 tax 4 48694
50033 49363.5 3 actein 10 tax 4 46204 50002 48033 48079.67 4
Two-way ANOVA V.R DF SS MS F p-value A 3 1.09E+11 3.64E+10 753.6035
1.35E-27 B 3 2.55E+10 8.51E+09 176.1761 1.04E-18 A .times. B 9
1.92E+10 2.13E+09 44.18546 1.53E-14 Error 29 1.4E+09 48318909 Total
44 1.55E+11 t-test (Least Significant Difference Method; LSD)
Combination Average t-value p-value DMSO 236368 actein .1 174798.3
10.8481 1.01E-11 actein 1 148202.7 15.53406 4.685953 1.36E-15
6.0678E-05 actein 10 106747.3 22.83817 11.99007 7.304113 4.37E-20
9.2448E-13 4.8E-08 tax.25 198416.3 actein .1 tax 166248.7 5.667697
3.98E-06 .25 actein 1 tax .25 152503.5 7.23546 2.166118 5.75E-08
0.03866239 actein 1 tax .25 104498.3 16.54763 10.87994 7.565194
2.6E-16 9.4171E-12 2.44E-08 tax1 118624 actein .1 tax 1 125924
1.150416 0.259371 actein 1 tax 1 99638 3.345188 4.142443 0.002284
0.00027139 actein 10 tax 1 75175.33 7.655323 7.997545 4.310135
1.93E-08 8.0625E-09 0.000171 tax4 37060.67 actein .1 tax 4 37437.33
0.066366 0.947542 actein 1 tax 4 49363.5 1.938819 1.879459 0.06231
0.07026431 actein 10 tax 4 48079.67 1.941464 1.875098 0.202321
0.061975 0.07088192 0.841079 Factor A is tax concentration; Factor
B is actein concentration; A .times. B is combination Bold numbers
represent significant t-value and p-value.
t = x 1 - x 2 M S ( error ) .times. ( 1 n 1 + 1 n 2 )
##EQU00001##
[0131] Similar experiments were performed on the combination of
actein with herceptin, doxorubicin, cisplatin, 5-fluorouracil, and
vinblastine on MD-MBA-453 cells, and on the combination of actein
plus tamoxifen on MCF7 cells. The same method was used to obtain
the CI values for these classes of chemotherapy agents (Tables 5a
and 5b).
[0132] Actein at concentrations achievable in vivo (0.2 or 2
.mu.g/ml) potentiates the effects of several chemotherapy agents at
clinically-relevant drug concentrations (Table 5a). Actein at 2
.mu.g/ml (2.8 .mu.M) enhances the effects of 5-FU (0.002-0.2
.mu.g/ml; 1.54 .mu.M), doxorubicin (0.2 .mu.g/ml; 0.34 .mu.M),
cisplatin (2 .mu.g/ml; 6.7 .mu.M) and tamoxifen (2 .mu.g/ml, 5.4
.mu.M). Actein at 0.2 or 2 .mu.g/ml enhances the effect of
herceptin (8 .mu.g/ml, 54 nM). At 2 .mu.g/ml, actein has an
additive effect on vinblastine (4 .mu.g/ml).
[0133] When black cohosh was extracted with MeOH, and partitioned
with EtOAc, hexane, and water, the triterpene glycosides were
present primarily in the EtOAc extract. When the EtOAc extract was
combined with doxorubicin or paclitaxel, synergy occurred with 2
.mu.g/ml actein, and with 0.02-0.2 .mu.g/ml (0.34 .mu.M)
doxorubicin or 4 nM paclitaxel (Table 5b).
TABLE-US-00005 TABLE 5 Combination index values for the combination
of: (a) actein with various chemotherapy drugs; herceptin,
tamoxifen, doxorubicin, cisplatin, 5-FU or vinblastine; and (b)
EtOAc fraction with doxorubicin or paclitaxel. (a) Actein
(.mu.g/mL) 5-FU (.mu.g/mL) 0.2 2 20 0.002 1.75 - - 0.51 + + + 0.23
+ + + 0.02 1.69 - - 0.45 + + + 0.17 + + + 0.2, 0.15 uM 1.69 - -
0.45 + + + 0.17 + + + Actein (.mu.g/mL) herceptin (.mu.g/mL) 0.2 2
20 0.08 1.15 - 1.12 - 1.13 - 0.8 1.20 - 1.17 - 1.18 - 8, 54 nM 0.35
+ + + 0.32 + + + 0.33 + + + Actein (.mu.g/mL) tamoxifen (.mu.g/mL)
0.2 2 20 0.5 1.47 - - 1.22 - 0.94 +/- 5 1.15 - 0.90 + 0.61 + + 50,
134 uM 1.07 +/- 0.82 + 0.54 + + + Actein (.mu.g/mL) cisplatin
(.mu.g/mL) 0.2 2 20 0.2 3.33 - - 1.93 - - 1.44 - - 2 2.11 - - 0.71
+ + 0.22 + + + 20, 67 uM 2.04 - - 0.64 + + 0.15 + + + Actein
(.mu.g/mL) vinblastine (.mu.g/mL) 0.2 2 20 0.4 4.40 - - 4.45 - -
4.08 - - 4 0.95 +/- 1.00 +/- 0.63 + + 40, 44 uM 0.95 +/- 1.00 +/-
0.63 + + (b) EtOAc (.mu.g/mL) doxorubicin (.mu.g/mL) 0.2 2 20 0.002
1.13 - 1.21 - 0.75 + + 0.02 0.43 + + + 0.51 + + + 0.05 + + + 0.2,
0.34 uM 0.43 + + + 0.50 + + + 0.04 + + + EtOAc (.mu.g/mL) taxol
(nM) 0.2 2 20 0.25 1.89 - - 1.86 - - 1.86 - - 1 1.08 +/- 1.05 +/-
1.05 +/- 4 0.79 + + 0.76 + + 0.76 + + Symbols: CI - - >1.3
antagonism - 1.1-1.3 moderate antagonism +/- 0.9-1.1 additive
effect + 0.8-0.9 slight synergism + + 0.6-0.8 moderate synergism +
+ + <0.6 synergism IC.sub.50 values were determined from the
combination of 3 concentrations of actein and 3 concentrations of
the specific chemotherapy agent and the solvent control, as
illustrated for the combination of actein and paclitaxel in Table
3.
TABLE-US-00006 TABLE 6 Combination index values for the combination
of actein with the EtOAc fraction and cimigenol with paclitaxel.
EtOAc (.mu.g/ml) actein (.mu.g/ml) EtOAc .2 EtOAc 2 EtOAc 20
actein.2 3.8166 -- 3.8166 -- 3.96666 -- actein 2 3.65 -- 3.65 --
3.8 -- actein 20 0.15126 +++ 0.15126 +++ 0.30126 +++ Cimigenol
(.mu.g/ml) Taxol (.mu.g/ml) cimi .2 cimi 2 cimi 20 tax .25 1.5023
-- 1.5023 -- 1.4665 -- tax 1 1.85714 -- 1.85714 -- 1.82142 -- tax 4
0.85714 + 0.85714 + 0.82142 +
TABLE-US-00007 TABLE 7 Combination index values for the combination
of actein with herceptin and the EtOAc fraction of black cohosh
with doxorubicin on BT474 human breast cancer cells. Actein
(.mu.g/mL) herceptin (.mu.g/mL) 0.2 2 20 0.8, 5.4 nM 3.14 -- 3.06
-- 3.06 -- 8 0.08 +++ 0 +++ 0 +++ 32 0.08 +++ 0 +++ 0 +++ EtOAc
(.mu.g/mL) doxorubicin (.mu.g/mL) 0.2 2 20 0.002 1.45 -- 1.79 --
0.79 ++ 0.02 0.67 ++ 1 +/- 0 +++ 0.2, 0.34 uM 0.67 ++ 1 +/- 0 +++
Symbols: CI -- >1.3 antagonism - 1.1-1.3 moderate antagonism +/-
0.9-1.1 additive effect + 0.8-0.9 slight synergism ++ 0.6-0.8
moderate synergism +++ <0.6 synergism IC.sub.50 values were
determined from the combination of 3 concentrations of actein and 3
concentrations of the specific chemotherapy agent and the solvent
control.
[0134] To further understand the effect of actein and the EtOAc
fraction of black cohosh, the investigators tested the effects on
BT474 human breast cancer cells (ER.sup.+, her2 overexpressing,
25-fold), which can form tumors in athymic mice. The investigators
obtained strong synergy when actein (0.2 or 2 .mu.g/ml) was
combined with herceptin (0.8 or 8 .mu.g/ml) and additive effects
when the EtOAc fraction (2 .mu.g/ml) was combined with doxorubicin
(0.02 .mu.g/ml, 34 nM).
[0135] Actein, or the fraction enriched for triterpene glycosides,
could be used in combination with agents, in single use (including
paclitaxel, herceptin, and tamoxifen), to treat breast cancer. If
actein or the triterpene glycoside fraction is free of significant
side effects, they could be used in combination with herceptin for
long-term treatment of patients with metastatic disease.
Effects of Actein, in Combination with Chemotherapy Agents on the
Distribution of Cells in the Cell Cycle
[0136] To understand the nature of the interaction of actein with
the different classes of chemotherapy agents, we determined the
effect of actein in combination with various chemotherapy agents on
the distribution of cells in the cell cycle. When the cells were
synchronized by serum starvation followed by serum stimulation,
treatment with actein induced a dose dependent increase in the
percent of cells in G1 at 48 hours (Table 8a). When actein (2 or 20
.mu.g/ml) was combined with paclitaxel (1 nM), or when actein (20
.mu.g/ml) was combined with doxorubicin (0.1 .mu.g/ml, nM) or 5 FU
(0.02 .mu.g/ml, nM), there was a synergistic increase in the
percent of cells in the subG.sub.1 phase at 48 hours, an indicator
of apoptosis (Table 8 b, c).
[0137] In the case of doxorubicin and 5FU, the addition of actein
to the chemotherapy agent resulted in an increase in cells in the
G1 phase of the cell cycle (Table 8). The inventors' results
indicate that it may be better to give the chemotherapy agents
before actein, in order to retain the block at S or G2/M that is
induced by some chemotherapy agents.
TABLE-US-00008 TABLE 8a Effect of actein alone and in combination
with chemotherapy agents on cell cycle distribution in MDA-MB-453
cells. Sub G1 (%) G1 (%) S (%) G2/M (%) dmso, 0.08% 2.1 74.3 10.5
13.6 actein, 20 .mu.g/ml 1.8 79.6 8.5 10.0 actein, 40 .mu.g/ml 2.2
83.8 4.9 9.0 a The cells were grown in DMEM + 0.25% FBS for 48 hrs
and then treated with actein at 20 .mu.g/ml or 40 .mu.g/ml and
analyzed at 48 hrs by DNA flow cytometry. The values indicate the %
of cells in the indicated phases of the cell cycle. The control
contains 0.08% DMSO.
TABLE-US-00009 TABLE 8b G1 Sub G1 (%) (%) S (%) G2/M (%) Dmso 1.0
70.6 11.8 17.0 Actein 2 .mu.g/mL 0.9 69.8 11.0 18.6 Actein 20
.mu.g/mL 1.6 70.8 9.7 18.2 Taxol 1 nM 1.0 71.0 10.8 17.0 Taxol 1 nM
+ Actein 2 .mu.g/mL 1.8 69.2 10.6 18.5 Taxol 1 nM + Actein 20
.mu.g/mL 2.8 70.1 8.6 18.9 b The cells were treated with 0, 2 or 20
.mu.g/ml (29.6 .mu.M) actein alone and in combination with
paclitaxel (1 nM) and analyzed at 48 hrs by DNA flow cytometry. The
values indicate the % of cells in the indicated phases of the cell
cycle. The control contains 0.044% DMSO.
TABLE-US-00010 TABLE 8c G1 Sub G1 (%) (%) S (%) G2/M (%) dmso,
0.08% 3.0 59.0 10.0 28.0 Actein, 20 .mu.g/mL 2.7 59.2 8.6 29.5
Doxorubicin, 0.1 .mu.g/mL 2.5 30.1 5.6 61.7 Doxorubicin + Actein,
20 .mu.g/mL 5.3 39.2 9.6 46.0 5-FU, 0.02 .mu.g/mL 3. 28.9 47.6 20.3
5-FU + Actein, 20 .mu.g/mL 6.8 38.0 34.1 21.2 c The cells were
treated with 0 or 20 .mu.g/ml (29.6 .mu.M) actein alone and in
combination with doxorubicin (0.1 .mu.g/ml, 0.17 .mu.M), 5-FU (0.02
.mu.g/ml, 0.15 .mu.M) and analyzed at 48 hrs by DNA flow cytometry.
The values indicate the % of cells in the indicated phases of the
cell cycle. The control contains 0.08% DMSO.
Effects of Actein on Proteins Involved in Carcinogenesis
[0138] The inventors' previous results indicated that actein
decreased the level of cyclin D1, cdk4, and the hyperphosphorylated
form of the pRB protein, and increased the level of p21.sup.cip1 in
MCF7 cells--changes that may contribute to the arrest in G1. The
level of the epidermal growth factor receptor (EGFR), which is
overexpressed in various cancers (Suzui et al., Growth inhibition
of human hepatoma cells by acyclic retinoid is associated with
induction of p21 (CIP1) and inhibition of expression of cyclin D1.
Cancer Research, 62:3997-4006, 2002), was not altered after
treatment with actein. There also was no consistent effect of
actein on the phosphorylated and activated form of EGFR (p-EGFR).
However, the inventors did see a significant decrease of p-EGFR
with the 40 .mu.g/ml dose at 24 h. Thus, the EGFR did not appear to
be a direct target for actein.
[0139] Since the Her2 overexpressing cells were the most sensitive
to growth inhibition by black cohosh extracts and components, the
inventors tested the effect of actein on the Her2 receptor and on
the phosphorylation and activation of the Her2 receptor (p-Her2)
(FIG. 8) in MDA-MB-453 human breast cancer cells (which express
both Her2 and p-Her2 at high levels). Actein at 20 .mu.g/ml caused
a slight decrease in the level of the Her2 protein at 3 and 24 h.
After exposure to actein at 20 or 40 .mu.g/ml, there was a small
effect on p-Her2 at 3 h. The inventors found that actein at 20 or
40 .mu.g/ml induced a dose-dependent decrease in the level of the
p-Her2 receptor at 24 h. It is not clear how actein inhibits
phosphorylation. For example, it is not clear whether actein binds
to and directly inhibits the kinase activity of the Her2 receptor,
analogous to the action of Iressa (Masuda et al.,
Epigallocatechin-3-gallate inhibits activation of HER-2/neu and
downstream signaling pathways in human head and neck and breast
carcinoma cells. Clin. Cancer Res., 9: 3486-91, 2003), or whether
it inhibits activation of the other component of the heterodimeric
complex.
[0140] As the synthetic triterpenoid
2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) is a ligand
for PPAR-.gamma. (Wang et al., 2000; Lapillonne et al, 2003), the
inventors next tested the effect of actein on PPAR-.gamma. (FIG.
18). After treatment with actein, the intensities of the
PPAR-.gamma. bands relative to the .beta.-actin bands were: 1.39 (3
h, 20 .mu.g/ml), 0.93 (3 h, 40 .mu.g/ml), 1.3 (24 h, 20 .mu.g/ml),
and 0.67 (24 h, 40 .mu.g/ml). Thus actein at 20 .mu.g/ml increased
the level of PPAR.gamma. at 3 and 24 hours. This anti-inflammatory
protein is therefore among the targets of actein.
Effects of Actein on Transcriptional Control of Specific Genes
[0141] To further determine the nature of the target of actein, the
inventors tested the effect of actein on molecules, such as cyclin
D1, that function downstream of active Her2-containing
heterodimers. Since the inventors found that actein induces
cell-cycle arrest at G1, it was of interest to examine the effects
of this compound on cellular levels of proteins that control
cell-cycle progression. Cyclin D1 was of particular interest,
because it plays a critical role in mediating the transition from
G1 to S, is overexpressed in about 50-60% of primary human breast
carcinomas (Joe et al., Resveratrol induces growth inhibition,
S-phase arrest, apoptosis, and changes in biomarker expression in
several human cancer cell lines. Clin. Cancer Res., 8:893-903,
2002), and is overexpressed in several human breast cancer cell
lines (Soh et al., Novel roles of specific isoforms of protein
kinase C in activation of the c-fos serum response element. Mol.
Cel. Bio., 19:1313-24, 1999).
[0142] Actein suppressed the level of cyclin D1 protein in
MDA-MB-453 cells. After treatment with actein, the intensities of
the cyclin D1 bands relative to the .beta.-actin bands were: 3 hr,
40 .mu.g/ml: 0.93; 24 hr, 20 .mu.g/ml: 1.3; 40 .mu.g/ml: 0.44. The
inventors further show that actein at 40 .mu.g/ml reduced the level
of cyclin D1 mRNA at 24 hours, 0.66-fold in MCF7 cells (FIG. 13)
and 0.56-fold in MDA-MB-453 cells (FIG. 14). The inventors next
examined the effect of actein on cyclin D1 transcriptional promoter
activity in MDA-MB-453 cells, using transient transfection reporter
assays (Soh et al., Novel roles of specific isoforms of protein
kinase C in activation of the c-fos serum response element. Mol.
Cell. Biol., 19:1313-24, 1999; Soh et al., Cyclic GMP mediates
apoptosis induced by sulindac derivatives via activation of c-Jun
NH2-terminal kinase 1. Clin. Cancer Res., 10:4136-41, 2000; Masuda
et al., Effects of epigallocatechin-3-gallate on growth, epidermal
growth factor receptor signaling pathways, gene expression, and
chemosensitivity in human head and neck squamous cell carcinoma
cell lines. Clin. Cancer Res., 7:4220-29, 2001). To accomplish
this, the inventors used luciferase promoter sequences that were
1745 bp upstream of the cyclin D1 gene. At 24 hours after exposure
to actein at 20 (0.87 fold) or 40 .mu.g/ml (0.093 fold), there was
a dose dependent decrease in promoter activity, compared to
.beta.-gal as a control (FIG. 9). This result, in addition to the
inventors' Western-blot data, suggests that actein inhibits the
expression of cyclin D1 at the level of transcription.
[0143] Since NF-kB is instrumental in controlling cell
proliferation, the inventors then explored the effect of actein on
NF-kB promoter activity. Actein at 20 .mu.g/ml induced an increase
(1.59 fold) and, at 40 .mu.g/ml, a decrease (0.12 fold), in
NF-.kappa.B promoter activity (FIG. 9). To understand the basis for
this effect, the inventors checked the effect of actein on the
level of the related proteins, I.kappa.B and I.kappa..kappa.B.
After treatment with actein, the intensities of the I.kappa.B bands
relative to the .beta.-actin bands were: 1.2 (3 h, 20 .mu.g/ml),
1.09 (3 h, 40 .mu.g/ml), 0.81 (24 h, 20 .mu.g/ml), and 0.53 (24 h,
40 .mu.g/ml) (FIG. 17). After treatment with actein, the
intensities of the I.kappa..kappa.B bands relative to the
.beta.-actin bands were: 1.79 (3 h, 20 .mu.g/ml), 1.78 (3 h, 40
.mu.g/ml), 0.48 (10 h, 20 .mu.g/ml), 0.59 (10 h, 40 .mu.g/ml), 1.06
(24 h, 20 .mu.g/ml), and 0.95 (24 h, 40 .mu.g/ml).
[0144] In summary, the EtOAc fraction of black cohosh: (1) inhibits
cell proliferation at .about.20 and 10 .mu.g/ml, in ER+ and ER-
human breast cancer cell lines, respectively; and (2) induces
cell-cycle arrest at G1 at low concentrations (.about.IC.sub.50),
and at G2/M at high concentrations (.about.3.times.IC.sub.50).
[0145] The triterpene glycoside fraction of black cohosh, and the
triterpene glycosides--actein, 23-epi-26-deoxyactein, cimifugoside,
and cimiracernoside A--inhibit the growth of human breast cancer
cells, and induce cell cycle arrest at G1.
[0146] In MCF7 cells, actein decreases the level of cyclin D1,
cdk4, and ppRb and increases the level of p21 and p27--changes
which lead to G1 arrest. It reduces the level of cyclin D1 mRNA and
promoter activity, thereby acting at the level of transcription.
Actein does not affect the level of EGFR, and, therefore, does not
specifically act through the estrogen receptor, the Her2 receptor,
or the EGFR receptor. Actein is capable of enhancing the effects of
tamoxifen on MCF7 breast cancer cells.
[0147] In MDA-MB-453 cells, actein decreases the level of p-Her2
and the level of cyclin D1 mRNA and promoter activity at 24 h. It
increases the level of p21 mRNA at 24 h. Its effects on the level
of NF-.kappa.B promoter activity is complex: actein increase the
level of NF-.kappa.B promoter activity at 20 .mu.g/ml while
decreases the level at 40 .mu.g/ml at 24 h. Actein is capable of
enhancing the effects of paclitaxel, herceptin, 5-FU, doxorubicin,
and cisplatin.
[0148] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be
appreciated by one skilled in the art, from a reading of the
disclosure, that various changes in form and detail can be made
without departing from the true scope of the invention in the
appended claims.
TABLE-US-00011 TABLE 9 Summary. 20 molecule assay 0 3 hr, - ug/ml
40 ug/ml 10 hr, - 20 ug/ml 40 ug/ml 24 hr, - 20 ug/ml 40 ug/ml
cyclin D1 promoter MDA-MB-453 1 0.87 0.093 CD1 RNA RT-PCR
MDA-MB-453 1 0.88 1 1 1.18 1.02 1 0.98 0.56 CD1 RNA RT-PCR MCF7 1
0.91 0.94 1 1.1 0.93 1 0.84 0.66 cyclin D1 MDA-MB-453 1 0.93 1 1.3
0.44 p-Her2 WB MDA-MB-453 1 0.71 0.86 1 0.81 0.53 p21 WB MCF7 1
1.47 1 1 1.75 1.37 1 0.94 0.78 RT-PCR MDA-MB-453 1 0.92 1.03 1 1.06
1.19 1 0.92 1.46 ppRB WB MCF7 1 1.51 1.59 1 0.61 0.64 1 0.8 0.43
PPAR-g 1 1.39 0.92 1 1.3 0.67 NF-kB promoter MDA-MB-453 1 1.59 0.12
ikb WB MDA-MB-453 1 1.2 1.09 1 0.81 0.53 ikkb WB MDA-MB-453 1 1.79
1.78 1 0.48 0.59 1 1.06 0.95
* * * * *