U.S. patent application number 11/730985 was filed with the patent office on 2008-03-20 for combination chemotherapy compositions and methods.
This patent application is currently assigned to NOVOGEN RESEARCH PTY LTD. Invention is credited to David Brown, Alan James Husband, Graham Edmund Kelly, Harriet Kluger, Gil Mor.
Application Number | 20080069900 11/730985 |
Document ID | / |
Family ID | 28679466 |
Filed Date | 2008-03-20 |
United States Patent
Application |
20080069900 |
Kind Code |
A1 |
Kelly; Graham Edmund ; et
al. |
March 20, 2008 |
Combination chemotherapy compositions and methods
Abstract
This invention relates to combination therapies involving
anticancer chemotherapeutic agents and isoflavones or analogues
thereof. The invention flier relates to compounds, compositions,
methods and therapeutic uses involving, containing, comprising,
including and/or for preparing platinum-isoflavonoid complexes
suitable for use in the combination therapies of the invention.
Inventors: |
Kelly; Graham Edmund;
(Northbridge, AU) ; Husband; Alan James;
(McMahon's Point, AU) ; Brown; David; (North Ryde,
AU) ; Kluger; Harriet; (New Haven, CT) ; Mor;
Gil; (New Haven, CT) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
NOVOGEN RESEARCH PTY LTD
|
Family ID: |
28679466 |
Appl. No.: |
11/730985 |
Filed: |
April 5, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10530176 |
Mar 9, 2006 |
|
|
|
PCT/AU03/01296 |
Oct 2, 2003 |
|
|
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11730985 |
Apr 5, 2007 |
|
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Current U.S.
Class: |
424/649 ;
435/375; 514/184; 514/210.16; 514/283; 514/456; 514/679;
514/734 |
Current CPC
Class: |
A61K 31/352 20130101;
A61K 33/24 20130101; C07F 15/0093 20130101; A61K 31/337 20130101;
A61P 39/06 20180101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/282 20130101;
A61K 31/12 20130101; A61K 33/24 20130101; A61K 31/12 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 31/555 20130101; A61K
2300/00 20130101; A61K 31/555 20130101; A61K 31/282 20130101; A61P
35/00 20180101; A61K 31/352 20130101; A61K 31/353 20130101; A61K
31/337 20130101; A61P 43/00 20180101; A61K 45/06 20130101; A61K
31/353 20130101 |
Class at
Publication: |
424/649 ;
435/375; 514/184; 514/210.16; 514/283; 514/456; 514/679;
514/734 |
International
Class: |
A61K 31/437 20060101
A61K031/437; A61K 31/05 20060101 A61K031/05; A61K 31/12 20060101
A61K031/12; A61K 31/337 20060101 A61K031/337; A61P 35/00 20060101
A61P035/00; C12N 5/06 20060101 C12N005/06; A61K 33/24 20060101
A61K033/24; A61K 31/352 20060101 A61K031/352; A61K 31/397 20060101
A61K031/397 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 2, 2002 |
AU |
2002951833 |
Claims
1. A method of increasing the sensitivity of melanoma cells or a
melanoma to a chemotherapeutic agent by contacting said cells or
melanoma with an isoflavonoid compound of formula (I): ##STR21## in
which R.sub.1, R.sub.2 and Z are independently hydrogen, hydroxy,
OR.sub.9, OC(O)R.sub.10, OS(O)R.sub.10, CHO, C(O)R.sub.10, COOH,
CO.sub.2R.sub.10, CONR.sub.3R.sub.4, alkyl, haloalkyl, arylalkyl,
alkenyl, alkynyl, aryl, heteroaryl, alkylaryl, alkoxyaryl, thio,
alkylthio, amino, alkylamino, dialkylamino, nitro or halo, or
R.sub.2 is as previously defined, and R.sub.1 and Z taken together
with the carbon atoms to which they are attached form a
five-membered ring selected from ##STR22## or R.sub.1 is as
previously defined, and R.sub.2 and Z taken together with the
carbon atoms to which they are attached form a five-membered ring
selected from ##STR23## and W is R.sub.1, A is hydrogen, hydroxy,
NR.sub.3R.sub.4 or thio, and B is selected from ##STR24## W is
R.sub.1, and A and B taken together with the carbon atoms to which
they are attached form a six-membered ring selected from ##STR25##
W, A and B taken together with the groups to which they are
associated are selected from ##STR26## W and A taken together with
the groups to which they are associated are selected from ##STR27##
and B is selected from ##STR28## wherein R.sub.3 is hydrogen,
alkyl, arylalkyl, alkenyl, aryl, an amino acid, C(O)R.sub.11 where
R.sub.11 is hydrogen, alkyl, aryl, arylalkyl or an amino acid, or
CO.sub.2R.sub.12 where R.sub.12 is hydrogen, alkyl, haloalkyl, aryl
or arylalkyl, R.sub.4 is hydrogen, alkyl or aryl, or R.sub.3 and
R.sub.4 taken together with the nitrogen to which they are attached
comprise pyrrolidinyl or piperidinyl, R.sub.5 is hydrogen,
C(O)R.sub.11 where R.sub.11 is as previously defined, or
CO.sub.2R.sub.12 where R.sub.12 is as previously defined, R.sub.6
is hydrogen, hydroxy, alkyl, aryl, amino, thio, NR.sub.3R.sub.4,
COR.sub.11 where R.sub.11 is as previously defined,
CO.sub.2R.sub.12 where R.sub.12 is as previously defined or
CONR.sub.3R.sub.4, R.sub.7 is hydrogen, C(O)R.sub.11 where R.sub.11
is as previously defined, alkyl, haloalkyl, alkenyl, aryl,
arylalkyl or Si(R.sub.13).sub.3 where each R.sub.13 is
independently hydrogen, alkyl or aryl, R.sub.8 is hydrogen,
hydroxy, alkoxy or alkyl, R.sub.9 is alkyl, haloalkyl, aryl,
arylalkyl, C(O)R.sub.11 where R.sub.11 is as previously defined, or
Si(R.sub.13).sub.3 where R.sub.13 is as previously defined,
R.sub.10 is hydrogen, alkyl, haloalkyl, amino, aryl, arylalkyl, an
amino acid, alkylamino or dialkylamino, the drawing "- - -"
represents either a single bond or a double bond, T is
independently hydrogen, alkyl or aryl, X is O, NR.sub.4or S, and Y
is ##STR29## wherein R.sub.14, R.sub.15 and R.sub.16 are
independently hydrogen, hydroxy, OR.sub.9, OC(O)R.sub.10,
OS(O)R.sub.10, CHO, C(O)R.sub.10, COOH, CO.sub.2R.sub.10,
CONR.sub.3R.sub.4, alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl,
aryl, heteroaryl, thio, alkylthio, amino, alkylamino, dialkylamino,
nitro or halo, or any two of R.sub.14, R.sub.15 and R.sub.16 are
fused together to form a cyclic alkyl, aromatic or heteroaromatic
structure, and pharmaceutically acceptable salts thereof
2. The method of claim 1 wherein the melanoma cells or melanoma
display resistance to the chemotherapeutic agent.
3. The method of claim 2, wherein the sensitivity of the melanoma
cells or melanoma to the chemotherapeutic agent is restored as a
result of the treatment.
4. The method of claim 1, wherein the compound of formula (I) is
administered to a subject in need of such treatment
5. The method of claim 1 wherein the chemotherapeutic agent is
selected from carboplatin, cisplatin, paclitaxel, docatexel,
gemcitabine, and topotecan.
6. The method of claim 5 wherein the chemotherapeutic agent is
carboplatin.
7. The method of claim 1 wherein the compound of formula (I) is
dehydroequol.
8. A method for the treatment or prevention of melanoma in a
subject, the method comprising administering to the subject a
therapeutically effective amount of a compound of formula (I) as
defined in claim 1 and a chemotherapeutic agent.
9. The method of claim 8 wherein the compound of formula (I) is
administered prior to the chemotherapeutic agent.
10. The method of claim 8 wherein the compound of formula (I) and
the chemotherapeutic agent are administered simultaneously.
11. The method of claim 8 wherein the therapy follows observed
resistance in the melanoma cells or melanoma to a chemotherapeutic
agent.
12. The method of claim 8 wherein the chemotherapeutic agent is
selected from carboplatin, cisplatin, paclitaxel, docataxel,
gemcitabine and topotecan.
13. The method of claim 12 wherein the chemotherapeutic agent is
carboplatin.
14. The method of claim 8 wherein the compound of formula (I) is
dehydroequol.
15. Combination therapy for the treatment or prevention of melanoma
comprising administering to a subject a therapeutically effective
amount of a compound of formula (I) as defined in claim 1 and a
chemotherapeutic agent.
16. A pharmaceutical composition for the treatment or prevention of
melanoma, the composition comprising at least one isoflavonoid
compound of formula (I) and at least one chemotherapeutic
agent.
17. The composition of claim 16 wherein the chemotherapeutic agent
is selected from: carboplatin, cisplatin, paclitaxel, docatexel,
gemcitabine and topotecan.
18. The composition of claim 16 wherein the compound of formula (I)
is dehydroequol.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation-In-Part of U.S.
application Ser. No. 10/530,176, filed Mar. 9, 2006; which is a 371
of PCT/AU03/01296, filed Oct. 2, 2003; the disclosures of each of
which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to combination therapies involving
anticancer chemotherapeutic agents and isoflavones or analogues
thereof. The invention further relates to compounds, compositions,
methods and therapeutic uses involving, containing, comprising,
including and/or for preparing platinum-isoflavonoid complexes
suitable for use in the combination therapies of the invention.
BACKGROUND
[0003] The regulation of cell division (mitosis) is of critical
importance to the normal growth and development of a multicellular
organism, as well as the homeostatic maintenance of tissues, and
the ability of certain cell types to respond appropriately to
environmental cues.
[0004] Oncogenesis is the process by which normal cells lose the
ability to regulate progression through the cell cycle via
"checkpoints" (mitosis), and the ability to undergo apoptosis due
to acquired or inherited damage to regulatory genes. Those cells
containing aberrant genetic information may undergo clonal
expansion and act as a site for additional genetic alteration, at
which time uncontrolled proliferation results in the formation of a
neoplasm.
[0005] Neoplasms are generally classified as benign or malignant.
Benign tumours proliferate locally and are composed of
differentiated cells resembling those of the tissue of origin, the
edge of the tumour remaining well defined, and usually
encapsulated. Malignant neoplasms (classically termed "cancers")
are not encapsulated and their edges are ill-defined, the cells are
also less well differentiated than the cells of origin, and show
increased mitotic activity.
[0006] Localised, chronic irritation or inflammation can also cause
cells to divide abnormally, resulting in abnormal growths or
cellular masses, or tumours. Reactive cellular growth responses to
clearly defined, chronic irritant stimulation are described as
metaplasias. In dysplasias, there is a disorganisation of the
pattern of squamous epithelium in tissues such as the skin,
oesophagus and uterus in response to chronic irritation or
inflammation.
[0007] Numerous compounds are commercially available as
chemotherapeutic agents for destruction of abnormally proliferating
cells in benign and malignant neoplasias, dyplasias and
metaplasias. Predicting the responsiveness of a given
tumour-related disease type to a particular drug is difficult, as
each disease type is different and may respond to different
treatments. Generally, clinical treatment of cancer and other
cellular proliferative disorders involves having different
chemotherapy treatment options for each condition.
[0008] An example of an important chemotherapeutic agent is the
platinum-based compound cisplatin (cis-diamminedichloroplatinum
(H); cis-Cl.sub.2(NH.sub.3)Pt). Cisplatin has a square planar
geometry, with each of the two chloride groups (and likewise, each
of the two amine groups) being adjacent, or cis, to each other.
[0009] Cisplatin was first approved for human use in the late
1970's and is prescribed for the treatment of a variety of tumours
including germ-cell, advanced bladder carcinoma, adrenal cortex
carcinoma, breast, testicular and ovarian cancer, head and neck
carcinoma and lung carcinoma.
[0010] Cisplatin is active against proliferating or cancerous cells
by binding to DNA and interfering with its repair mechanism,
eventually leading to cell death. It is thought that the first step
in the cellular process is that a molecule of water replaces one of
the chloride ions of cisplatin. The resulting intermediate
structure can then bind to a single nitrogen on a DNA nucleotide.
Following that, the second chloride is also replaced by another
water molecule and the platinum agent then binds to a second
nucleotide. Binding studies of cisplatin with DNA have indicated a
preference for nitrogen 7 on two adjacent guanines on the same
strand. It also binds to adenine and across strands to a lesser
extent.
[0011] The binding of cisplatin to DNA causes production of
intrastrand cross-links and formation of DNA adducts. The adducts
or cisplatin-DNA complexes attract the attention of DNA repair
proteins which become irreversibly bound, The resulting distortion
to the shape of the DNA by the binding of cisplatin prevents
effective repair and hence, cell death.
[0012] Other well known chemotherapeutic agents include
carboplatin, the taxanes such as paclitaxel, docetaxel,
gemcitabine, 5-fluorouracil, methotrexate and the
tetracyclines.
[0013] Patients undergoing cancer chemotherapy often have to
contend with quite severe and debilitating side effects due to the
toxicity of the active agents. Common side effects of chemotherapy
are nausea and vomiting. Other side effects include temporary
reduction in bone marrow function, numbness or tingling in hands or
feet, changes in hearing, temporary taste alterations, loss of
appetite, diarrhoea and allergic reactions.
[0014] Chemotherapy regimes are further complicated by the efficacy
of currently available chemotherapeutic agents against various
cancers or other tumour types sometimes being insufficient. For
example, some cancer cells have developed natural tolerance against
the therapeutic agents. Further, some therapeutic or prophylactic
agents exert side effects, or can induce the development of
tolerance in abnormally dividing cells during clinical use, leading
to a situation in which certain tumour types become multiply drug
resistant. Multidrug resistance thus remains a main complication of
long-term successful tumour chemotherapy.
[0015] Melanoma is one of the most prevalent cancers in Australia
and the incidence of melanoma is on the rise around the world.
Indeed, in the United States the incidence of melanoma is rising
faster than that of any other malignancy. However, melanoma is
typically resistant to standard chemotherapy and radiation therapy.
A number of chemotherapeutic agents, including platinum-based and
taxane drugs have been used to treat melanoma but with
disappointing response rates. Our understanding of the resistance
mechanisms employed by melanoma to avoid the cytotoxic effects of
conventional therapeutics is limited, as is our ability to devise
alternative therapeutics to overcome resistance." The increased
incidence of melanoma, compounded by the lack of effective therapy,
in particular once the disease has metastasized, underscores the
need for improved methods of treating patients with melanoma.
[0016] Accordingly there is a strong need to identify new, improved
and/or alternative pharmaceutical compositions, agents and
treatment regimes against chemoresistance, mutated growth or
proliferation of cells in cancer and other age-related diseases.
There is a further need for chemotherapeutic agents which address
undesirable side effects associated with known agents. There is
also an everpresent need for physicians to have at their disposal
alternative therapeutic options for the treatment of malignancies
that display innate or acquired resistance to currently available
therapeutics. Agents which can act synergistically with other
chemotherapeutics thereby improving efficacy are highly sought
after. Agents that enhance the efficacy of conventional
therapeutics, in conjunction with reducing dosage and treatment
regimens, and enhance the targeting of malignant cells, will
hopefully result in fewer side-effects commonly associated with
standard chemotherapy.
[0017] It is an object of the present invention to provide
pharmaceutical compositions and methods for the treatment,
amelioration or prophylaxis of cancer and diseases associated with
oxidant stress. The present invention also seeks to provide
pharmaceutical compositions and methods for targeting neoplastic
cells for treatment, which compositions and methods provide
improved cell activity in terms of targeting function, improved
delivery of toxic agents and/or improvement or restoration of
chemosensitivity.
SUMMARY OF THE INVENTION
[0018] This application now describes new treatment regimes and
chemotherapeutic compositions and compounds. The invention is based
on the totally unexpected activity of isoflavonoid compounds in
restoring or, addressing the chemo-selectivity or activity of
anticancer agents, synergistic compositions including same and
novel isoflavonoid-drug complexes.
[0019] According to an aspect of the present invention there is
provided a method of increasing the sensitivity of cancer cells or
a tumour to a chemotherapeutic agent by contacting said cells or
tumour with an isoflavonoid compound of formula (I) as set out
below.
[0020] Compounds of the general formula (I) are the isoflavonoid
compounds represented by the formula: ##STR1## in which [0021]
R.sub.1, R.sub.2 and Z are independently hydrogen, hydroxy,
OR.sub.9, OC(O)R.sub.10, OS(O)R.sub.10, CHO, C(O)R.sub.10, COOH,
CO.sub.2R.sub.10, CONR.sub.3R.sub.4, alkyl, haloalkyl, arylalkyl,
alkenyl, alkynyl, aryl, heteroaryl, alkylaryl, alkoxyaryl, thio,
alkylthio, amino, alkylamino, dialkylamino, nitro or halo, or
[0022] R.sub.2 is as previously defined, and R.sub.1 and Z taken
together with the carbon atoms to which they are attached from a
five-membered ring selected from ##STR2## [0023] R.sub.1 is as
previously defined, and R.sub.2 and Z taken together with the
carbon atoms to which they are attached form a five-membered ring
selected from ##STR3## and [0024] W is R.sub.1, A is hydrogen,
hydroxy, NR.sub.3R.sub.4 or thio, and B is selected from ##STR4## W
is R.sub.1, and A and B taken together with the carbon atoms to
which they are attached form a six-membered ring selected from
##STR5## [0025] W, A and B taken together with the groups to which
they are associated are selected from ##STR6## [0026] W and A taken
together with the groups to which they are associated are selected
from ##STR7## and B is selected from ##STR8## wherein [0027]
R.sub.3 is hydrogen, alkyl, arylalkyl, alkenyl, aryl, an amino
acid, C(O)R.sub.11 where R.sub.11 is hydrogen, alkyl, aryl,
arylalkyl or an amino acid, or CO.sub.2R.sub.12 where R.sub.12 is
hydrogen, alkyl, haloalkyl, aryl or arylalkyl, [0028] R.sub.4 is
hydrogen, alkyl or aryl, or [0029] R.sub.3 and R.sub.4 taken
together with the nitrogen to which they are attached comprise
pyrrolidinyl or piperidinyl, [0030] R.sub.5 is hydrogen,
C(O)R.sub.11 where R.sub.11 is as previously defined, or
CO.sub.2R.sub.12 where R.sub.12 is as previously defined, [0031]
R.sub.6 is hydrogen, hydroxy, alkyl, aryl, amino, thio,
NR.sub.3R.sub.4, COR.sub.11 where R.sub.11 is as previously
defined, CO.sub.2R.sub.12 where R.sub.12 is as previously defined
or CONR.sub.3R.sub.4, [0032] R.sub.7 is hydrogen, C(O)R.sub.11
where R.sub.11 is as previously defined, alkyl, haloalkyl, alkenyl,
aryl, arylalkyl or Si(R.sub.13).sub.3 where each R.sub.13 is
independently hydrogen, alkyl or aryl, [0033] R.sub.8 is hydrogen,
hydroxy, alkoxy or alkyl, [0034] R.sub.9 is alkyl, haloalkyl, aryl,
arylalkyl, C(O)R.sub.11 where R.sub.11 is as previously defined, or
Si(R.sub.13).sub.3 where R.sub.13 is as previously defined, [0035]
R.sub.10 is hydrogen, alkyl, haloalkyl, amino, aryl, arylalkyl, an
amino acid, alkylamino or dialkylamino, [0036] the drawing
represents either a single bond or a double bond, [0037] T is
independently hydrogen, alkyl or aryl, [0038] X is O, NR.sub.4 or
S, and [0039] Y is: ##STR9## wherein [0040] R.sub.14, R.sub.15 and
R.sub.16 are independently hydrogen, hydroxy, OR.sub.9,
OC(O)R.sub.10, OS(O)R.sub.10, CHO, C(O)R.sub.10, COOH,
CO.sub.2R.sub.10, CONR.sub.3R.sub.4, alkyl, haloalkyl, arylalkyl,
alkenyl, alkynyl, aryl, heteroaryl, thio, alkylthio, amino,
alkylamino, dialkylamino, nitro or halo, or any two of R.sub.14,
R.sub.15 and R.sub.16 are fused together to form a cyclic alkyl,
aromatic or heteroaromatic structure, and pharmaceutically
acceptable salts thereof
[0041] In a preferred embodiment the cancer cells or tumour are
pre-treated with a compound of formula (I), prior to treatment with
the chemotherapeutic agent.
[0042] In another embodiment, the compound of formula (I) is
administered concurrently with the. chemotherapeutic agent.
[0043] In a further embodiment the compound of formula (I) is
administered after resistance to a chemotherapeutic agent is
observed in cancer cells and tumours, and in particular after
multidrug resistance is observed.
[0044] In a further embodiment the sensitivity of the cancer cells
or tumour to the chemotherapeutic agent is restored or
regenerated.
[0045] In an embodiment, the cancer cells are melanoma cells and
the tumour is a melanoma.
[0046] According to another aspect there is provided a combination
therapy comprising administering to a subject a therapeutically
effective amount of a compound of formula (I) and a
chemotherapeutic agent.
[0047] Combination therapy in accordance with the invention is
typically for the treatment or prevention of cell proliferation and
cancers including benign prostatic hypertrophy; melanoma; breast
cancer; uterine cancer; ovarian cancer; testicular cancer; large
bowel cancer; endometrial cancer; prostatic cancer; uterine cancer;
and diseases associated with oxidant stress including cancer,
myocardial infarction stroke, arthritis, sunlight induced skin
damage or cataracts (for convenience hereafter referred to as the
"therapeutic indications").
[0048] In a preferred embodiment the administration of the compound
of formula (I) precedes the administration of the chemotherapeutic
agent. Alternatively, the administration is concurrent. In a
further embodiment the combination therapy follows observed
resistance by cancer cells and tumours to a chemotherapeutic agent
or agents.
[0049] In a preferred embodiment the subject cell growth is
proliferation, and the subject down-regulation is killing off the
proliferating cells. The condition being treated is typically
cancer, more typically a metastatic cancer. The cancer may be
selected from melanoma, breast cancer, prostatic cancer, testicular
cancer, ovarian cancer, uterine cancer and/or colorectal cancer,
and more preferably is ovarian cancer, prostatic cancer or
pancreatic cancer.
[0050] In a further aspect of the present invention there is
provided a method for the treatment of cancer in a subject, the
method comprising administering to the subject a therapeutically
effective amount of a compound of formula (I) and a
chemotherapeutic agent.
[0051] According to a further aspect of the present invention there
is provided a method for increasing the sensitivity of melanoma
cells or a melanoma to a chemotherapeutic agent by contacting said
cells or melanoma with an isoflavonoid compound of formula (I).
[0052] In an embodiment, the chemotherapeuatic agent is
carboplatin. In an embodiment, the compound of formula (I) is
dehydroequol.
[0053] According to a further aspect of the preset invention there
is provided a method for the treatment of melanoma in a subject,
the method comprising administering to the subject a
therapeutically effective amount of a compound of formula (I) and a
chemotherapeutic agent.
[0054] In further aspects of the invention there is provided
methods for the manufacture of medicaments for the above stated
methods of the invention and pharmaceutical agents useful for
same.
[0055] This application also describes new therapeutic compositions
and complexes comprising platinum-based pharmaceutical agents. The
invention is based on the totally unexpected biological activity of
new platinum-isoflavonoid complexes and of isoflavonoid compounds
of formula (I) which form synergistic compositions or complexes
with platinum-based chemotherapeutic agents.
[0056] Accordingly, a further aspect of the invention provides a
pharmaceutical composition for the treatment or prevention of cell
proliferation and cancers, the composition comprising at least one
isoflavonoid compound of formula (I) and at least one
chemotherapeutic agent.
[0057] In an embodiment, the cancer is melanoma. In an embodiment,
the chemotherapeutic agent is selected from: carboplatin,
cisplatin, paclitaxel, docatexel, gemcitabine and topotecan. In an
embodiment, the compound of formula (I) is dehydroequol.
[0058] The compositions and platinum-isoflavonoid complexes are
important targeting agents for the delivery of toxic signals to
cells. The compositions and methods of the invention are directed
to treating a condition in a subject, which condition is
characterised by the undesirable, detrimental or otherwise unwanted
growth or proliferation of cells.
[0059] Also disclosed herein are platinum-isoflavonoid complexes
and analogues thereof described by general formula (II): ##STR10##
in which [0060] R.sub.A, R.sub.B, R.sub.C, and R.sub.D are
independently halo, hydroxy, XR.sub.E, alkoxy, OC(O)R.sub.F,
OS(O)R.sub.F, thio, alkylthio, amino, alkylamino or dialkylamino,
[0061] X is O, NR.sub.F or S, and [0062] R.sub.F is hydrogen,
alkyl, arylalkyl, alkenyl, aryl or an amino acid, wherein [0063] at
least one of R.sub.A, R.sub.B, R.sub.C, and R.sub.D, and preferably
only R.sub.A, is XR.sub.E where R.sub.E is an isoflavonoid compound
represented by general formula (I) set out above or is derived from
or is a radical or ion of the isoflavonoid compound (I) and ligates
to the platinum through any one or more of the heteroatoms X or a
radical of the heteroatoms defined as part of R.sub.E or
alternatively by a double bond on the isoflavonoid compound (I) and
[0064] when R.sub.A is XR.sub.E, R.sub.B, R.sub.C and/or R.sub.D
together may form part of a bidentate or tridentate ligand of
general formulae (B) and (T) respectively ##STR11## [0065] wherein
L represents a ligating atom chosen from N, O and S, [0066] n is
from 0 to 8, and [0067] each R6 is independently as defined above
or may together form part of a cyclic alkyl, aromatic or
heteroaromatic structure, [0068] which platinum-isoflavonoid
complexes include pharmaceutically acceptable salts thereof.
[0069] It has also surprisingly been found by the inventors that
platinum-isoflavonoid complexes of the general formula (II) have
particular utility and effectiveness in the treatment or prevention
of the therapeutic indications noted above.
[0070] Thus also disclosed herein is a method for, the treatment or
prevention of the therapeutic indications described above which
method comprises administering to a subject a therapeutically
effective amount of one or more platinum-isoflavonoid complexes of
the formula (II) as defined above.
[0071] Also disclosed herein is a method of treating a condition in
a mammal, which condition is characterised by the undesirable,
detrimental or otherwise unwanted growth of cells, said method
comprising administering to said mammal an effective amount a
platinum-isoflavonoid complex of formula (II) for a time and under
conditions sufficient to down-regulate the growth of said
cells.
[0072] In a preferred embodiment the subject cell growth is
proliferation, and the subject down-regulation is killing off the
proliferating cells. The condition being treated is typically
cancer, more typically a metastatic cancer. The cancer may be
selected from melanoma, breast cancer, prostatic cancer, testicular
cancer, ovarian cancer, uterine cancer and/or colorectal cancer,
and more preferably is ovarian cancer, prostatic cancer or
pancreatic cancer.
[0073] Also disclosed herein is a method of down-regulating the
growth of cells, said method comprising contacting said cells with
an effective amount of a platinum-isoflavonoid complex of formula
(II).
[0074] In a preferred embodiment the subject cell growth is
proliferation, and the subject down-regulation is killing off the
proliferating cells.
[0075] Also disclosed herein is the use of platinum-isoflavonoid
complexes of the formula (II) for the manufacture of a medicament
for the treatment or prevention of one or more of the therapeutic
indications.
[0076] Also disclosed herein is the use of one or more
platinum-isoflavonoid complexes of the formula (II) in the
treatment or prevention of one or more of the therapeutic
indications.
[0077] Also disclosed herein is an agent for the treatment or
prevention of the therapeutic indications which comprises one or
more platinum isoflavonoid complexes of the formula (II) either
alone or in association with one or more carriers or
excipients.
[0078] Also disclosed herein is a therapeutic composition which
comprises one or more platinum-isoflavonoid complexes of the
formula (II) in association with one or more pharmaceutical
carriers and/or excipients.
[0079] Also disclosed herein is a drink or food-stuff, which
contains one or more platinum-isoflavonoid complexes of the formula
(II).
[0080] Also disclosed herein are compositions comprising a platinum
complex of the general formula (IIa), ##STR12## in which [0081]
R.sub.G, R.sub.H, R.sub.I,and R.sub.J are independently halo,
hydroxy, alkoxy, OC(O)R.sub.K, OS(O)R.sub.K, thio, alkylthio,
amino, alkylamino or dialkylamino, [0082] X is O, NR.sub.K or S,
and [0083] R.sub.K is hydrogen, alkyl, arylalkyl, alkenyl, aryl or
an amino acid, or a pharmaceutically acceptable salt thereof, and
an isoflavonoid compound of general formula (I) as defined
above.
[0084] These compositions comprising a platinum complex of the
formula (IIa) and an isoflavonoid compound of the formula (I) are
found to have particular utility, effectiveness and synergism in
the treatment or prevention of the therapeutic indications set out
above.
[0085] Thus also disclosed herein is a method for the treatment or
prevention of the therapeutic indications which comprises
administering to a subject a therapeutically effective amount of
compositions comprising a platinum complex of the formula (IIa) in
conjunction with an isoflavonoid compound of formula (I).
[0086] Also disclosed herein is the combined use of a platinum
complex of the formula (IIa) and an isoflavonoid compound of the
formula,(I) in the manufacture of a medicament for the treatment or
prevention of the therapeutic indications.
[0087] Also disclosed herein is the use of a platinum complex of
the formula (IIa) and an isoflavonoid compound of the formula (I)
in the treatment or prevention of the therapeutic indications.
[0088] Also disclosed herein is a kit comprising a platinum complex
of the formula (IIa) and an isoflavonoid compound of the formula
(I) either alone or in association with one or more carriers or
excipients.
[0089] Also disclosed herein is an agent for the treatment, or
prevention of the therapeutic indications which comprises a
composition comprising a platinum complex of the formula (IIa) and
an isoflavonoid compound of the formula (I) either alone or in
association with one or more carriers or excipients.
[0090] Throughout this specification and the claims which follow,
unless the text requires otherwise, the word "comprise", and
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated integer or step or group of
integers or steps but not the exclusion of any other integer or
step or group of integers or steps.
BRIEF DESCRIPTION OF THE FIGURES
[0091] FIG. 1 represents the cell viability of various cancer cell
lines over different concentrations of carboplatin.
[0092] FIG. 2 represents the cell viability of various cancer cell
lines over different concentrations of paclitaxel.
[0093] FIG. 3 represents the cell viability of various cancer cell
lines over different concentrations of carboplatin following
dehydroequol pre-treatment.
[0094] FIG. 4 represents the cell viability of various cancer cell
lines over different concentrations of paclitaxel following
dehydroequol pre-treatment.
[0095] FIG. 5 represents a Western Blot analysis of carboplatin or
paclitaxel treatment resistant ovarian cancer CP70 cells with and
without dehydroequol pre-treatment.
[0096] FIG. 6 represents tumour mass comparison of dehydrequol and
cisplatin when delivered as single active agents or combination
therapy with the 5% HPBCD vehicle control group.
[0097] FIG. 7 represents tumour volume comparison of dehydrequol
and cisplatin when delivered as single active agents or combination
therapy with the 5% HPBCD vehicle control group.
[0098] FIG. 8 represents a body weight comparison as an indicator
of toxicity in each dehydroequol, cisplatin or combination
treatment group in comparison with the HPBCD 5% vehicle
control.
[0099] FIG. 9 represents unpaired t-tests showing differences in
expression of XIAP between benign, primary, metastatic, and all
malignant (primary and metastatic) melanoma specimens.
[0100] FIG. 10 represents a cell viability assay showing the effect
of dehydroequol on three melanoma cell lines, YUGEN8, YUMAC, and
YUSAC. Each experiment was performed in triplicate and viability
was determined by the CellTiter 96 Aqueous Once Solution Cell
Proliferation Assay. Cells were treated with 10 ug/mL of
dehydroequol for 24 hours.
[0101] FIG. 11 represents the effect of dehydroequol (Pxd) on YUMAC
(A)and YUSAC (B) cells. Cells were treated with a single dose of
dehydroequol (10 ug/mL) for varying time points (0, 4, 8, and 24
hours), XIAP expression was determined by western blot analysis.
Caspase-3,-8, and -9 activities were determined by the Caspase-Glo
3, 8, and 9 assays, respectively.
[0102] FIG. 12 represents cell viability assays demonstrating the
effect of dehydroequol on melanoma cells. (A) Cells were treated
with increasing doses of Carboplatin (50-200 ug/mL) alone or (B)
cells were pretreated with 10 ug/mL dehydroequol for 4 hours
followed by treatment with increasing doses of Carboplatin. Each
experiment was performed in triplicate and viability was determined
by the CellTiter 96 Aqueous One Solution Cell Proliferation Assay,
reported as a percentage of viable cells relative to untreated
cells.
[0103] FIG. 13 represents the effect of pretreatment with
dehydroequol on the apoptotic cascade. YUMAC cells received either
no treatment, 200 ug/mL of Carboplatin for 24 hours, 10 ug/mL of
dehydroequol for 4 hours, or 10 ug/mL of dehydroequol for 4 hours
followed by 200 ug/mL Carboplatin for 24 hours. Both pro- and
antiapoptotic protein expression was determined by Western blot
analysis. Activity of caspases -3,-8, and -9 were determined using
the Caspase-Glo 3, 8, and 9 assays, respectively.
[0104] FIG. 14 represents the in vivo sensitizing effect of
dehydroequol on A2780 mouse xenograft model. (A) Tumor mass
measured in animals treated with vehicle (Group 1), dehydroequol
(Pxd) 25 mg/kg (Group 2), Topotecan 2 mg/kg (Group 3), Topotecan 1
mg/kg (Group 4), Pxd 25 mg/kg +Topotecan 2 mg/kg (Group 5), or Pxd
12.5 mg/kg+Topotecan 1 mg/kg (Group 6). *p<0.01 group 5 vs 3 and
group 6 vs 3;**p<0.01 group 2 vs 1 (B) Comparative mean terminal
tumor mass taken from the same groups of animals. Inset shows
tabulated mean tumor mass data and calculated %T/C values.
*p<0.01 (group 4 vs 3); **p<0.01 group 6 vs 4);***p<0.01
group 2 vs 1. Eight animals were tested in each group.
DETAILED DESCRIPTION OF THE INVENTION
[0105] The terms "isoflavonoid", "isoflavonoid" and "isoflavone" as
used herein are to be taken broadly to include ring-fused
benzopyran molecules having a pendent phenyl group from the pyran
ring based on a 1,2-diphenylpropane system. Thus, the classes of
compounds generally referred to as isoflavones, isoflavenes,
isoflavans, isoflavanones, isoflavanols and the like are
generically referred to herein as isoflavones, isoflavone
derivatives or isoflavonoid compounds.
[0106] The term "alkyl" is taken to mean both straight chain and
branched chain alkyl groups such as methyl, ethyl, propyl,
isopropyl, butyl, isobutyl, secbutyl, teriary butyl, and the like.
The alkyl group has 1 to 10 carbon atoms, preferably from 1 to 6
carbon atoms, more preferably methyl, ethyl propyl or isopropyl.
The alkyl group may optionally be substituted by one or more of
fluorine, chlorine, bromine, iodine, carboxyl,
C.sub.1-C.sub.4-alkoxycarbonyl,
C.sub.1-C.sub.4-alkylamino-carbonyl,
di-(C.sub.1-C.sub.4-alkyl)-amino-carbonyl, hydroxyl,
C.sub.1-C.sub.4-alkoxy, formyloxy,
C.sub.1-C.sub.4-alkyl-carbonyloxy, C.sub.1-C.sub.4-alkylthio,
C.sub.3-C.sub.6-cycloalkyl or phenyl.
[0107] The term "aryl" is taken to include phenyl and naphthyl and
may be optionally substituted by one or more C.sub.1-C.sub.4-alkyl,
hydroxy, C.sub.1-C.sub.4-alkoxy, carbonyl,
C.sub.1-C.sub.4-alkoxycarbonyl, C.sub.1-C.sub.4-alkylcarbonyloxy or
halo.
[0108] The term "halo" is taken to include fluoro, chloro, bromo
and iodo, preferably fluoro and chloro, more preferably fluoro.
Reference to for example "haloalkyl" will include monohalogenated,
dihalogenated and up to perhalogenated alkyl groups. Preferred
haloalkyl groups are trifluoromethyl and pentafluoroethyl.
[0109] The term "pharmaceutically acceptable salt" refers to an
organic or inorganic moiety that carries a charge and that can be
administered in association with a pharmaceutical agent, for
example, as a counter-cation or counter-anion in a salt.
Pharmaceutically acceptable cations are known to those of skilled
in the art, and include but are not limited to sodium, potassium,
calcium, zinc and quaternary amine. Pharmaceutically acceptable
anions are known to those of skill in the art, and include but are
not limited to chloride, acetate, citrate, bicarbonate and
carbonate.
[0110] The term "pharmaceutically acceptable derivative" or
"prodrug" refers to a derivative of the active compound that upon
administration to the recipient, is capable of providing directly
or indirectly, the parent compound or metabolite, or that exhibits
activity itself. Prodrugs are included within the scope of the
present invention.
[0111] As used herein, the terms "treatment" and "prevention" and
the like are to be considered in their broadest context. In
particular, the term "treatment" does not necessarily imply that an
animal is treated until total recovery. Accordingly, "treatment"
includes amelioration of the symptoms or severity of a particular
condition or preventing or otherwise reducing the risk of
developing a particular condition.
[0112] As used herein the term "therapeutically effective amount"
includes within its meaning a non-toxic but sufficient amount of an
agent(s) or compound(s) to provide the desired therapeutic or
preventative effect. The exact amount required will vary from
subject to subject depending on factors such as the species being
treated, the age and general condition of the subject, the severity
of the condition being treated, the particular agent(s) being
administered and the mode of administration and so forth. Thus, it
is not possible to specify an exact "therapeutically effective
amount". However, for any given case, an appropriate
"therapeutically effective amount" may be determined by one of
ordinary skill in the art using only routine experimentation.
[0113] Preferred isoflavonoid compounds of formula (I) are selected
from general formulae (III)-(IX), and more preferably are selected
from general formulae (IV)-(IX): ##STR13## in which [0114] R.sub.1,
R.sub.2, R.sub.5, R.sub.6, R.sub.14, R.sub.15, W and Z are as
defined above, more preferably [0115] R.sub.1, R.sub.2, R.sub.14,
R.sub.15, W and Z are independently hydrogen, hydroxy, OR.sub.9,
OC(O)R.sub.10, C(O)R.sub.10, COOH, CO.sub.2R.sub.10, alkyl,
haloalkyl, arylalkyl, aryl, thio, alkylthio, amino, alkylamino,
dialkylamino, nitro or halo, [0116] R.sub.5 is hydrogen,
C(O)R.sub.11 where R.sub.11 is hydrogen, alkyl, aryl, or an amino
acid, or CO.sub.2R.sub.12 where R.sub.12 is hydrogen, alkyl or
aryl, [0117] R.sub.6 is hydrogen, hydroxy, alkyl, aryl, COR.sub.11
where R.sub.11 is as previously defined, or CO.sub.2R.sub.12 where
R.sub.12 is as previously defined, [0118] R.sub.9 is alkyl,
haloalkyl, arylalkyl, or C(O)R.sub.11 where R.sub.11 is as
previously defined, and [0119] R.sub.10 is hydrogen, alkyl, amino,
aryl, an amino acid, alkylamino or dialkylamino, more preferably
[0120] R.sub.1 and R.sub.14 are independently hydroxy, OR.sub.9,
OC(O)R.sub.10 or halo, [0121] R.sub.2, R.sub.15, W and Z are
independently hydrogen, hydroxy, OR.sub.9, OC(O)R.sub.10,
C(O)R.sub.10, COOH, CO.sub.2R.sub.10, alkyl, haloalkyl, or halo,
[0122] R.sub.5 is hydrogen, C(O)R.sub.11 where R.sub.11 is hydrogen
or alkyl, or CO.sub.2R.sub.12 where R.sub.12 is hydrogen or alkyl,
[0123] R.sub.6 is hydrogen or hydroxy, [0124] R.sub.9 is alkyl,
arylalkyl or C(O)R.sub.11 where R.sub.11 is as previously defined,
and [0125] R.sub.10 is hydrogen or alkyl, and more preferably
[0126] R.sub.1 and R.sub.14 are independently hydroxy, methoxy,
benzyloxy, acetyloxy or chloro, [0127] R.sub.2, R.sub.15, W and Z
are independently hydrogen, hydroxy, methoxy, benzyloxy, acetyloxy,
methyl, trifluoromethyl or chloro, [0128] R.sub.5 is hydrogen or
CO.sub.2R.sub.12 where R .sub.12 is hydrogen or methyl, and [0129]
R.sub.6 is hydrogen.
[0130] Particularly preferred isoflavonoid compounds of formula (I)
are selected from: ##STR14## ##STR15## ##STR16## ##STR17##
[0131] In a further embodiment the preferred isoflavonoid compounds
are the isoflav-3-ene and isoflavan compounds of general formula
(VI), and more preferred are the 3-ene compounds of the general
formula (VIa): ##STR18## in which [0132] R.sub.1, R.sub.2, R.sub.6,
R.sub.14, R.sub.15, W and Z are as defined above; more preferably
[0133] R.sub.1, R.sub.2, R.sub.14, R.sub.15, W and Z are
independently hydrogen, hydroxy, OR.sub.9, OC(O)R.sub.10,
C(O)R.sub.10, COOH, CO.sub.2R.sub.10, alkyl, haloalkyl, arylalkyl,
aryl, thio, alkylthio, amino, alkylamino, dialkylamino, nitro or
halo, [0134] R.sub.6 is hydrogen, hydroxy, alkyl, aryl, COR.sub.11
where R.sub.11 is as previously defined, or CO.sub.2R.sub.12 where
R.sub.12 is as previously defined, [0135] R.sub.9 is alkyl,
haloalkyl, arylalkyl, or C(O)R.sub.11 where R.sub.11 is as
previously defined, and [0136] R.sub.10 is hydrogen, alkyl, amino,
aryl, an amino acid, alkylamino or dialkylamino, more preferably
[0137] R.sub.1 is hydroxy, OR.sub.9, OC(O)R.sub.10 or halo, [0138]
R.sub.2, R.sub.14, R.sub.15, W and Z are independently hydrogen,
hydroxy, OR.sub.9, OC(O)R.sub.10, C(O)R.sub.10, COOH,
CO.sub.2R.sub.10, alkyl, haloalkyl, or halo, [0139] R.sub.6 is
hydrogen, [0140] R.sub.9 is alkyl, arylalkyl or C(O)R.sub.11 where
R.sub.11 is as previously defined, and [0141] R.sub.10 is hydrogen
or alkyl, and more preferably [0142] R.sub.1 is hydroxy, methoxy,
benzyloxy, acetyloxy or chloro, [0143] R.sub.2, R.sub.14, R.sub.15,
W and Z are independently hydrogen, hydroxy, methoxy, benzyloxy,
acetyloxy, methyl, trifluoromethyl or chloro, and [0144] R.sub.6 is
hydrogen, [0145] including pharmaceutically acceptable salts and
derivatives thereof.
[0146] In a most preferred embodiment of the invention the
isoflavonoid compound is dehydroequol, Cpd. 12. As such, particular
reference is made to dehydroequol in the description, Examples
which follow and accompanying drawings however this is not to be
taken as being unnecessarily limiting on the disclosure of the
invention provided herein.
[0147] Chemotherapeutic agents are generally grouped as
DNA-interactive agents, antimetabolites, tubulin-interactive
agents, hormonal agents, other agents such as asparaginase or
hydroxyurea. Each of the groups of chemotherapeutic agents can be
further divided by type of activity or compound. Chemotherapeutic
agents used in combination with the isoflavonoid compound of
formula (I) of the present invention, or salts thereof of the
present invention, may be selected from any of these groups but are
not limited thereto. For a detailed discussion of the
chemotherapeutic agents and their method of administration, see
Dorr, et al, Cancer Chemotherapy Handbook, 2d edition, pages 15-34,
Appleton and Lang (Connecticut, 1994) herein incorporated by
reference.
[0148] DNA-interactive agents include alkylating agents, e.g.
cisplatin, cyclophosphamide, altretamine; DNA strand-breakage
agents, such as bleomycin; intercalating topoisomerase I and II
inhibitors, e.g., topotecan, dactinomycin and doxorubicin);
nonintercalating topoisomerase I and II inhibitors such as,
etoposide and teniposide; the DNA minor groove binder plicamydin,
for example, and nucleoside analogs which inhibit DNA synthesis,
such as gemcitabine and flurouracil.
[0149] The alkylating agents form covalent chemical adducts with
cellular DNA, RNA, or protein molecules, or with smaller amino
acids, glutathione, or similar chemicals. Generally, alkylating
agents react with a nucleophilic atom in a cellular constituent,
such as an amino, carboxyl, phosphate, or sulfhydryl group in
nucleic acids, proteins, amino acids, or in glutathione. The
mechanism and the role of these alkylating agents in cancer therapy
is not well understood.
[0150] Typical alkylating agents include, but are not limited to,
nitrogen mustards, such as chlorambucil, cyclophosphamide,
isofamide, mechlorethamine, melphalan, uracil mustard; aziridine
such as thiotepa; methanesulphonate esters such as busulfan;
nitroso ureas, such as carmustine, lomustine, streptozocin;
platinum complexes, such as cisplatin, carboplatin; bioreductive
alkylator, such as mitomycin, and procarbazine, dacarbazine and
altretamine.
[0151] DNA strand breaking agents include bleomycin, for
example.
[0152] DNA topoisomerase II inhibitors include the following
intercalators, such as amsacrine, dactinomycin, daunorubicin,
doxorubicin (adriamycin), idarubicin, and mitoxantrone;
nonintercalators, such as etoposide and teniposide, for
example.
[0153] Antimetabolites interfere with the production of nucleic
acids by one of two major mechanisms. Certain drugs inhibit
production of deoxyribonucleoside triphosphates that are the
immediate precursors for DNA synthesis, thus inhibiting DNA
replication. Certain of the compounds are analogues of purines or
pyrimidines and are incorporated in anabolic nucleotide pathways.
These analogues are then substituted into DNA or RNA instead of
their normal counterparts.
[0154] Antimetabolites useful herein include, but are not limited
to, folate antagonists such as methotrexate and trimetrexate;
pyrimidine antagonists, such as fluorouracil, fluorodeoxyuridine,
CB3717, azacitidine, cytarabine, and floxuridine; purine
antagonists include mercaptopurine, 6-thioguanine, fludarabine,
pentostatin; and ribonucleotide reductase inhibitors include
hydroxyurea.
[0155] Tubulin interactive agents act by binding to specific sites
on tubulin, a protein that polymerizes to form cellular
microtubules. Microtubules are critical cell structure units. When
the interactive agents bind the protein, the cell can not form
microtubules. Tubulin interactive agents include the vinca
alkaloids vincristine and vinblastine, paclitaxel (Taxol) and
docetaxel, for example.
[0156] Hormonal agents are also useful in the treatment of cancers
and tumors. They are used in hormonally susceptible tumors and are
usually derived from natural sources. Hormonal agents include, but
are not limited to, estrogens, conjugated estrogens and ethinyl
estradiol and diethylstilbesterol, chlorttianisen and idenestrol;
progestins such as hydroxyprogesterone caproate,
medroxyprogesterone, and megestrol; and androgens such as
testosterone, testosterone propionate; fluoxymesterone, and
methyltestosterone.
[0157] Adrenal corticosteroids are derived from natural adrenal
cortisol or hydrocortisone. They are used because of their
anti-inflammatory benefits as well as the ability of some to
inhibit mitotic divisions and to halt DNA synthesis. These
compounds include, but are not limited to, prednisone,
dexamethasone, methylprednisolone, and prednisolone.
[0158] Leutinizing hormone releasing hormone agents or
gonadotropin-releasing hormone antagonists are used primarily the
treatment of prostate cancer. These include leuprolide acetate and
goserelin acetate. They prevent the biosynthesis of steroids in the
testes.
[0159] Antihormonal antigens include, for example, antiestrogenic
agents such as tamoxifen, antiandrogen agents such as flutamide,
and antiadrenal agents such as mitolane and aminoglutethimide.
[0160] Further agents include the following: hydroxyurea appears to
act primarily through inhibition of the enzyme ribonucleotide
reductase, and asparaginase is an enzyme which converts asparagine
to nonfunctional aspartic acid and thus blocks protein synthesis in
the tumour.
[0161] Preferred chemotherapeutic agents for use in the subject
invention are cisplatin, carboplatin, taxol (paclitaxel),
docataxel, fluorouracil, gemcitabine, fluxuridine, cyclophosphamide
ifosfamide, hexamethylmelamine, estramustine, mitomycin, topotecan
and docetaxel.
[0162] Compounds of formula (I) also e chemotherapeutic activity
and in this regard particular reference can be made to
dehydroequol, Cpd. 12.
[0163] Preferred bidentate and tridentate platinum ligands of the
present inventions include those commonly known in the art. For
example, suitable bidentate ligands may be selected from
ethylene-1,2-diamine and 1,10-phenathraline and other ligands well
known in the art.
[0164] Preferred platinum complexes are halo and amino substituted,
more preferably chloro and amine substituted, more preferably
cis-dichlorodiamino substituted. Preferred platinum-isoflavonoid
complexes are preferably halo and amino substituted, more
preferably cis-dichloroamino substituted or cis-diaminochloro
substituted.
[0165] Compounds of the present invention have particular
application in the treatment of diseases associated with or
resulting from estrogenic effects, androgenic effects, vasolidatory
and spasmodic effects, inflammatory effects and oxidative
effects.
[0166] The amount of compounds of formulae (I), (II) or (I) and
(IIa) which are required in a therapeutic treatment according to
the invention will depend upon a number of factors, which include
the specific application, the nature of the particular compound
used, the condition being treated, the mode of administration and
the condition of the patient. Compounds of formulae I or Ia and II
may be administered in a manner and amount as is conventionally
practised. See, for example, Goodman and Gilman, The
Pharmacological Basis of Therapeutics, 1299 (7th Edition, 1985).
The specific dosage utilised will depend upon the condition being
treated, the state of the subject, the route of administration and
other well known factors as indicated above. In general, a daily
dose per patient may be in the range of 0.1 mg to 10 g; typically
from 0.5 mg to 1 g; preferably from 50 mg to 200 mg. Importantly
the synergistic relationship of the isoflavonoid compounds of
general formula (I) and the chemotherapeutic agent allow for
significant reductions in dosage regimes of relatively toxic drugs
such as cisplatin, paclitaxel and carboplatin for example.
[0167] Other preferred dosage regimes and amounts are set out in
the Examples and accompanying drawings.
[0168] The production of a pharmaceutical composition for the
treatment of the therapeutic indications herein described (for
convenience hereafter referred to as the "active compounds") are
typically admixed with one or more pharmaceutically or
veterinarially acceptable carriers and/or excipients as are well
known in the art.
[0169] The carrier must, of course, be acceptable in the sense of
being compatible with any other ingredients in the formulation and
must not be deleterious to the subject. The carrier or excipient
may be a solid or a liquid, or both, and is preferably formulated
with the compound as a unit-dose, for example, a tablet, which may
contain from 0.5% to 59% by weight of the active compound, or up to
100% by weight of the active compound. One or more active compounds
may be incorporated in the formulations of the invention, which may
be prepared by any of the well known techniques of pharmacy
consisting essentially of admixing the components, optionally
including one or more accessory ingredients.
[0170] The formulations of the invention include those suitable for
oral, rectal, optical, buccal (for example, sublingual), parenteral
(for example, subcutaneous, intramuscular, intradermal, or
intravenous) and transdermal administration, although the most
suitable route in any given case will depend on the nature and
severity of the condition being treated and on the nature of the
particular active compound which is being used.
[0171] Formulation suitable for oral administration may be
presented in discrete units, such as capsules, sachets, lozenges,
or tablets, each containing a predetermined amount of the active
compound; as a powder or granules; as a solution or a suspension in
an aqueous or non-aqueous liquid; or as an oil-in-water or
water-in-oil emulsion. Such formulations may be prepared by any
suitable method of pharmacy which includes the step of bringing
into association the active compound and a suitable carrier (which
may contain one or more accessory ingredients as noted above). In
general, the formulations of the invention are prepared by
uniformly and intimately admixing the active compound with a liquid
or finely divided solid carrier, or both, and then, if necessary,
shaping the resulting mixture such as to form a unit dosage. For
example, a tablet may be prepared by compressing or moulding a
powder or granules containing the active compound, optionally with
one or more accessory ingredients. Compressed tablets may be
prepared by compressing, in a suitable machine, the compound of the
free-flowing, such as a powder or granules optionally mixed with a
binder, lubricant, inert diluent, and/or surface active/dispersing
agent(s). Moulded tablets may be made by moulding, in a suitable
machine, the powdered compound moistened with au inert liquid
binder.
[0172] Formulations suitable for buccal (sublingual) administration
include lozenges comprising the active compound in a flavoured
base, usually sucrose and acacia or tragacanth; and pastilles
comprising the compound in an inert base such as gelatin and
glycerin or sucrose and acacia.
[0173] Compositions of the present invention suitable for
parenteral administration conveniently comprise sterile aqueous
preparations of the active compounds, which preparations are
preferably isotonic with the blood of the intended recipient. These
preparations are preferably administered intravenously, although
administration may also be effected by means of subcutaneous,
intramuscular, or intradermal injection. Such preparations may
conveniently be prepared by admixing the compound with water or a
glycine buffer and rendering the resulting solution sterile and
isotonic with the blood. Injectable formulations according to the
invention generally contain from 0.10/% to 60% w/v of active
compound(s) and are administered at a rate of 0.1 ml/minute/kg or
as appropriate. Parenteral administration is a preferred route of
administration for the compounds of the present invention.
[0174] Formulations suitable for rectal administration are
preferably presented as unit dose suppositories. These may be
prepared by admixing the active compound with one or more
conventional solid carriers, for example, cocoa butter, and then
shaping the resulting mixture.
[0175] Formulations or compositions suitable for topical
administration to the skin preferably take the form of an ointment,
cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which
may be used include Vaseline, lanoline, polyethylene glycols,
alcohols, and combination of two or more thereof. The active
compound is generally present at a concentration of from 0.1% to
0.5% w/w, for example, from 0.5% to 2% w/w. Examples of such
compositions include cosmetic skin creams.
[0176] Formulations suitable for transdermal administration may be
presented as discrete patches adapted to remain in intimate contact
with the epidermis of the recipient for a prolonged period of time.
Such patches suitably contain the active compound as an optionally
buffered aqueous solution of, for example, 0.1 M to 0.2 M
concentration with respect to the said active compound.
[0177] Formulations suitable for transdermal administration may
also be delivered by iontophoresis (see, for example,
Pharmaceutical Research 3 (6), 318 (1986)) and typically take the
form of an optionally buffered aqueous solution of the active
compound. Suitable formulations comprise citrate or bis/tris buffer
(pH 6) or ethanol/water and contain from 0.1 M to 0.2 M active
ingredient.
[0178] The active compounds may be provided in the form of food
stuffs, such as being added to, admixed into, coated, combined or
otherwise added to a food stuff, The term food stuff is used in its
widest possible sense and includes liquid formulations such as
drinks including dairy products and other foods, such as health
bars, desserts, etc. Food formulations containing compounds of the
invention can be readily prepared according to standard
practices.
[0179] Therapeutic methods, uses and compositions may be for
administration to humans and other animals, including mammals such
as companion and domestic animals (such as dogs and cats) and
livestock animals (such as cattle, sheep, pigs and goats), birds
(such as chickens, turkeys, ducks) and the like.
[0180] The active compound or pharmaceutically acceptable
derivatives prodrugs or salts thereof can also be co-administered
with other active materials that do not impair the desired action,
or with materials that supplement the desired action, such as
antibiotics, antifungals, antiinflammatories, or antiviral
compounds. The active agent can comprise two or more isoflavones or
derivatives thereof in combination or synergistic mixture. The
active compounds can also be administered with lipid lowering
agents such as probucol and nicotinic acid; platelet aggregation
inhibitors such as aspirin; antithrombotic agents such as coumadin;
calcium channel blockers such as verapamil, diltiazem, and
nifedipine; angiotensin converting enzyme (ACE) inhibitors such as
captopril and enalapril, and .beta.-blockers such as propanolol,
terbutalol, and labetalol. The compounds can also be administered
in combination with nonsteriodal antiinflammatories such as
ibuprofen, indomethacin, aspirin, fenoprofen, mefenamic acid,
flufenamic acid and sulindac. The compounds can also be
administered with corticosteroids.
[0181] The co-administration may be simultaneous or sequential.
Simultaneous administration may be effected by the compounds being
in the same unit dose, or in individual and discrete unit doses
administered at the same or similar time. Sequential administration
may be in any order as required and typically will require an
ongoing physiological effect of the first or initial active agent
to be current when the second or later active agent is
administered, especially where a cumulative or synergistic effect
is desired.
[0182] The isoflavones of formula (I) for use in the present
invention may be derived from any number of sources readily
identifiable to a person skilled in the art. Preferably, they are
obtained in the form of concentrates or extracts from plant
sources. Again, those skilled in the art will readily be able to
identify suitable plant species, however, for example, plants of
particular use in the invention include leguminous plants. More
preferably, the isoflavone extract is obtained from chickpea,
lentils, beans, red clover or subterranean clover species and the
like.
[0183] Isoflavone extracts may be prepared by any number of
techniques known in the art. For example, suitable isoflavone
extracts may be prepared by water/organic solvent extraction from
the plant source. It will be appreciated that an isoflavone extract
may be prepared from any single tissue of a single species of plant
or a combination of two or more different tissues thereof.
Similarly, an extract may be prepared from a starting material
which contains a heterogeneous mixture of tissues from two or more
different species of plant.
[0184] Generally, where an isoflavone extract is prepared from
plant material, the material may be comminuted or chopped into
smaller pieces, partially comminuted or chopped into smaller pieces
and contacted with water and an organic solvent, such as a water
miscible organic solvent. Alternatively, the plant material is
contacted with water and an organic solvent without any
pre-treatment. The ratio of water to organic solvent may be
generally in the range of 1:10 to 10:1 and may, for example,
comprise equal proportions of water and solvent, of 1% to 30% (v/v)
organic solvent. Any organic solvent or a mixture of such solvents
may be used. The organic solvent may preferably be a C2-10, more
preferably a C1-4 organic solvent (such as methanol, chloroform,
ethanol, propanol, propylene glycol, erythrite, butanol,
butanediol, acetonitrile, ethylene glycol, ethyl acetate, glycidol,
glycerol dihydroxyacetone or acetone). Optionally the water/organic
solvent mixture may include an enzyme which cleaves isoflavone
glycosides to the algycone form. The mixture may be vigorously
agitated so as to form an emulsion. The temperature of the mix may
range, for example, from an ambient temperature to boiling
temperature. Exposure time may range from one hour to several
weeks. One convenient extraction period is twenty-four hours at
90.degree. C. The extract may be separated from undissolved plant
material and the organic solvent removed, such as by distillation,
rotary evaporation, or other standard procedures for solvent
removal. The resultant extract containing water soluble and
non-water soluble components may be dried to give an
isoflavone-containing extract, which may be formulated with one or
more pharmaceutically acceptable carriers, excipients and/or
auxiliaries according to the invention.
[0185] An extract made according to the description provided in the
previous paragraphs may contain small amounts of oil which include
isoflavones in their aglycone form (referred to herein as
isoflavones). This isoflavone enriched oil, may be subject to HPLC
to adjust the isoflavone ratios, or, if it is at the desired
isoflavone ratio, may be dried, for example in the presence of
silica, and be formulated with one or more carriers, excipients
and/or auxiliaries to give an isoflavone containing extract.
Alternatively, the isoflavones contained in said small amounts of
oil may be further concentrated by addition to the oil of a
non-water soluble organic solvent such as hexane, heptane, octane
acetone or a mixture of one or more of such solvents. One example
is 80% hexane, 20% acetone w/w having high solubility for oils but
low solubility for isoflavones. The oil readily partitions into the
organic solvent, and an enriched isoflavone containing extract
falls out of solution. The recovered extract may be dried, for
example in an oven at 50.degree. C. to about 120.degree. C., and
formulated with one or more pharmaceutically acceptable carriers,
excipients and/or auxiliaries.
[0186] It will be appreciated that the present invention also
contemplates the production of suitable isoflavones, functional
derivatives, equivalents or analogues thereof, by established
synthetic techniques well known in the art. See, for example, Chang
et al. (1994) which discloses methods appropriate for the synthesis
of various isoflavones.
[0187] International Patent Applications WO 98/08503 and WO
00/49009 (which are incorporated herein in their entirety by
reference) and references cited therein also provide general
synthetic methods for the preparation of isoflavonoid compounds for
use in the present invention.
[0188] General methods known in the art may also be employed by
those skilled in the art of chemical synthesis for constructing the
platinum complexes depicted m formula (II), and by reference to the
general schemes 1 and 2 below.
[0189] Chemical functional group protection, deprotection, synthons
and other techniques known to those skilled in the art may be used
where appropriate in the synthesis of the compounds of the present
invention. ##STR19## ##STR20##
[0190] The inventors have found a surprising synergy between the
compounds of formula (I), and in particular the isoflav-3-ene
compounds of formula (VIa), with known chemotherapeutic agents. The
isoflavonoid compounds of the invention are found to restore or at
least improve chemosensitivity to previously resistant cancer cell
lines. In particular, dehydroequol (12, DHE) is found to exhibit
synergistic interaction with cisplatin, carboplatin, topotecan and
paclitaxel with various established cancer cell lines, in
particular the ovarian cancer cell lines Cp70 and A27A0. Synergism
was also observed with prostate cancer cell lines DU145 and PC3 and
pancreatic cell line HPAC.
[0191] These results are further elucidated in the examples which
follow. These results show that combination chemotherapy with the
isoflavonoid compounds with established anticancer agents are
useful in the treatment of proliferation of cancer cells and
neoplastic tumours by reducing the IC.sub.50 of standard
chemotherapy. Administration of the isoflavonoid compounds
described herein either simultaneously, sequentially or as a
pre-treatment to standard chemotherapies increases the sensitivity
of the cancer cells and tumours to chemotoxic agents.
[0192] The Examples show the efficacy of combination chemotherapy
with dehydroequol as a treatment for epithelial ovarian cancer
cells by such reduction of the IC50 of standard chemotherapy. This
thereby increases sensitivity of the cancer cells to chemotoxic
agents. The results of these tests and trial are important as
ovarian cancer is the fourth leading cause of cancer death and the
most lethal of the gynaecologic malignancies. Recent new therapies
have led to some improvement in the five year survival, yet there
has been no improvement in the overall survival. Be main
limitations of therapy in ovarian cancer patients are
chemoresistance and side-effects. The combination chemotherapy and
isoflavonoid pre-treatment addresses the survival rates of patients
undergoing the chemotherapy, and in particular those patients with
ovarian cancer. Without wishing to be limited to theory, it is
believed that the isoflavone derivative dehydroequol induces
apoptosis in ovarian cancer cells by specifically removing the
blockers of apoptosis.
[0193] The invention is further described with reference to the
following non-limiting examples.
EXAMPLE 1
Dehydroequol-Cisplatin Synergy in vitro
[0194] The effect of a composition comprising the platinum complex
cisplatin and the isoflavonoid compound dehydroequol (compound No.
12) on various cancer cell lines was assessed on culture plates.
Cell viability was determined using CellTiter.COPYRGT.. Apoptosis
was evaluated using Hoechst 33342 dye.
[0195] It was found that the amount of cisplatin needed to kill a
set number of cancer cells is less when in admixture with an
isoflavonoid compound as compared to a control with cisplatin
alone. This example demonstrates the surprising synergy between
cisplatin and the isoflavonoid compounds of the present invention.
Dehydroequol was found to exhibit a strong synergistic interaction
with cisplatin in cell lines derived from ovarian (A2780, Cp70),
prostate (DU145 and PC3) and pancreatic (HPAC) cancers. Table 1
below shows that the IC.sub.50 of cisplatin against the mentioned
cell lines is markedly lowered by co-incubating representative
cells with a sub-IC.sub.50 level (2 .mu.M) of dehydroequol.
TABLE-US-00001 TABLE 1 Effect of concurrent exposure to
dehydroequol and cisplatin on the IC.sub.50 levels on nominated
cancer cell lines IC.sub.50 (uM) Cisplatin IC.sub.50 (uM) + Cell
line Cisplatin dehydroequol 2 uM dehydroequol A2780 3.0 1.7
<0.001 CP70 10.4 1.5 0.1 HPAC 34.5 50.0 7.7 PC3 0.4 9.6 0.001
DU145 5.0 5.9 0.1
EXAMPLE 2
Dehydroequol-Cisplatin, Dehydroequol-Carboplatin and
Dehydroequol-Paclitaxel Synergy in vitro and in vivo
Methods
[0196] The in vitro studies were performed using ovarian cancer
cells isolated from ascites using an immunomagnetic assay and
established ovarian cancer cell lines CP70 and A2780. Cell
viability was determined using CellTiter.COPYRGT.. Apoptosis was
evaluated using Hoechst 33342 dye. The in vivo effect was tested by
injecting CP70 subcutaneously into nude mice. Animals received
daily oral administration of dehydroequol, 10 or 20 mg/kg for 8
days alone or in combination with cisplatin 0.5 mg/kg. After 8 days
the animals were sacrificed and the tumour volume was measured.
[0197] The IC50 for carboplatin ranged from 60 .mu.g/ml to greater
than 100 .mu.g/ml (FIG. 1).
[0198] The IC50 for paclitaxel in the paclitaxel resistant cell
line, R182, was greater than 2 .mu.M (FIG. 2).
[0199] Pre-treatment with dehydroequol (10 .mu.g/ml) for two hours
significantly reduced the IC50 for carboplatin (0.5 .mu.g/ml+/-0.5)
and paclitaxel (0.05 .mu.M) (FIGS. 3 and 4).
[0200] Western blot analysis demonstrated that resistant ovarian
cancer cells expressed high levels of active XIAP (X-linked
inhibitor of apoptosis). Additionally, the active form of caspase 3
in chemoresistant cells was not detected. Caspase 3 activation was
observed in the chemoresistant cells only after pretreatment with
dehydroequol (FIG. 5).
[0201] FIGS. 6 and 7 depict the results of the next study, where 20
mg/kg dehydroequol (DHE) 5% HPBCD was compared to delivery of
cisplatin and to a combination of dehydroequol and cisplatin. The
20 mg/kg cisplatin dosage regimen inhibited tumour proliferation
but the data was not significantly different from cisplatin (1
mg/kg) and dehydroequol (20 mg/kg) controls. Importantly and
somewhat surprisingly, the lower dose 10 mg/kg dehydroequol-0.5
mg/kg cisplatin combination regimen inhibited tumour proliferation
more markedly than that over the 20 mg/kg dehydroequol-1 mg/kg
cisplatin (%T/C=14.7) regimen and the data were significantly
different from single agent controls (FIGS. 6 and 7).
[0202] Dehydroequol treatment for 48 hours (h) induced 60-80%
decrease in cell viability in carboplatin and paclitaxel resistant
cells. Pre-treatment with pH alone for 2 h decreased cell viability
by 20%. Furthermore, pretreatment (2 h) with pH in chemoresistant
cells followed by carboplatin or paclitaxel for 48 h resulted in a
30% and 50% significant decrease in cell viability, respectively,
Hoechst stain confirmed the presence of apoptosis in the treated
cells. In vivo, cisplatin (0.5 mg/kg) had no effect on tumour size
while the combination of pH (10 mg/kg) and cisplatin 0.5 mg/kg)
reduced tumour mass by 75% (p=0.05).
EXAMPLE 3
Toxicity--Dehydroequol and Cisplatin
[0203] No overt signs of toxicity were noted at any of the dosage
regimens used as shown in FIG. 8. Fluctuations in body mass were
within ethically acceptable boundaries.
EXAMPLE 4
XIAP Expression in Human Melanoma Tumours
[0204] The differences in XIAP expression between benign and
malignant melanoma tissue was assessed.
Methods
[0205] The melanoma tissue microarrays were constructed using a
total of 232 primary melanomas, 15 local recurrences and 299
metastatic cores, each measuring 0.6 mm in diameter. The cohort was
constructed from paraffin-embedded, formalin-fixed tissue blocks
obtained from the Yale University Department of Pathology Archives.
Specimens and clinical information were collected under the
guidelines and approval of a Yale University Institutional Review
Board. Age at diagnosis ranged from 18 to 91 years (mean age 52.4
years). The cohort included 55% males and 45% females, A
pathologist reviewed slides from all of the blocks to select
representative areas of invasive tumor to be cored. The cores were
placed on the tissue microarray using a Tissue Microarrayer
(Beecher Instruments, Silver Spring, Md.). The tissue microarrays
were then cut to 0.5 .mu.m sections and placed on glass slides
using an adhesive tape-transfer system (Instrumedics, Inc.,
Hackensack, N.J.) with UV cross-linking. Similarly a tissue
microarray was made containing cores from 540 benign nevi. The
nevus array contained 31 metastatic specimens from patients that
were also represented on the melanoma array. Both arrays contained
identical cell lines. The overlapping metastatic specimens and cell
lines were used for normalization of the scores obtained from the
benign and malignant arrays.
[0206] Staining was performed for automated analysis of melanoma
specimens. Slides were deparaffinized in xylene, and transferred
though two changes of 100% ethanol. For antigen retrieval, the
slides were boiled in a pressure cooker containing 6.5 mM sodium
citrate (pH 6.0). Endogenous peroxidase activity was blocked in a
mixture of methanol and 2.5% hydrogen peroxide for thirty minutes
at room temperature. To reduce non-specific background staining,
slides were incubated at room temperature for 30 minutes in 0.3%
bovine serum albumin/1X Trisbuffered saline. Slides were incubated
at 4.degree. C. overnight in a humidity tray with a primary mouse
anti-human XIAP antibody (BD Transduction Laboratories) diluted
1:50. To create a tumor mask, slides were simultaneously incubated
overnight with a primary rabbit anti-human S100 antibody diluted
1:500. Slides were rinsed three times in 1X Tris-buffered
saline/0.05% Tween-20 and incubated for 1 hour at room temperature
with goat anti-mouse HRP to identify the target and goat
anti-rabbit IgG conjugated to Alexa 546 to identify the S100 mask.
The slides were washed again as above and incubated for ten minutes
with Cy5 directly conjugated to tyramide (Perkin Elmer, Boston,
Mass.) at a dilution of 1:50 for primary antibody identification.
The slides were rinsed again and coverslips were mounted with
ProLong Gold antifade reagent, which contained
4,6-diamidine-2-phenylindole (DAPI) to identify the nuclei.
[0207] Images were acquired using automated, quantitative analysis.
Briefly, areas of tumor were distinguished from stroma by creating
a mask with the S100 signal tagged with Alexa 546. Coalescence of
S100 at the cell surface was used to identify the
membrane/cytoplasm compartment within the tumor mask, while
4,6-diamidino-2-phenylindole (DAPI) was used to identify the
nuclear compartment within the tumor mask. The target marker, XIAP,
was visualized with Cy5 (red). Cy5 was used because its emission
peak is outside the color spectrum of tissue autoflourescence.
Multiple monochromatic, high resolution (1024.times.1024 pixel
0.5-.mu.m) grayscale images were obtained for each histospot, using
the 10.times. objective of an Olympus AX-51 epifluorescence
microscope (Olympus, Melville, N.Y.) with an automated microscope
stage and digital image acquisition driven by custom program and
macro-based interfaces with IPLabs software (Scanalytics Inc.,
Fairfax, Va.).
[0208] Two images (one in-focus and one out-of-focus) were taken of
the compartment specific tags and the target marker. A percentage
of the out-of-focus image was subtracted from the in-focus image
for each pixel, representing the signal to noise ratio of the
image. An algorithm described as RESA (Rapid Exponential
Subtraction Algorithm) was used to subtract the out-of-focus
information in a uniform fashion for the entire microarray.
Subsequently, the PLACE algorithm (Pixel Locale Assignment for
Compartmentalization of Expression) was used to assign each pixel
in the image to a specific subcellular compartment and the signal
in each location is calculated. Pixels that cannot accurately be
assigned to a compartment were discarded. The data were saved and
subsequently expressed as the average signal intensity per-unit of
compartment area. For the nuclear and membrane/cytoplasmic
compartments, the image was measured on a scale of 0-255, and
expressed as target signal intensity relative to the compartment
area.
[0209] The JMP5 (SAS Institute Inc., Cary, N.C.) software package
was used for data analyses. Continuous AQUA scores of target
expression were divided by the median score and associations with
clinical and pathological parameters were completed using the
Chi-Square test. Comparison of expression in malignant and benign
specimens, as well as comparisons between primary and metastatic
specimens was performed with unpaired t-tests.
Results
[0210] Unpaired t-tests showed that expression was significantly
higher in malignant versus benign tissue cores (P<0.0001), as
shown in FIG. 9. Moreover, the expression of XIAP was significantly
higher in metastatic specimens than in primary specimens
(P<0.0001), as shown in FIG. 9. The median AQUA scores for XIAP
were 9.922 in nevi specimens. 20.56 in primary lesions and 26.98 in
metastatic lesions.
[0211] Table 2 demonstrates the association between high XIAP
expression and commonly used clinical and pathological variables.
High XIAP was associated with advanced stage (metastatic) disease
and thick lesions over 2 mm (p=0.0003 and p=0.0264, respectively).
TABLE-US-00002 TABLE 2 Association between high XIAP expression and
other prognostic clinical and pathological variables.
Clincial/pathological Chi-Square Variable Value P-value Disease
Stage 13.071 0.003 (metastatic vs. primary) Breslow (>2 mm)
4.927 0.0264 Clark Level (IV-V) 3.559 0.1687 Age (<40 years)
0.338 0.5613 Gender (male) 1.800 0.1798 Presence of Ulceration
2.016 0.3650
EXAMPLE 5
Sensitisation of Melanoma Cells to Carboplatin by Dehydroequol and
Association with XIAP Levels
Methods
[0212] Low passage (passage 3-18) melanoma cell strains were
excised from tumors of patients treated at the Yale Cancer Center,
obtained form the Cell Culture facility of the Yale Skin Disease
Research Core Center (YSDRCC). The melanoma cell strain YUMAC was
from an in transit metastasis from a male patient, YUSAC2 from a
soft tissue metastasis from a male patient, and YUGEN8 from a brain
metastasis of a female patient. 501 mel, 624 mel and 888 mel were
provided by Dr. Steven A. Rosenberg, National Cancer Institute,
Bethesda, Md. The mm127 cell line was provided by Dr Peter Parsons
of the Queensland Institute of Medical Research. Cells where grown
in F12 medium with 10% F13S at 37.degree. C. in 5% CO.sub.2.
Carboplatin was purchased from Sigma Chemical Co. (St. Louis,
Mo.).
[0213] Cell viability was evaluated using the CellTiter 96 Aqueous
One Solution Cell Proliferation Assay according to the
manufacturer's instructions (Promega, USA). Briefly
4.times.10.sup.4 cells per well were plated in triplicate in a
96-well microtitre plate. Cells were grown to 60% confluency, at
which stage the media was replaced with reduced serum
phenol-depleted OPTI-MEM (Gibco.TM., Invitrogen Corp, Grand Island,
N.Y.), and incubated for 4 hours prior to treatment. Following
treatment, 20 .mu.L of CellTiter 96 Aqueous One Solution was added
to each well and the plate was incubated at 37.degree. C. for 2
hours. Optical densities were measured at 490 nm. Values of treated
cells were compared to untreated cells and reported as percent
viability.
[0214] For western blot analysis cells were plated in 35 mm.sup.2
Petri dishes and grown to 60% confluency for treatment. Following
treatment, cells were lysed in Radioimmunoprecipitation (RIPA)
buffer containing the protease inhibitor cocktail, Complete (Roche
Diagnostics, Mannheim, Germany). For phospo-AKT analysis lysis was
performed in 10X cell lysis buffer (Cell Signaling, Beverly,
Mass.). Protein concentrations were calculated by the BCA
(Bicinchoninic Acid) assay (Pierce Biotechnology, Rockford, Ill.).
Protein (20 .mu.g) was separated in a sample buffer [2.5% SDS, 10%
glycerol, 5% .beta.-mercapto-ethanol, 0.15 M Tris (pH=6.8) and
0.01% bromophenol blue] and subjected to SDS-polyacrylamide gel
electrophorisis using precast 12% polyacrylamide gels and
transferred to pure nitrocellulose membranes. To inhibit
non-specific binding, membranes were blocked in 5% powdered milk
for 1 hour at room temperature. The membranes were washed three
times in PBS with 0.5% tween (PBS-T) for 10 minutes per wash and
incubated with primary antibodies diluted in PBS-T with 1% milk, in
a 50 mL falcon tube on a rotator overnight at 4.degree. C. The
following primary antibodies and concentrations were used: mouse
anti-XIAP (BD Transduction Laboratories) at 1:1000, mouse
anti-Caspase 2 (BD Biosciences) at 1:1,000, rabbit anti-Bid (Cell
Signaling, Beverly, Mass.) at 1:2500, and rabbit anti-actin (Sigma)
at 1:100. Following primary incubation, membranes were washed as
described above and incubated with horse anti-mouse or horse
anti-rabbit peroxidase (Vector Laboratories, Burlingame, Calif.),
diluted 1:10,000, for 1 hour at room temperature. Membranes were
washed again in PBS-T as above and washed three times in ddH20, 10
minutes per wash. Finally, proteins were visualized using enhanced
chemiluminescence.
[0215] Following drug treatment of cells, 10 .mu.g of protein in 50
.mu.L of ddH.sub.2O was combined with equilibrated Caspase-Glo.TM.
3/7, 8, or 9 reagents (Promega). After incubation for 1 hour at
room temperature, luminescence was measured using a TD 20/20
Luminometer (Turner Designs, Sunnyvale, Calif.). Blank values were
subtracted and fold increase in activity was calculated based on
activity measured from untreated cells. Each sample was measured in
duplicate.
Results
[0216] The effect of dehydroequol on three patient-derived melanoma
cell strains was evaluated. A concentration of 10 .mu.g/ml
dehydroequol was used and cell viability determined by the
CellTiter 96.RTM. Aqueous One Solution Cell Proliferation Assay. As
shown in FIG. 10, two of the three cell strains (MAC and YUGEN8)
were sensitive to 10 .mu.g/ml of dehydroequol, while the third cell
strain (YUSAC2) was resistant. Surprisingly, YUSAC2 cells grew more
in the presence of dehydroequol than in media alone.
[0217] The effect of dehydroequol on XIAP expression and function
was studied in one of the two dehydroequol sensitive cell strains,
YUMAC, and in the resistant strain (YUSAC2). Exposure of YUMAC
cells to dehydroequol decreased the level of XIAP at 4 hours
compared with pretreament levels. A further decrease in XIAP levels
was seen at 8 hours post treatment and no expression was observed
at 24 hours (FIG. 11a). It was then evaluated whether changes on
XIAP expression affects other component of the apoptotic cascade.
As shown in FIG. 11a, activity of caspases 3, 8 and 9 was increased
at 24 hours (FIG. 11a). With the YUSAC2 cells, the degree XIAP
degradation and resultant caspase activation was much less than
what was observed with YUMAC cells (FIG. 11b).
[0218] YUMAC, YUSAC2 and YUGEN8 cells were also evaluated for
sensitivity to Carboplatin. The cells were treated with increasing
doses of Carboplatin, ranging from 50-200 1.mu.g/ml for 24 hours,
and cell viability was determined by the CellTiter 96.RTM. Assay.
As shown in FIG. 12a, all three cell strains were resistant to
Carboplatin, with YUSAC2 demonstrating the most resistance., YUMAC
and YUGEN8 were also relatively resistant to Carboplatin; over 70%
of the cells were viable with the highest concentration of the
drug. As with dehydroequol, YUSAC2 cells grew more in the presence
of Carboplatin than in medium alone. The IC.sub.50 for all three
cell strains was greater than 200 .mu.g/ml.
[0219] As demonstrated in Example 2, resistance of ovarian cancer
cells to Carboplatin is associated with high XIAP levels, and that
this resistance can be reversed by pretreatment with dehydroequol.
In the present study the inventors assessed whether pretreatment of
melanoma cells with dehydroequol reverses the baseline resistance
to Carboplatin and whether this reversal is associated with XIAP
degradation. Melanoma cells were pre-treated with 10 .mu.g/ml of
dehydroequol for 4 hours, dehydroequol was removed from the media
and the cells were treated with increasing doses of Carboplatin
(50-200 .mu.g/ml) for an additional 24 hours. As shown in FIG. 12b,
pretreatment with dehydroequol sensitized the YUMAC and YUGEN8
cells to Carboplatin, as demonstrated by a decrease in the
IC.sub.50 of Carboplatin for these cell strains to <50 .mu.g/ml
and <200 .mu.g/ml, respectively. Pretreatment with dehydroequol
also decreased the resistance of YUSAC2 to Carboplatin, but to a
much lesser degree with the IC.sub.50 for Carboplatin remaining at
>200 .mu.g/ml.
[0220] In order to further characterize the effect of dehydroequol
on melanoma cells, YUMAC cells were pretreated with a shorter
exposure to dehydroequol (2 hours) followed by treatment with
Carboplatin. The effect on apoptotic mediators, including
procaspase-2, BID (a member of the mitochondrial pathway that is
activated after caspase 2 activation) and caspase-3 was assessed.
Treatment with Carboplatin alone had no effect on XIAP levels, or
any other component of the apoptotic cascade. Treatment with
dehydroequol for 2 hours decreased XIAP expression and induced
caspase 2 activation. However, when Carboplatin was added after
dehydroequol pre-treatment, XIAP degradation was observed, followed
by caspase 2 and Bid activation, and a significant increase on the
activity of caspase 3/7 (FIG. 13).
EXAMPLE 6
Dehydroequol-topotecan Co-administration Effectively Reduces Tumor
Kinetics in an A2780 Xenograft Model of EOC
[0221] To determine the effects of topotecan and dehydroequol on
epithelial ovarian cancer (EOC) cells in vivo, a mouse xenograft
model was established using A2780 cells by inoculating 5 wk old
male Balb/c nude mice s.c. with 1.times.10.sup.6 A780 cells
bilaterally (100 .mu.L midway between the axillary and inguinal
region) along the dorsal surface. Therapy commenced 8-10 days
post-inoculation and individual mouse weight was measured everyday,
dehydroequol was formulated as a suspension in 1% carboxymethyl
cellulose (CMC) and topotecan was formulated in PBS.
[0222] Animals were randomly assigned to 6 groups: Group 1 received
1% CMC qdx10 (control group); Group 2 received 25 mg/kg
dehydroequol (p.o.) qdx10; Group 3 received 2 mg/kg topotecan
(i.p.) qdx5; Group 4 received 1 mg/kg topotecan (i.p.) qdx5; Group
5 received combination dehydroequol (25 mg/kg, p.o., qdx10) and
topotecan (2 mg/kg, i.p., qdx5); and Group 6 received dehydroequol
(12.5. mg/kg, p.o., qdx10) plus topotecan (1 mg/kg, i.p., qdx5).
Dehydroequol dosed orally at 25 mg/kg strongly retarded A2780 tumor
proliferation and significantly reduced terminal tumor burden
(%T/C=45.7) (FIGS. 14A-B). Similarly, topotecan also significantly
reduced tumor proliferation and terminal tumor burden when dosed at
both 1 and 2 mg/kg (%T/C=31.6 and 16.9 respectively). More
importantly, animals that received combination
dehydroequol/topotecan (25 and 2 mg/kg, respectively) had
significantly reduced tumor kinetics and terminal tumor burden
(%T/C=8.15) at levels below that of the corresponding monotherapy
controls. Furthermore, animals that received combination
dehydroequol/topotecan at doses half that of the monotherapy
controls (12.5 mg/kg dehydroequol and 1 mg/kg topotecan) had
significantly reduced tumor proliferation kinetics and terminal
tumor burden (%T/C=-14.25) when compared to monotherapy controls
(FIGS. 14A-B). These data indicate that dehydroequol and topotecan
dosed in combination act synergistically in reducing overall tumor
burden using the A2780 ovarian cancer tumor model. Additionally,
the animals receiving the low-dose combination did not show
significant weight loss (data not shown) indicating that
dehydroequol-topotecan combination is able to act synergistically
in preventing tumor proliferation with minimal effect on weight
loss. Further, animals receiving the high-dose combination showed
less myelosuppression than animals receiving topotecan monotherapy
(data not shown), suggesting that dehydroequol may protect against
topotecan-induced myelosuppression.
[0223] These examples highlight the utility of the isoflavonoid
compounds of formula (I) in combination with chemotherapeutic
agents, and the compounds of formula (II) or (IIa) and (I) as
therapeutic agents for inducing sensitivity to chemoresistant
cancer cells and tumours to low levels of chemotherapy and to the
general down regulation of cell proliferation and the treatment,
amelioration, defence against, prophylaxis and/or prevention of the
therapeutic indications.
[0224] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The inventions also includes all of the steps, features,
compositions and compounds referred to or indicated in the
specification, individually or collectively, and any and all
combinations of any two or more of said steps or features.
[0225] The reference to any prior art in this specification is not,
and should not be taken as, an acknowledgment or any form of
suggestion that that prior art forms part of the common general
knowledge in the field of endeavour.
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