U.S. patent application number 12/609761 was filed with the patent office on 2010-03-18 for small molecule antagonists of bcl2 family proteins.
This patent application is currently assigned to The Regents of the University of Michigan. Invention is credited to Shaomeng Wang, Liang Xu, Dajun Yang.
Application Number | 20100069344 12/609761 |
Document ID | / |
Family ID | 34807403 |
Filed Date | 2010-03-18 |
United States Patent
Application |
20100069344 |
Kind Code |
A1 |
Wang; Shaomeng ; et
al. |
March 18, 2010 |
SMALL MOLECULE ANTAGONISTS OF BCL2 FAMILY PROTEINS
Abstract
The present invention relates to naturally occurring and
chemically synthesized small molecule antagonists of Bcl-2 family
proteins. In particular, the present invention provides gossypol
compounds (e.g., isomers, enantiomers, racemic compounds,
metabolites, derivatives, pharmaceutically acceptable salts, in
combination with acids or bases, and the like) and methods of using
these compounds as antagonists of the anti-apoptotic effects of
Bcl-2 family member proteins (e.g., Bcl-2, Bcl-X.sub.L, and the
like). The present invention also provides compositions comprising
gossypol compounds and optionally one or more additional
therapeutic agents (e.g., anticancer/chemotherapeutic agents). The
present invention also provides methods for treating diseases and
pathologies (e.g., neoplastic diseases) comprising administering a
composition comprising gossypol compounds and optionally one or
more additional therapeutic agents (e.g.,
anticancer/chemotherapeutic agents) and/or techniques (e.g.,
radiation therapies, surgical interventions, and the like) to a
subject or in vitro cells, tissues, and organs.
Inventors: |
Wang; Shaomeng; (Saline,
MI) ; Yang; Dajun; (Rockville, MD) ; Xu;
Liang; (Ann Arbor, MI) |
Correspondence
Address: |
Casimir Jones, S.C.
2275 DEMING WAY, SUITE 310
MIDDLETON
WI
53562
US
|
Assignee: |
The Regents of the University of
Michigan
Ann Arbor
MI
Georgetown University
Washington
DC
|
Family ID: |
34807403 |
Appl. No.: |
12/609761 |
Filed: |
October 30, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12242380 |
Sep 30, 2008 |
|
|
|
12609761 |
|
|
|
|
10729156 |
Dec 5, 2003 |
7432304 |
|
|
12242380 |
|
|
|
|
10158769 |
May 30, 2002 |
|
|
|
10729156 |
|
|
|
|
PCT/US02/17206 |
May 30, 2002 |
|
|
|
10158769 |
|
|
|
|
60293983 |
May 30, 2001 |
|
|
|
Current U.S.
Class: |
514/179 |
Current CPC
Class: |
A61P 35/02 20180101;
A61P 35/00 20180101; A61P 31/10 20180101; A61P 31/12 20180101; A61K
45/06 20130101; A61P 31/18 20180101; A61P 31/04 20180101; A61K
31/11 20130101; A61K 2300/00 20130101; A61K 31/11 20130101 |
Class at
Publication: |
514/179 |
International
Class: |
A61K 31/573 20060101
A61K031/573; A61P 35/00 20060101 A61P035/00 |
Claims
1.-51. (canceled)
52. A pharmaceutical composition comprising a gossypol compound and
prednisone.
53. The pharmaceutical composition of claim 52 further comprising a
pharmaceutically acceptable carrier.
52. The pharmaceutical composition of claim 52 formulated as a
tablet, pill, or capsule for oral ingestion by a subject in need
thereof.
55. A kit comprising the pharmaceutical composition of claim 52 and
instructions for administering said pharmaceutical composition to a
subject in need thereof.
56. The kit of claim 55 further comprising one or more anticancer
agents.
57. The kit of claim 56 further comprising instructions for
administering said one or more anticancer agents to said
subject.
58. The kit of claim 56, wherein said anticancer agent is selected
from the group consisting of docetaxel and paclitaxel.
59. The kit of claim 58 wherein said anticancer agent is
docetaxel.
60. The pharmaceutical composition of any one of claims 52-54
wherein said gossypol compound is (-)-gossypol.
61. The kit of any one of claims 55-59 wherein said pharmaceutical
composition comprises (-)-gossypol.
Description
[0001] This application is a continuation in part of U.S. patent
application Ser. No. 10/158,769 filed May 30, 2002, and
PCT/US02/17206 filed May 30, 2002, both of which claim priority to
U.S. Provisional Patent Application Ser. No. 60/293,983, filed May
30, 2001, the contents of each of which are fully incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to naturally occurring and
chemically synthesized small molecule antagonists of Bcl-2 family
proteins. In particular, the present invention provides gossypol
compounds (e.g., isomers, enantiomers, racemic compounds,
metabolites, derivatives, pharmaceutically acceptable salts, in
combination with acids or bases, and the like) and methods of using
these compounds as antagonists of the anti-apoptotic effects of
Bcl-2 family member proteins (e.g., Bcl-2, Bcl-X.sub.L, and the
like). The present invention also provides compositions comprising
gossypol compounds and optionally one or more additional
therapeutic agents (e.g., anticancer/chemotherapeutic agents). The
present invention also provides methods for treating diseases and
pathologies (e.g., neoplastic diseases) comprising administering a
composition comprising gossypol compounds and optionally one or
more additional therapeutic agents (e.g.,
anticancer/chemotherapeutic agents) and/or techniques (e.g.,
radiation therapies, surgical interventions, and the like) to a
subject or in vitro cells, tissues, and organs.
BACKGROUND OF THE INVENTION
[0003] Multicellular organisms use a process called apoptosis to
instruct damaged or unnecessary cells to destroy themselves for the
good of the organism. Control of the apoptotic process is very
important for the normal development of the organism. For example,
fetal development of fingers and toes requires the controlled
removal, by apoptosis, of excess interconnecting tissues, as does
proper formation of neural synapses within the brain. Careful
control of apoptosis is also important to adult organisms, for
instance, controlled apoptosis is responsible for the sloughing of
the inner lining of the uterus (the endometrium) at the start of
menstruation.
[0004] Apoptosis not only plays an important role in tissue
sculpting during fetal development and normal cellular maintenance,
it is also the primary defense against rogue cells that threaten
the well being of the entire organism. For instance, in the cell
mediated immune response, effector cells (e.g., cytotoxic T
lymphocytes "CTLs") destroy virus-infected host cells by inducing
the infected host cells to undergo apoptosis. The organism
subsequently relies in turn upon the apoptotic process to destroy
the effector cells when they are no longer needed. Autoimmunity is
prevented by the CTLs inducing apoptosis in each other and even in
themselves. Defects in this process are associated with a variety
of autoimmune diseases such as lupus erythematosus and rheumatoid
arthritis.
[0005] Multicellular organisms use the apoptotic process to
instruct cells with damaged nucleic acids (e.g., DNA) to destroy
themselves prior to becoming cancerous. However, some
cancer-causing viruses prevent apoptosis in transformed cells. For
example, several human papilloma viruses (HPVs) are implicated in
causing cervical cancer by suppressing apoptotic removal of
transformed cells through the production of a protein, E6, which
inactivates the p53 apoptosis promoter. Epstein-Barr virus (EBV),
the causative agent of mononucleosis and Burkitt's lymphoma, a
solid tumor of B lymphocytes, produces a first protein similar to
Bcl-2, and a second that causes transformed cells to increase
production of Bcl-2. The expression of various Bcl-2 family
proteins helps virus-transformed cells resist apoptosis. Still
other viruses manipulate the cell's apoptotic machinery without
directly resulting in the development of a cancer. For example,
destruction of the immune system in individuals infected with the
human immunodeficiency virus (HIV) is thought to progress through
infected CD4+ T cells (about 1 in 100,000) instructing their sister
cells to undergo apoptosis. Faulty regulation of the apoptotic
machinery has also been implicated in various degenerative
conditions and vascular diseases.
[0006] Some cancers that arise by non-viral means have also
developed mechanisms to escape destruction by apoptosis. Melanoma
cells, for instance, avoid apoptosis by inhibiting the expression
of the gene encoding the apoptosis effector protein Apaf-1. Other
cancers, especially lung and colon, secrete elevated levels of
soluble decoy molecules that bind FasL, inhibiting it from binding
to Fas. CTLs are thus prohibited from destroying these cancer
cells. Other cancer cells express high levels of FasL, again,
avoiding destruction by the CTLs.
[0007] It is apparent that the controlled regulation of the
apoptotic process and the apoptotic machinery is vital to the
survival of multicellular organisms. Typically, the biochemical
changes that occur in a cell instructed to undergo apoptosis occur
in an orderly procession. However, as shown above, flawed
regulation of these process can cause serious harm.
[0008] There have been various attempts to use small molecules to
control and restore regulation of the apoptotic machinery in
aberrant cells (e.g., cancer cells). Generally, these attempts have
had limited success as treatments for the underlying diseases for a
number of reasons, including high toxicity, low bioavailability,
high costs, and the like. What is needed are improved methods and
compositions for regulating apoptosis in subjects afflicted with
diseases and conditions that are characterized by faulty regulation
of the apoptotic process.
SUMMARY OF THE INVENTION
[0009] It is generally accepted in the field of molecular oncology
that most, if not all, malignant cancer cells harbor (at a minimum)
two derangements that lead to the malignant phenotype: a
proliferative lesion, causing cells to multiply inappropriately,
and an apoptotic lesion, that prevents the cell(s) from executing
the apoptosis program in response to either the detection, within
the cell, of these genetic abnormalities (e.g., up-regulation of a
growth or mitosis oncogene like Ras or Myc), or the pharmacological
effects of cell death-inducing cancer therapeutic drugs or
radiation therapy. The apoptotic lesion confers on the cells a
survival advantage in the face of either further accumulated
oncogenic lesions, or exposure to pharmacologically effective
levels of cancer therapeutic drugs or radiation therapy.
[0010] A number of apoptotic lesions have been described in tumor
cells (e.g. loss of p53, decreased Apaf-1, increased IAPs,
decreased caspases), both in vitro and in vivo, most notably
enhanced expression and accumulation of proteins of the
anti-apoptotic Bcl-2 gene family. Bcl-2 is the prototypical member
of this family, which includes Bcl-X.sub.L, Mcl-1, A1, and Boo/Diva
proteins. Bcl-2 is a human oncogene that prevents the activation of
the apoptosis program in many cells, and when expressed at
inappropriately high levels in cancerous or pre-cancerous cells,
confers on them a selective advantage. Bcl-2 and Bcl-X.sub.L are
overexpressed in many types of human cancer (e.g., breast,
prostate, colorectal, lung, etc.), including Non-Hodgkin's
lymphoma, which is caused by a chromosomal translocation (t14, 18)
that leads to overexpression of Bcl-2, suggesting that many cancer
cell types depend on the elevated levels of Bcl-2 and/or
Bcl-X.sub.L to survive the other cellular derangements that
simultaneously both define them as cancerous or pre-cancerous cells
and cause them to attempt to execute the apoptosis pathway. Also,
increased expression of Bcl-2 family proteins has been recognized
as a basis for the development of resistance to cancer therapeutic
drugs and radiation that act in various ways to induce cell death
in tumor cells.
[0011] The induction of apoptosis in cancer cells or their
supporting cells (e.g., neovascular cells in the tumor vasculature)
is thought to be a universal mechanism of action for virtually all
of the effective cancer therapeutic drugs or radiation therapies on
the market or in practice today. The present invention contemplates
that exposure of humans suffering from cancer to therapeutically
effective amounts of drug(s) (e.g., small molecules) that inhibit
the function(s) of Bcl-2 and Bcl-X.sub.L kills cancer cells or
supporting cells outright (those cells whose continued survival is
dependent on the overactivity of Bcl-2 or Bcl-X.sub.L) or to render
such cells as a population more susceptible to the cell
death-inducing activity of cancer therapeutic drugs or radiation
therapies. The present invention contemplates that inhibitors of
Bcl-2/Bcl-X.sub.L satisfy an unmet need for the treatment of
multiple cancer types, either when administered as monotherapy to
induce apoptosis in cancer cells dependent on Bcl-2/ Bcl-X.sub.L
function, or when administered in a temporal relationship with
other cell death-inducing cancer therapeutic drugs or radiation
therapies so as to render a greater proportion of the cancer or
supportive cells vulnerable to executing the apoptosis program
compared to the corresponding proportion of cells in a subject
treated only with the cancer therapeutic drug or radiation therapy
alone.
[0012] During the course of the development of the present
invention, gossypol was found to bind to a key binding site (the
BH3-binding site) in both Bcl-2 and Bcl-X.sub.L, to which the
natural protein antagonists of Bcl-2/Bcl-X.sub.L, including Bax,
Bak, Bad, Bim, NOXA, and PUMA bind. Thus, particularly preferred
embodiments provide compositions and methods comprising gossypol
compounds (e.g., (-)-gossypol, (-)-gossypol acetic acid, and the
like) having Bcl-2/B Bcl-X.sub.L inhibitory activity, and that
cause cells that depend for their survival, at least in part, on
Bcl-2 and/or Bcl-X.sub.L to execute the apoptosis program and die.
The present invention is not limited to a particular mechanism.
Indeed, an understanding of the mechanism is not necessary to
practice (make and use) the present invention. Nonetheless, it is
contemplated that two classes of such Bcl-2/Bcl-X-dependent cells
are 1) a first class of cells that are internally deranged to such
an extent that the "flux" through the apoptosis pathway would be
sufficient, were it not for the elevated levels of Bcl-2 and/or
Bcl-X.sub.L, to trigger execution of the apoptosis program; and 2)
a second class of cells whose apoptosis program has been stimulated
in response to a cancer therapeutic drug or radiation but below a
threshold that has been set in that cell by the elevated levels of
Bcl-2/Bcl-X.sub.L. Either class of cells, by virtue of being
dependent on Bcl-2, Bcl-X or both for their survival, can be killed
by an effective amount of a Bcl-2/Bcl-X.sub.L inhibiting compound
(e.g., (-)-gossypol, (-)-gossypol acetic acid, and the like).
[0013] Indeed, gossypol compounds (e.g., (-)-gossypol) can induce
the death of tumor cells in vitro and can reduce tumor burden in
mice bearing human tumor xenografts (See, Examples). In addition,
gossypol compounds (e.g., (-)-gossypol), by virtue of reducing the
activity of Bcl-2 and/or Bcl-X.sub.L in cancer cells or supporting
cells, increases the proportion of cells in a subject that will
respond to the cell-damaging effects of cancer therapeutic drugs or
radiation therapy by executing the apoptosis program, leading to a
greater tumor response in subjects treated in combination with
gossypol and the cancer therapeutic drug or radiation therapy
compared to those treated with chemo/radiation alone. This enhanced
tumor response will be reflected in any of a number of clinically
desirable endpoints, including tumor shrinkage and/or loss, time to
tumor progression (TTP), or survival. In additional preferred
embodiments, gossypol compounds (e.g., (-)-gossypol), in
combination with any of a number of cancer therapeutic drugs or
radiation, produces added tumor reductions over chemo/radiation
alone (See, Examples). In some examples, gossypol compounds (e.g.,
(-)-gossypol) produce "synergistic" apoptosis (in vitro isobologram
Examples) or tumor responses (in vivo Examples). The in vivo
synergism even leads, in some cases, to regression of tumors that
would not regress with either agent alone.
[0014] From these observations, combination treatment of human
subjects with a therapeutically effective amount of a gossypol
compound (e.g., (-)-gossypol) and an approved course of cancer
therapeutic drugs or radiation, produces a greater tumor response
and clinical benefit in such subjects compared to those treated
with gossypol compound or cancer drugs/radiation alone. It is
contemplated that gossypol (e.g., (-)-gossypol) acts either to kill
cells outright or to increase the proportion of cancer or
supporting cells that respond to the apoptosis-inducing effects of
drugs/radiation by executing the apoptosis program. Put another
way, because gossypol compounds lower the apoptotic threshold of
all cells that express Bcl-2 and/or Bcl-X.sub.L, the proportion of
cells that successfully execute the apoptosis program in response
to the apoptosis-inducing activity of cancer drugs/radiation is
increased. Alternatively, gossypol compounds can be used to allow
administration of a lower, and therefore less toxic and more
tolerable, dose of a cancer therapeutic drug or radiation to
produce the same tumor response/clinical benefit as the
conventional dose of the drug/radiation alone. Since the doses for
all approved cancer drugs and radiation treatments are known, the
present invention contemplates the various combinations of them
with gossypol compounds. Also, since gossypol compounds act at
least in part by inhibiting Bcl-2 and/or Bcl-X.sub.L, the exposure
of cancer and supporting cells to a therapeutically effective
amount of gossypol can be temporally linked to coincide with the
attempts of cells to execute the apoptosis program in response to
the cancer drug or radiation therapy. Thus, in some embodiments,
administering the compositions and methods of the present invention
in view of certain temporal relationships, which can be tested in
clinical trials, provides especially efficacious therapeutic
practices.
[0015] The present invention relates to naturally occurring and
chemically synthesized small molecule antagonists of Bcl-2 family
proteins. In particular, the present invention provides gossypol
compounds (e.g., isomers, enantiomers, racemic compounds,
metabolites, derivatives, pharmaceutically acceptable salts, in
combination with acids or bases, and the like) and methods of using
these compounds as antagonists of the anti-apoptotic effects of
Bcl-2 family member proteins (e.g., Bcl-2, Bcl-X.sub.L, and the
like). The present invention also provides compositions comprising
gossypol compounds and optionally one or more additional
therapeutic agents (e.g., anticancer/chemotherapeutic agents). The
present invention also provides methods for treating diseases and
pathologies (e.g., neoplastic diseases) comprising administering a
composition comprising gossypol compounds and optionally one or
more additional therapeutic agents (e.g.,
anticancer/chemotherapeutic agents) and/or techniques (e.g.,
radiation therapies, surgical interventions, and the like) to a
subject or in vitro cells, tissues, and organs.
[0016] The term cancer is generally used to described hundreds of
neoplastic diseases and neoplasias. The neoplastic growths can be
benign or malignant. There are three broad types of cancer:
carcinomas, sarcomas, and hematologic malignancies (more commonly
known as lymphomas and leukemias). Each type of cancer can affect
almost any organ or part of the body. Carcinomas originate in the
outer layer of cells of the skin and internal membranes (e.g.,
breasts, lungs, intestines, skin, prostate, etc.). Sarcomas arise
from connective tissue such as bone, muscle, cartilage and blood
vessels. Lymphomas and leukemias, hematologic cancers, arise in the
blood or blood-forming organs such as the spleen, lymph nodes and
bone marrow.
[0017] Cancer cells include tumor cells, neoplastic cells,
malignant cells, metastatic cells, and hyperplastic cells.
Neoplastic cells can be benign or malignant. Neoplastic cells are
benign if they do not invade or metastasize. A malignant cell is
one that is able to invade and/or metastasize. Hyperplasia is a
pathologic accumulation of cells in a tissue or organ, without
significant alteration in structure or function.
[0018] Malignant tumors are generally referred to as being either
primary or secondary. Primary tumors arise directly in the tissue
in which they are found. Secondary tumors, or metastases, are
tumors that originated elsewhere in the body, but have now spread,
to a distant tissues and organs. There are some malignancies that
are predisposed to spreading to the skeleton. Prostrate cancer and
breast carcinoma typically metastasize to bone. Another frequent
site of tumor metastasis is the brain.
[0019] The common routes for tumor metastasis are direct growth
into adjacent structures, spread through the vascular or lymphatic
systems, and tracking along tissue planes and body spaces (e.g.,
peritoneal fluid, cerebrospinal fluid, etc.). Clinically, most
patients die from metastatic disease.
[0020] The present invention is not limited to any particular
mechanism. Indeed, an understanding of any particular mechanism is
unnecessary to practice (make and use) the compositions and methods
of the present invention. Nonetheless, it is believed that the
molecular mechanisms involved in metastatic tumor maintenance are
different from those involved in primary tumor maintenance. The
present invention contemplates that elucidation of the cellular
mechanisms associated with metastatic cancer maintenance and
metastasis provides insight into the development of new effective
anticancer treatments.
[0021] Malignant tumor progression, in many cases, is correlated
with increased migratory capacity involving, at least in part,
altered metalloproteolytic activity. Tumor invasion is thought to
rely on the modification of cell adhesion and the proteolysis of
extracellular matrix components. Bcl-2 is though to have specific
effects on the molecules involved in cancer cell migration and
invasion (See, V. Amberger, et al., Cancer Res., 58:149-158
(1998)). Cancer cells that express Bcl-2 proteins may be more
invasive than other cancer cells. Bcl-2 proteins are also thought
to enhance cancer cell migration and invasion by altering the
expression of metalloproteinases and their inhibitors. Wick et al.
(W. Wick, et al., FEBS Lett., 440:419-424 (1998)) reported that
ectopic expression of Bcl-2 in two glioma cell lines significantly
enhanced migration and invasion in a Matrigel-coated membrane
invasion assay (See, S. Mohanam, et al., Cancer Res. 53:4143-4147
(1993)) and a fetal rat brain confrontation assay (See, P.
Pedersen, et al., Cancer Res., 53:5158-5165 (1993)). Bcl-2
expression is also thought to lead to activation and/or increase of
matrix metalloproteinases (e.g., MMP-2, MMP-9) or the cell surface
urokinase-type plasminogen activator (u-PA), and reductions of
metalloproteinases tissue inhibitors (TIMPs).
[0022] Successful migration and invasion of cancer cells requires
the ability to survive, or to become resistant to, the endogenous
apoptotic death program signals once the cancer cell has detached
from the primary tumor tissue. The present invention is not limited
to any particular mechanism. Indeed, an understanding of any
particular mechanism is unnecessary to practice (make and use) the
compositions and methods of the present invention. Nonetheless, the
present invention contemplates that overexpression of
anti-apoptotic Bcl-2 proteins provides tumor cells with a mechanism
for surviving in new and non-permissive environments (e.g.,
metastatic sites), and contributes to the organospecific pattern of
clinical metastatic cancer spread. It is further contemplated that
overexpression of Bcl-X.sub.L counteracts the proapoptotic signals
in the cancer cells' microenvironment, thus favoring successful
development of metastases. The bcl-X.sub.L gene is further thought
to play a role in breast cancer dormancy by promoting the survival
of cells in metastatic foci in specific organs (See, Nuria Rubio,
Lab Invest, 81:725-734 (2001)). For example, in human breast
carcinomas, the overexpression of anti-apoptotic Bcl-X.sub.L
protein is thought to increase metastatic potential by providing,
at least in part, increased resistance to cytokines, overriding
apoptotic signals, enhancing anchorage-independent growth (e.g.,
caused by a modified interaction with the extracellular matrix),
and increasing cell survival in the circulation (Fernandez et al.,
Cell Death Differ., 7:350-359 (2000)). It has been shown that a
number of cell adhesion molecules play a role in metastasis and
that integrins are especially involved in tumorigenic spread.
Integrins are implicated in cell-cell and cell-extracellular matrix
(ECM) interactions, signaling, sensing cellular microenvironment,
and in moderating cellular activities including, but not limited
to, migration, differentiation, survival and tissue (re)modeling in
both normal and pathological states. The present invention
contemplates that anti-apoptotic proteins such as Bcl-2 and/or
Bcl-X.sub.L regulate cell-cell interactions (See, J. Reed, Nature,
387:773-776 (1997)). Down-regulation of cell surface integrins by
antibodies could lead to induction of apoptosis. For example, Bcl-2
expression is up-regulated by .alpha..sub.5.beta..sub.1 integrins
preventing apoptosis when cells are detached from the matrix (See,
S. Frisch and E. Ruoslahti, Curr. Opin. Cell Biol., 9:701-706
((1997)). Expression of Bcl-2 is contemplated to promote the
metastatic potential of the human breast cancer cell line MCF7 in
vivo and migratory and invasive properties in vitro (See, D. Del
Bufalo, et al., FASEB J., 11:947-953 (1997)).
[0023] In some embodiments, the present invention provides methods
of inhibiting tumor metastasis in a subject, comprising
administering to the subject a gossypol compound (e.g.,
(-)-gossypol) that decreases the survival of metastatic cells by
inhibiting cellular activity of Bcl-2/Bcl-X.sub.L proteins. In
certain other embodiments, the present invention provides methods
of treating (e.g., ameliorating and/or preventing) cancer
metastasis comprising administering to a subject having a cancer
metastasis a therapeutically effective amount of a gossypol
compound (e.g., (-)-gossypol), and optionally one or more
anticancer and/or anti-neoplastic agents. The present invention is
not intended to be limited to administering any particular gossypol
compound, or compounds for the prevention (or retarding) of tumor
metastasis. Indeed, a number of gossypol compounds are contemplated
as being useful in the preventing, attenuating, or retarding of
tumor metastasis including, but not limited to, (.+-.)-gossypol;
(-)-gossypol; (+)-gossypol; (.+-.)-gossypolone; (-)-gossypolone;
(+)-gossypolone; (.+-.)-gossypol acetic acid; (-)-gossypol acetic
acid; (+)-gossypol acetic acid; (.+-.)-ethyl gossypol; (-)-ethyl
gossypol; (+)-ethyl gossypol; (.+-.)-hemigossypolone;
(-)-hemigossypolone; (+)-hemigossypolone; Schiff's base of
(.+-.)-gossypol; Schiff's base of (-)-gossypol; Schiff's base of
(+)-gossypol; Schiff's base of (.+-.)-gossypolone; Schiff's base of
(-)-gossypolone; Schiff's base of (+)-gossypolone; Schiff's base of
(.+-.)-gossypol acetic acid; Schiff's base of (-)-gossypol acetic
acid; Schiff's base of (+)-gossypol acetic acid; Schiff's base of
(.+-.)-ethyl gossypol; Schiff s base of (-)-ethyl gossypol;
Schiff's base of (+)-ethyl gossypol; Schiff's base of
(.+-.)-hemigossypolone; Schiff's base of (-)-hemigossypolone;
Schiff's base of (+)-hemigossypolone, (.+-.)-apogossypol,
(-)-apogossypol, (+)-apogossypol, (.+-.)-apogossypol acetic acid,
(-)-apogossypol acetic acid, (+)-apogossypol acetic acid,
(.+-.)-ethyl apogossypol, (-)-ethyl apogossypol, (+)-ethyl
apogossypol, and the like. The present invention further
contemplates that a range of additional (second) chemotherapeutic,
anticancer, or anti-neoplastic agents, radiation therapies, and/or
surgical interventions can optionally be combined (in any temporal
order) with gossypol compounds to prevent or retard tumor
metastasis in a subject. In this regard, the present invention
describes various exemplary additional (second) agents and
therapies that are useful in certain embodiments of the present
invention directed to tumor metastasis.
[0024] An important goal in oncology is to optimize the use of
available treatment options (e.g., chemotherapy, radiation therapy,
surgery, and the like) to achieve maximum obtainable therapeutic
effect while preserving organs and the subject's general quality of
life.
[0025] Bcl-2 is the founding member of a family of proteins and was
first isolated as the product of an oncogene. The Bcl-2 family of
proteins now includes both anti-apoptotic molecules such as Bcl-2
and Bcl-X.sub.L and pro-apoptotic molecules such as Bax, Bak, Bid,
and Bad. Bcl-2 and Bcl-X.sub.L are thought to be important
regulators of Bcl-2 family mediated apoptosis.
[0026] In preferred embodiments, the administration of gossypol
compounds is contemplated to provide an effective treatment of
neoplastic conditions and other disorders that involve either the
aberrant hyperproliferation or defective apoptosis of cells (e.g.,
tumor cells).
[0027] In other preferred embodiments, the present invention
provides methods of treatment or prophylaxis of cancers in a
subject comprising administering to the subject a gossypol compound
in an amount effective to inhibit Bcl-2 and/or Bcl-X.sub.L, thus
inducing apoptosis and suppressing tumor growth and/or
proliferation. Preferably, a gossypol compound is administered in
conjunction with another agent or treatment, such as a
chemotherapeutic agent (e.g., a tumor cell apoptosis promoting
agent) or radiation. The present invention is not limited to any
particular mechanism. Indeed, an understanding of any particular
mechanism is unnecessary to practice (make and use) the methods and
compositions of the present invention. Nonetheless, it is
contemplated that increasing apoptosis in target cells (e.g.,
pathogenic cells including, but not limited to, cancer cells)
reestablishes normal apoptotic control associated with basal
expression of Bcl-2 and/or Bcl-X.sub.L and/or another
anti-apoptotic Bcl-2 family protein (e.g. Bcl-w).
[0028] The methods of the present invention are particularly well
suited for the treatment of cancers characterized by overexpression
of Bcl-2 family proteins including, but not limited to,
[0029] Bcl-2 and/or Bcl-X.sub.L.
[0030] In some preferred embodiments, the methods of the present
invention provide effective amounts of gossypolone to a patient
having a condition characterized by the overexpression of Bcl-2
family proteins, and optionally one or more anticancer or
anti-neoplastic agent including, but not limited to radiation
therapy.
[0031] In one preferred embodiment, the present invention provides
a method of modulating apoptosis in a cell comprising: providing a
cell, wherein the cell overexpresses a Bcl-2 family protein; a
gossypol compound; and treating the cell with an effective amount
of the gossypol compound under conditions such that apoptosis in
the cell is modulated.
[0032] The methods of the present invention are not intended to be
limited to administration of any particular gossypol compounds.
Indeed, the present invention contemplates the administration of a
number of gossypol enantiomers, metabolites, derivatives, and
pharmaceutically acceptable salts, as well as Schiff's bases of
these compounds. For example, gossypol compounds suitable for use
in the present invention include, but are not limited to,
(.+-.)-gossypol; (-)-gossypol; (+)-gossypol; (.+-.)-gossypolone;
(-)-gossypolone; (+)-gossypolone; (.+-.)-gossypol acetic acid;
(-)-gossypol acetic acid; (+)-gossypol acetic acid; (.+-.)-ethyl
gossypol; (-)-ethyl gossypol; (+)-ethyl gossypol;
(.+-.)-hemigossypolone; (-)-hemigossypolone; (+)-hemigossypolone;
Schiff's base of (.+-.)-gossypol; Schiff's base of (-)-gossypol;
Schiff's base of (+)-gossypol; Schiff's base of (.+-.)-gossypolone;
Schiff's base of (-)-gossypolone; Schiff s base of (+)-gossypolone;
Schiff s base of (.+-.)-gossypol acetic acid; Schiff's base of
(-)-gossypol acetic acid; Schiff's base of (+)-gossypol acetic
acid; Schiff's base of (.+-.)-ethyl gossypol; Schiff's base of
(-)-ethyl gossypol; Schiff's base of (+)-ethyl gossypol; Schiff's
base of (.+-.)-hemigossypolone; Schiff's base of
(-)-hemigossypolone; Schiff's base of (+)-hemigossypolone,
(.+-.)-apogossypol, (-)-apogossypol, (+)-apogossypol,
(.+-.)-apogossypol acetic acid, (-)-apogossypol acetic acid,
(+)-apogossypol acetic acid, (+)-ethyl apogossypol, (-)-ethyl
apogossypol, (+)-ethyl apogossypol, and the like.
[0033] In preferred embodiments, the present invention provides
administering the (-)-gossypol enantiomer to a patient having a
condition characterized by overexpression of a Bcl-2 family
protein. In some embodiments, the overexpressed Bcl-2 family
proteins contemplated include, but are not limited to, Bcl-2,
Bcl-X.sub.L, Mcl-1, Bcl-w, A1/BFL-1, BOO-DNA, Bcl-6, Bcl-8, and
Bcl-y. In still some other embodiments, the overexpressed Bcl-2
family proteins have pro-apoptotic activity. In yet other
embodiments, the overexpressed Bcl-2 family proteins have
anti-apoptotic activity.
[0034] In some embodiments, the compositions and methods of the
present invention are used to treat diseased cells, tissues,
organs, or pathological conditions and/or disease states in a
subject organism (e.g., a mammalian subject including, but not
limited to, humans and veterinary animals), or in in vitro and/or
ex vivo cells, tissues, and organs. In this regard, various
diseases and pathologies are amenable to treatment or prophylaxis
using the present methods and compositions. A non-limiting
exemplary list of these diseases and conditions includes, but is
not limited to, breast cancer, prostate cancer, lymphoma, skin
cancer, pancreatic cancer, colon cancer, melanoma, malignant
melanoma, ovarian cancer, brain cancer, primary brain carcinoma,
head-neck cancer, glioma, glioblastoma, liver cancer, bladder
cancer, non-small cell lung cancer, head or neck carcinoma, breast
carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung
carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma,
bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon
carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid
carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal
carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal
cortex carcinoma, malignant pancreatic insulinoma, malignant
carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant
hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic
leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia,
chronic myelogenous leukemia, chronic granulocytic leukemia, acute
granulocytic leukemia, hairy cell leukemia, neuroblastoma,
rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential
thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma,
soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia,
and retinoblastoma, and the like, T and B cell mediated autoimmune
diseases; inflammatory diseases; infections; hyperproliferative
diseases; AIDS; degenerative conditions, vascular diseases, and the
like. In some embodiments, the cancer cells being treated are
metastatic.
[0035] In some embodiments, infections suitable for treatment with
the compositions and methods of the present invention include, but
are not limited to, infections caused by viruses, bacteria, fungi,
mycoplasma, prions, and the like. The present invention is not
intended to be limited, however, to treating of any particular
infections or infectious agents.
[0036] In one preferred embodiment, the present invention provides
methods of modulating cell division in a tissue comprising:
providing a tissue, wherein the tissue overexpresses a Bcl-2
protein; a gossypol compound; an anticancer agent; and treating the
tissue with effective amounts of the gossypol compound and the
anticancer agent under conditions such that cell division is
modulated. In some of these embodiments, the present invention
contemplates gossypol compounds bind to Bcl-2 family proteins thus
modulating cell division. In still further embodiments, the methods
optionally comprise one or more antineoplastic and/or
anti-hyperproliferative chemotherapeutic agents (e.g., small or
large molecule drugs, polypeptides, polynucleotides, synthetic or
naturally occurring chemical compounds, and the like), or therapies
(e.g., radiation therapy, surgical interventions, etc.).
[0037] In yet another embodiment, the present invention provides
methods of treating a subject (e.g., a patient) comprising
administering a gossypol compound to a subject overexpressing a
Bcl-2 family protein. In a preferred example of these embodiments,
the gossypol compound binds to a Bcl-2 family protein.
[0038] Some embodiments of the present invention are directed to
providing methods of treating a subject comprising administering a
gossypol compound and one or more anticancer agents to a subject
overexpressing a Bcl-2 family protein.
[0039] A number of suitable anticancer agents are contemplated for
use in the methods of the present invention. Indeed, the present
invention contemplates, but is not limited to, administration of
numerous anticancer agents such as: agents that induce apoptosis;
polynucleotides (e.g., anti-sense, ribozymes, siRNA); polypeptides
(e.g., enzymes and antibodies); biological mimetics (e.g., gossypol
or BH3 mimetics); agents that bind (e.g., oligomerize or complex)
with a Bcl-2 family protein such as Bax; alkaloids; alkylating
agents; antitumor antibiotics; antimetabolites; hormones; platinum
compounds; monoclonal or polyclonal antibodies (e.g., antibodies
conjugated with anticancer drugs, toxins, defensins, etc.), toxins,
radionuclides; biological response modifiers (e.g., interferons
(e.g., IFN-.alpha., etc.) and interleukins (e.g., IL-2, etc.),
etc.); adoptive immunotherapy agents; hematopoietic growth factors;
agents that induce tumor cell differentiation (e.g.,
all-trans-retinoic acid, etc.); gene therapy reagents (e.g.,
antisense therapy reagents and nucleotides); tumor vaccines;
angiogenesis inhibitors; proteosome inhibitors: NF kappa .beta.
modulators; anti-CDK compounds; HDAC inhibitors; and the like.
Numerous other examples of chemotherapeutic compounds and
anticancer therapies suitable for co-administration with the
disclosed gossypol compounds are known to those skilled in the
art.
[0040] In preferred embodiments, anticancer agents comprise agents
that induce or stimulate apoptosis. Agents that induce apoptosis
include, but are not limited to, radiation (e.g., X-rays, gamma
rays, UV); kinase inhibitors (e.g., Epidermal Growth Factor
Receptor (EGFR) kinase inhibitor, Vascular Growth Factor Receptor
(VGFR) kinase inhibitor, Fibroblast Growth Factor Receptor (FGFR)
kinase inhibitor, Platelet-derived Growth Factor Receptor (PDGFR)
kinase inhibitor, and Bcr-Abl kinase inhibitors such as GLEEVEC);
antisense molecules; antibodies (e.g., HERCEPTIN, RITUXAN, ZEVALIN,
and AVASTIN); anti-estrogens (e.g., raloxifene and tamoxifen);
anti-androgens (e.g., flutamide, bicalutamide, finasteride,
aminoglutethamide, ketoconazole, and corticosteroids);
cyclooxygenase 2 (COX-2) inhibitors (e.g., celecoxib, meloxicam,
NS-398, and non-steroidal anti-inflammatory drugs (NSAIDs));
anti-inflammatory drugs (e.g., butazolidin, DECADRON, DELTASONE,
dexamethasone, dexamethasone intensol, DEXONE, HEXADROL,
hydroxychloroquine, METICORTEN, ORADEXON, ORASONE, oxyphenbutazone,
PEDIAPRED, phenylbutazone, PLAQUENIL, prednisolone, prednisone,
PRELONE, and TANDEARIL); and cancer chemotherapeutic drugs (e.g.,
irinotecan (CAMPTOSAR), CPT-11, fludarabine (FLUDARA), dacarbazine
(DTIC), dexamethasone, mitoxantrone, MYLOTARG, VP-16, cisplatin,
carboplatin, oxaliplatin, 5-FU, doxorubicin, gemcitabine,
bortezomib, gefitinib, bevacizumab, TAXOTERE or TAXOL); cellular
signaling molecules; ceramides and cytokines; and staurosporine,
and the like.
[0041] In still other embodiments, the compositions and methods of
the present invention provide gossypol compounds and at least one
anti-hyperproliferative or antineoplastic agent(s) selected from
alkylating agents, antimetabolites, and natural products (e.g.,
herbs and other plant and/or animal derived compounds).
[0042] Alkylating agents suitable for use in the present
compositions and methods include, but are not limited to: 1)
nitrogen mustards (e.g., mechlorethamine, cyclophosphamide,
ifosfamide, melphalan (L-sarcolysin); and chlorambucil); 2)
ethylenimines and methylmelamines (e.g., hexamethylmelamine and
thiotepa); 3) alkyl sulfonates (e.g., busulfan); 4) nitrosoureas
(e.g., carmustine (BCNU); lomustine (CCNU); semustine
(methyl-CCNU); and streptozocin (streptozotocin)); and 5) triazenes
(e.g., dacarbazine (DTIC;
dimethyltriazenoimid-azolecarboxamide).
[0043] In some embodiments, antimetabolites suitable for use in the
present compositions and methods include, but are not limited to:
1) folic acid analogs (e.g., methotrexate (amethopterin)); 2)
pyrimidine analogs (e.g., fluorouracil (5-fluorouracil; 5-FU),
floxuridine (fluorode-oxyuridine; FudR), and cytarabine (cytosine
arabinoside)); and 3) purine analogs (e.g., mercaptopurine
(6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and
pentostatin (2'-deoxycoformycin)).
[0044] In still further embodiments, chemotherapeutic agents
suitable for use in the compositions and methods of the present
invention include, but are not limited to: 1) vinca alkaloids
(e.g., vinblastine (VLB), vincristine); 2) epipodophyllotoxins
(e.g., etoposide and teniposide); 3) antibiotics (e.g.,
dactinomycin (actinomycin D), daunorubicin (daunomycin;
rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and
mitomycin (mitomycin C)); 4) enzymes (e.g., L-asparaginase); 5)
biological response modifiers (e.g., interferon-alfa); 6) platinum
coordinating complexes (e.g., cisplatin (cis-DDP) and carboplatin);
7) anthracenediones (e.g., mitoxantrone); 8) substituted ureas
(e.g., hydroxyurea); 9) methylhydrazine derivatives (e.g.,
procarbazine (N-methylhydrazine; MIH)); 10) adrenocortical
suppressants (e.g., mitotane (o,p' -DDD) and aminoglutethimide);
11) adrenocorticosteroids (e.g., prednisone); 12) progestins (e.g.,
hydroxyprogesterone caproate, medroxyprogesterone acetate, and
megestrol acetate); 13) estrogens (e.g., diethylstilbestrol and
ethinyl estradiol); 14) antiestrogens (e.g., tamoxifen); 15)
androgens (e.g., testosterone propionate and fluoxymesterone); 16)
antiandrogens (e.g., flutamide): and 17) gonadotropin-releasing
hormone analogs (e.g., leuprolide).
[0045] In still other embodiments, the present invention provides
methods of treating cancer in a subject comprising administering to
a patient having a condition characterized by overexpression of a
Bcl-2 family protein an effective amount of a gossypol
compound.
[0046] Additional embodiments are directed to methods of treating
cancer in a subject comprising administering to a subject having
cancer, wherein the cancer is characterized by overexpression of a
Bcl-2 family protein, an effective amount of a gossypol compound
and one or more anticancer agents.
[0047] Still other methods are directed to treating cancer in a
subject comprising administering to a patient having cancer,
wherein the cancer is characterized by resistance to cancer
therapies (e.g., chemoresistant, radiation resistant, hormone
resistant, and the like), an effective amount of a gossypol
compound.
[0048] In some embodiments, the present invention provides methods
of treating cancer in a subject comprising administering to a
patient having cancer, wherein the cancer is characterized by
overexpression of a Bcl-2 family protein, a dose of a gossypol
compound sufficient to reduce the overexpression of the Bcl-2
protein.
[0049] In some embodiments of the present invention, methods of
treating cancer in a subject comprising administering to a patient
having cancer, wherein the cancer is characterized by
overexpression of a Bcl-2 family protein, a dose of a gossypol
compound and one or more anticancer agents sufficient to reduce the
overexpression of the Bcl-2 protein are described.
[0050] In still some other embodiments, the present invention
provides methods of treating a hyperproliferative disease, wherein
the hyperproliferative disease is characterized by the
overexpression of an anti-apoptotic Bcl-2 family protein (e.g.,
Bcl-2 or Bcl-X.sub.L), in a subject comprising administering to a
subject a dose of a gossypol compound sufficient to inhibit the
function of the anti-apoptotic Bcl-2 protein and/or to reduce the
overexpression of the protein. In some of these embodiments, the
methods further comprise administering one or more
hyperproliferative and/or anti-neoplastic therapeutic agents to the
subject.
[0051] Some other embodiments of the present invention provide
pharmaceutical compositions comprising: a gossypol compound; and
instructions for administering the gossypol compound to a subject,
the subject characterized by overexpression of a Bcl-2 family
protein (e.g., an anti-apoptotic Bcl-2 family member protein).
Additional embodiments provide pharmaceutical compositions
comprising: a gossypol compound; one or more anticancer agents; and
instructions for administering the gossypol compound and the one or
more anticancer agents to a subject.
[0052] Further embodiments of the present invention provide
pharmaceutical compositions comprising: a gossypol compound;
optionally one or more anticancer agents; and instructions for
administering the gossypol compound to a subject, the subject
characterized by resistance to a cancer therapy. In preferred
embodiments, the instructions included with these kits meet U.S.
Food and Drug Administration rules, regulations, and suggestions
for the administration, preparation, and distribution of
therapeutic kits, compounds, and methods. The instructions
optionally also satisfy the domestic regulations placed on
therapeutic kits, compounds, and methods, by countries and
jurisdictions other than the U.S.
[0053] In yet another embodiment, the present invention provides
methods of screening a gossypol compound and a test compound
comprising: providing: a gossypol compound; a test compound; a
first group of cells; and contacting the first group of cells with
the gossypol compound and the test compound; and observing the
effects of contacting the first group of cells with the gossypol
compound and the test compound. In some of these embodiments, the
present invention further provides the additional step of comparing
the effects observed in the first cells against a second group of
the cells contacted with the gossypol compound alone, or with the
test compound alone. Effects that may be observed include, but are
not limited to, changes in cell proliferation, changes in apoptotic
status, and changes in the expression of Bcl-2 family proteins
(e.g., Bcl-2 and/or Bcl-X.sub.L), and the like. In still other
embodiments, the present invention further contemplates additional
methods for selling test compounds screened/identified by the above
methods. In some of these embodiments, test compounds may be
offered for sale by a third party in one or more forms (e.g., a
kit, including instructions for administering the test compound to
a patient). The present invention further provides kits comprising
a gossypol compound, one or more chemotherapeutic agents, and
instructions for administering the gossypol compound and the
chemotherapeutic agents to a subject. In certain of these
embodiments, the gossypol compound is (-)-gossypol and the
chemotherapeutic agent is selected from docetaxel, TAXOL,
cisplatin, and combinations thereof. The present invention is not
limited however to kits comprising (-)-gossypol and docetaxel,
TAXOL, cisplatin, and combinations thereof.
[0054] The present invention further provides a method of treating
or ameliorating a hyperproliferative (or neoplastic) disease in a
subject comprising administering to the subject a therapeutically
effective dose of a gossypol compound and one or more second agent
selected from a chemotherapeutic agent and radiation. In other
embodiments, the present invention provides a method of treating or
ameliorating a hyperproliferative (or neoplastic) disease in a
subject comprising administering to the subject a therapeutically
effective dose of a gossypol compound and one or more second agent
selected from a chemotherapeutic agent and radiation, with the
proviso that a combination of (.+-.)-gossypol, heat, and radiation
is not administered. In some embodiments, the one or more second
agents comprise anti-neoplastic agents.
[0055] In some methods, a gossypol compound and a chemotherapeutic
agent and/or radiation are administered simultaneously. In some
other embodiments, a gossypol compound and a chemotherapeutic agent
and/or radiation are administered sequentially. In still some other
embodiments, a gossypol compound is administered prior to
chemotherapeutic agent(s) and/or radiation. In yet other
embodiments, a gossypol compound is administered after
chemotherapeutic agent(s) and/or radiation.
[0056] The present invention further provides methods, wherein a
gossypol compound and a chemotherapeutic agent or radiation are
administered with different periodicities, different durations,
different concentrations, and/or different administration
routes.
[0057] Additional embodiments provide methods wherein a gossypol
compound and a chemotherapeutic agent and/or radiation have a
synergistic therapeutic effect in a subject or in vitro or ex vivo
cells, tissues, or organs.
[0058] In some embodiments, the subject being treated is an animal
such as a mammal, fish, or bird. In some embodiments, the mammal
being treated is a human. In some other embodiments, the mammal
being treated is laboratory animal (e.g., rodent (e.g., mouse, rat,
gerbil, rabbit), monkey, dog, pig, cat, etc.). In still some other
embodiments, the mammal is a veterinary animal (e.g., dog, cat,
horse, cow, pig, goat, sheep, etc.).
[0059] In certain preferred methods, a gossypol compound is
provided to a subject in a dose that sensitizes the subject to
treatment by one or more second agents. The present invention
provides compositions and methods directed at therapeutic treatment
of resistant diseases (e.g., cancer). Diseases that are
specifically contemplated by the present invention include, but are
not limited to, chemotherapy resistant diseases (e.g., cancers) and
radiation therapy resistant diseases (e.g., cancers). In
particularly preferred embodiments, the administration of gossypol
compound(s), and optionally one or more chemotherapeutic agents
(e.g., anticancer drug) or therapeutic methods (e.g., radiation
therapy) sensitizes the disease (e.g., disease cells) to
treatment.
[0060] In some embodiments, the hyperproliferative (or neoplastic)
disease is a cancer (e.g., breast cancer, prostate cancer,
pancreatic cancer, colon cancer, lung cancer, lymphoma, melanoma,
or head-neck cancer). The present invention contemplates treating
metastatic cancers.
[0061] The present invention further provides compositions (e.g.,
pharmaceutical formulations) and methods for treating diseases
(e.g., cancer) the use of which in a subject results in the
regression of the disease. In other embodiments, the use of the
compositions and methods of the present invention in a subject
having a disease (e.g., cancer) results in the arrest or stasis of
a disease.
[0062] The present invention further provides a pharmaceutical
composition for the treatment of tumors characterized in that it
comprises a gossypol compound and an additional therapeutic agent.
Similarly, also provided are pharmaceutical compositions comprising
a gossypol compound and an additional therapeutic agent, wherein
the pharmaceutical composition is useful as an anti-tumor
therapy.
[0063] In certain pharmaceutical compositions the gossypol compound
is selected from the group comprising (-)-gossypol, (+)-gossypol,
(-)-gossypolone, (+)-gossypolone, (-)-gossypol acetic acid,
(+)-gossypol acetic acid, (-)-ethyl gossypol, (+)-ethyl gossypol,
(-)-hemigossypolone, (+)-hemigossypolone, a Schiff's base of
(-)-gossypol, a Schiff's base of (+)-gossypol, a Schiff's base of
(-)-gossypolone, a Schiff's base of (+)-gossypolone, a Schiff's
base of (-)-gossypol acetic acid, a Schiff's base of (+)-gossypol
acetic acid, a Schiff's base of (-)-ethyl gossypol, a Schiff's base
of (+)-ethyl gossypol, a Schiff's base of (-)-hemigossypolone, and
a Schiff's base of (+)-hemigossypolone, (-)-apogossypol,
(+)-apogossypol, (-)-apogossypol acetic acid, (+)-apogossypol
acetic acid, (-)-ethyl apogossypol, (+)-ethyl apogossypol, or the
racemate of any of the above enantiomeric pairs.
[0064] In still other pharmaceutical compositions and therapeutic
methods the target tumor is selected from the group consisting of
breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic
cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer,
brain cancer, primary brain carcinoma, head-neck cancer, glioma,
glioblastoma, liver cancer, bladder cancer, non-small cell lung
cancer, head or neck carcinoma, breast carcinoma, ovarian
carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms' tumor,
cervical carcinoma, testicular carcinoma, bladder carcinoma,
pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic
carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal
carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell
carcinoma, endometrial carcinoma, adrenal cortex carcinoma,
malignant pancreatic insulinoma, malignant carcinoid carcinoma,
choriocarcinoma, mycosis fungoides, malignant hypercalcemia,
cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic
lymphocytic leukemia, chronic granulocytic leukemia, acute
granulocytic leukemia, acute myelogenous leukemia, chronic
myelogenous leukemia, hairy cell leukemia, neuroblastoma,
rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential
thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma,
soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia,
and retinoblastoma.
[0065] The present invention further provides pharmaceutical
compositions, wherein an additional therapeutic agent (one or more
second agents) is selected from the group consisting of agents that
induce apoptosis, pro-apoptotic Bcl-2 proteins, polynucleotides,
polypeptides, photodynamic compounds, radiodynamic compounds,
radionuclides, radioactive elements, gamma ray emitters, beta
particle emitters, drugs, biological mimetics, alkaloids,
alkylating agents, antibiotics, antimicrobials, antifungals,
antimetabolites, hormones, platinum compounds, monoclonal
antibodies, toxins, defensins, interferons, interleukins, adoptive
immunotherapy agents, hematopoietic growth factors, agents that
induce tumor cell differentiation, gene therapy reagents, antisense
molecules, kinase inhibitors, vascular growth factor receptor
kinase inhibitor, fibroblast growth factor receptor kinase
inhibitor, platelet-derived growth factor receptor kinase
inhibitor, GLEEVEC, anti-estrogens, anti-androgens, cyclooxygenase
2 (COX-2) inhibitors, non-steroidal anti-inflammatory drugs
(NSAIDs), chemotherapeutic drugs, nucleotide analogue reverse
transcriptase inhibitors, nucleoside analogue reverse transcriptase
inhibitors, non-nucleoside reverse transcriptase inhibitors,
protease inhibitors, and combinations thereof. In certain
embodiments, the additional therapeutic agent (or one or more
second agent) is selected from the group consisting of
3,7,11,15-tetramethyl-2,6,10,14-hexadecatraen-1-ol, a Bcl-2 family
protein (e.g., Bax, Bak, Bid, Bad), DNA, RNA, ribozymes, RNAse,
siRNAs, enzymes, .sup.111In-oxine, .sup.59Fe, .sup.67Cu, .sup.125I,
.sup.99Te, .sup.51Cr, .sup.32P, .sup.3H, .sup.35S, .sup.4C,
IFN-.alpha., IL-2, all-trans-retinoic acid, EGFR, VGFR, FGFR,
PDGFR, STI-571, GLEEVEC, HERCEPTIN, RITUXAN, raloxifene, tamoxifen,
flutamide, bicalutamide, finasteride, aminoglutethamide,
ketoconazole, corticosteroids, celecoxib, meloxicam, NS-398,
irinotecan CPT-11, fludarabine, dacarbazine, dexamethasone,
mitoxantrone, MYLOTARG, VP-16, 5-FU, cisplatin, carboplatin,
gemcitabine, doxorubicin, TAXOTERE, TAXOL, tenofovir disoproxil
fumarate, zidovudine, lamivudine, abacavir, zalcitabine,
didanosine, stavudine, nevirapine, delavirdine, efavirenz,
saquinavir (SQV (HGC)), saquinavir (SQV (SGC)), ritonavir,
indinavir, nelfinavir, amprenavir, mechlorethamine,
cyclophosphamide, ifosfamide, melphalan, L-sarcolysin,
chlorambucil, hexamethylmelamine, thiotepa, busulfan, carmustine,
lomustine, semustine, streptozocin, dacarbazine, methotrexate,
fluorouracil, floxuridine, cytarabine, mercaptopurine, thioguanine,
pentostatin, vinblastine, vincristine, etoposide, teniposide,
dactinomycin, daunorubicin, bleomycin, plicamycin, mitomycin,
L-asparaginase, hydroxyurea, procarbazine, mitotane,
aminoglutethimide, prednisone, hydroxyprogesterone caproate,
medroxyprogesterone acetate, megestrol acetate, diethylstilbestrol,
ethinyl estradiol, testosterone propionate, fluoxymesterone,
flutamide, and leuprolide, and combinations thereof.
[0066] In still some other embodiments, the present invention
provides compositions and methods for preventing (or attenuating)
the onset or spread of a hyperproliferative disease. In some other
embodiments, the present invention provides compositions and
methods for preventing (or attenuating) the onset or spread of a
neoplastic disease. In some preferred embodiments, the present
invention provides methods of preventing (or attenuating) cancers
in a subject comprising administering to the subject a gossypol
compound (e.g., (-)-gossypol, (-)-gossypol acetic acid, etc.) in an
amount effective to inhibit Bcl-2 family protein (e.g., Bcl-2
and/or Bcl-X.sub.L). In some of these embodiments, the Bcl-2 family
proteins contemplated include, but are not limited to, Bcl-2,
Bcl-X.sub.L, Mcl-1, Bcl-w, A1/BFL-1, BOO-DIVA, Bcl-6, Bcl-8, and
Bcl-y.
[0067] Preferably, methods of preventing (or attenuating)
hyperproliferative and/or neoplastic diseases comprise a gossypol
compound administered in conjunction with another agent or
treatment, such as an anticancer agent, an anti-neoplastic agent
(e.g., a tumor cell apoptosis promoting agent), or radiation
therapy. The present methods of preventing hyperproliferative
and/or neoplastic diseases are not limited to the administration of
any particular gossypol compound. Indeed, the present invention
contemplates that a number of gossypol compounds can be
administered to a subject to prevent (or attenuate)
hyperproliferative and/or neoplastic diseases including, but not
limited to, (.+-.)-gossypol; (-)-gossypol; (+)-gossypol;
(.+-.)-gossypolone; (-)-gossypolone; (+)-gossypolone;
(.+-.)-gossypol acetic acid; (-)-gossypol acetic acid; (+)-gossypol
acetic acid; (.+-.)-ethyl gossypol; (-)-ethyl gossypol; (+)-ethyl
gossypol; (.+-.)-hemigossypolone; (-)-hemigossypolone;
(+)-hemigossypolone; Schiff's base of (.+-.)-gossypol; Schiff's
base of (-)-gossypol; Schiff's base of (+)-gossypol; Schiff's base
of (.+-.)-gossypolone; Schiff's base of (-)-gossypolone; Schiff's
base of (+)-gossypolone; Schiff's base of (.+-.)-gossypol acetic
acid; Schiff's base of (-)-gossypol acetic acid; Schiff's base of
(+)-gossypol acetic acid; Schiff's base of (.+-.)-ethyl gossypol;
Schiff's base of (-)-ethyl gossypol; Schiff's base of (+)-ethyl
gossypol; Schiff's base of (.+-.)-hemigossypolone; Schiff s base of
(-)-hemigossypolone; Schiff s base of (+)-hemigossypolone,
(.+-.)-apogossypol, (-)-apogossypol, (+)-apogossypol,
(.+-.)-apogossypol acetic acid, (-)-apogossypol acetic acid,
(+)-apogossypol acetic acid, (.+-.)-ethyl apogossypol, (-)-ethyl
apogossypol, (+)-ethyl apogossypol, and the like.
[0068] Similarly, the present compositions and methods of
preventing (or attenuating) a hyperproliferative and/or neoplastic
disease are not limited to any particular additional (second)
chemotherapeutic, anticancer, or anti-neoplastic agents or
therapies. The present invention contemplates that any of the
exemplary therapeutics described herein (or referenced herein) may
find use in certain embodiments.
[0069] Those skilled in the art can determine the amount of
attenuation or whether prevention of a hyperproliferative and/or
neoplastic disease has occurred upon use of the compositions and
methods of the present invention in a subject, or in in vitro or ex
vivo cells, tissues, and organs using standard protocols in
comparison to nonpathological subjects, cells, tissues, and
organs.
[0070] Still further embodiments of the present invention provide
the use of a gossypol compound and an additional therapeutic agent
in the manufacture of a medicament for the treatment of a
neoplastic and/or hyperproliferative disease.
[0071] Other embodiments of the present invention specifically
contemplate chemical intermediates, and formulations of compounds
(e.g., gossypol compounds and optionally one or more
chemotherapeutic agents) used in medicaments, in the manufacture of
medicaments, kits for the administration of medicaments, or
diagnostic tests and other applications related thereto, and other
beneficial formulations.
[0072] Also provided are uses of the compositions and methods of
the present invention for the preparation of therapeutics,
medicaments, and other therapeutic applications.
[0073] In yet other embodiments, the present invention provides
methods and compositions according to any of the claims or
substantially as described in any of the Examples or various
embodiments disclosed herein.
[0074] Other advantages, benefits, and preferable embodiments of
the present invention will be apparent to those skilled in the
art.
DESCRIPTION OF THE FIGURES
[0075] The following figures form part of the specification and are
included to further demonstrate certain aspects and embodiments of
the present invention. The present invention is not intended to be
limited however to the embodiments specifically recited in these
figures.
[0076] The following figures form part of the specification and are
included to further demonstrate certain aspects and embodiments of
the present invention. The present invention is not intended to be
limited however to the embodiments specifically recited in these
figures.
[0077] FIG. 1 shows a sequence alignment of Bcl-2 (SEQ ID NO:1) and
Bcl-X.sub.L (SEQ ID NO:2).
[0078] FIG. 2A shows a ribbon representation of the overall Bcl-2
structure in complex with the Bak BH3 peptide modeled from the
structure of Bcl-X.sub.L in complex with Bak BH3 peptide. FIG. 2B
shows a detailed representation of the BH3 binding site in
Bcl-2.
[0079] FIG. 3 shows gossypol directly inhibits binding between Bak
BH3 peptide and Bcl-2, and between Bak BH3 peptide and Bcl-X.sub.L
proteins in certain fluorescence polarization (FP) based binding
assays.
[0080] FIG. 4 shows the results of competitive inhibition assays
using racemic gossypol, (-)-gossypol, and (+)-gossypol to directly
block binding between Bid 21-residue BH3 peptide and Bcl-2.
[0081] FIG. 5 shows the results of a competitive inhibition assay
using racemic gossypol, (-)-gossypol, and (+)-gossypol to directly
block binding between Bad 25-residue BH3 peptide and
Bcl-X.sub.L.
[0082] FIG. 6A shows the results of a FP-based binding assay of
racemic gossyplone to Bcl-X.sub.L in one embodiment of the present
invention. FIG. 6B shows the results of a FP-based binding assay of
a ethyl Schiff's base of (-)-gossypol to Bcl-X.sub.L (time-course)
in one embodiment of the present invention.
[0083] FIG. 7 shows the results of several cancer cell lines and
one normal cell line that express various levels of Bcl-2 and/or
Bcl-X.sub.L proteins in one embodiment of the present
invention.
[0084] FIGS. 8A and 8B show the results of cell based assays in
various embodiments of the present invention.
[0085] FIG. 9 shows the results of cell based assays in various
embodiments of the present invention.
[0086] FIG. 10 shows the results of cell based assays in various
embodiments of the present invention.
[0087] FIGS. 11A and 11B show the results of the interactions
between (-)-gossypol and Bcl-X.sub.L protein using .sup.15N
Heteronuclear Single Quantum Coherence Spectroscopy (HSQC) NMR
methods in various embodiments of the present invention. FIG. 11C
shows the three-dimensional structural representation of
(-)-gossypol in complex with Bcl-X.sub.L protein based upon NMR
experimental data and computational modeling in one embodiment of
the present invention. The Bcl-X.sub.L protein is represented in a
ribbon model and the (-)-gossypol is represented in a stick
model.
[0088] FIG. 12 shows the results of cell based assays in one
embodiment of the present invention.
[0089] FIG. 13 shows the results of cell based assays in one
embodiment of the present invention.
[0090] FIG. 14 shows the results of cell based assays in one
embodiment of the present invention.
[0091] FIG. 15 shows the results of in vivo animal xenograft based
assays in one embodiment of the present invention.
[0092] FIG. 16 shows the results of cell based assays in various
embodiments of the present invention.
[0093] FIGS. 17A and 17B show the results of cell based assays in
various embodiments of the present invention.
[0094] FIG. 18 shows the results of in vivo animal xenograft based
assays in various embodiments of the present invention.
[0095] FIG. 19 shows the results of in vivo animal xenograft based
assays in various embodiments of the present invention.
[0096] FIG. 20 shows the results of in vivo animal xenograft based
assays in one embodiment of the present invention.
[0097] FIG. 21 shows the results of cell based assays (inhibition
of cell growth in several head-neck cancer cell lines and three
fibroblast cell lines) by (-)-gossypol in one embodiment of the
present invention.
[0098] FIG. 22 show the results of Western blotting analysis of the
protein levels of Bcl-2, Bcl-X.sub.L and Bcl-X.sub.S in several
head-neck cancer cell lines and one fibroblast cell line in various
embodiments of the present invention.
[0099] FIG. 23 show the results of cell growth inhibition by
(-)-gossypol in a panel of head-neck cancer cell lines and one
fibroblast cell line as determined by an MTT assay (right Y-axis)
and its relationship with the ratio of Bcl-X.sub.L/Bcl-X.sub.S
(left Y-axis) in various embodiments of the present invention.
[0100] FIGS. 24A-24C show the results of apoptosis induction
studies using (-)-gossypol in 6 cell lines as determined by the
TUNEL assay (UM-SCC-1, UM-SCC-6, UM-SCC-12, UM-SCC-14A, fibroblast
1 and fibroblast 2) in various embodiments of the present
invention.
[0101] FIG. 25 shows the chemical structures of gossypol,
gossyplone, Schiff's bases of gossypol and Schiff's bases of
gossypolone, (-)-gossypol and (+)-gossypol in various embodiments
of the present invention.
[0102] FIG. 26 shows the results of a saturation curve of
Bcl-X.sub.L protein to Bad 25-residue BH3 peptide.
[0103] FIG. 27 shows the results of saturation curve of Bcl-2
protein binding to Bid 21-residue BH3 peptide.
[0104] FIGS. 28A and 28B show the results of nuclear magnetic
resonance (NMR) based binding assays of (-)-gossypol and
(+)-gossypol to Bcl-X.sub.L, respectively.
[0105] FIG. 29 shows the results of cell based assays in various
embodiments of the present invention.
[0106] FIG. 30 shows the results of cell based assays of gossypol,
(-)-gossypol, and (+)-gossypol in various embodiments of the
present invention.
[0107] FIG. 31 shows the results of cell based assays in one
embodiment of the present invention.
[0108] FIGS. 32A and 32B shows the results of cell based assays in
one embodiment of the present invention.
[0109] FIG. 33 shows the results of cell based colony formation
assays in one embodiment of the present invention.
[0110] FIG. 34 shows the results of cell based assays in various
embodiments of the present invention.
[0111] FIGS. 35A and 35B show the results of cell based assays in
one embodiment of the present invention.
[0112] FIG. 36 shows the results of in vivo animal xenograft based
assays in one embodiment of the present invention.
[0113] FIG. 37 shows the results of in vivo animal xenograft based
assays in one embodiment of the present invention.
[0114] FIG. 38 shows the results of in vivo animal xenograft based
assays in one embodiment of the present invention.
[0115] FIG. 39 shows the results of cell based assays of the
inhibition of cell growth in 2 prostate cancer cell lines PC-3 and
LnCaP by racemic gossypol, (-)-gossypol, and (+)-gossypol in
various embodiments of the present invention.
[0116] FIG. 40 shows the results of cell based assays in one
embodiment of the present invention.
[0117] FIG. 41 shows the results of Western blotting analysis of
several Bcl-2 family proteins in one embodiment of the present
invention.
[0118] FIG. 42 shows the results of cell based assays in one
embodiment of the present invention.
[0119] FIGS. 43A and 43B show the results of in vivo animal
xenograft based assays in various embodiments of the present
invention.
[0120] FIG. 44 shows the results of cell based assays in one
embodiment of the present invention.
[0121] FIG. 45 describes the competitive binding curve of racemic
apogossypol in directly blocking binding between Bad 25-residue BH3
peptide and Bcl-2 using an in vitro fluorescence polarization-based
assay.
[0122] FIG. 46 describes the competitive binding curve of racemic
apogossypol in directly blocking the binding between Bad 21-residue
BH3 peptide and Bcl-X.sub.L protein using an in vitro fluorescence
polarization-based assay.
DEFINITIONS
[0123] To facilitate an understanding of the present invention, a
number of terms and phrases are defined below.
[0124] As used herein, the term "gossypol compound" refers to
enantiomers, isomers, derivatives, metabolites, Schiff's bases,
combinations with acids or bases, and pharmaceutically acceptable
salts of the gossypol molecule. Accordingly, gossypol compounds
include, but are not limited to, (.+-.)-gossypol; (-)-gossypol;
(+)-gossypol; (t)-gossypolone; (-)-gossypolone; (+)-gossypolone;
(.+-.)-gossypol acetic acid; (-)-gossypol acetic acid; (+)-gossypol
acetic acid; (.+-.)-ethyl gossypol; (-)-ethyl gossypol; (+)-ethyl
gossypol; (.+-.)-hemigossypolone; (-)-hemigossypolone;
(+)-hemigossypolone; Schiff's base of (.+-.)-gossypol; Schiff's
base of (-)-gossypol; Schiff's base of (+)-gossypol; Schiff's base
of (.+-.)-gossypolone; Schiff's base of (-)-gossypolone; Schiff's
base of (+)-gossypolone; Schiff's base of (.+-.)-gossypol acetic
acid; Schiff's base of (-)-gossypol acetic acid; Schiff's base of
(+)-gossypol acetic acid; Schiff's base of (.+-.)-ethyl gossypol;
Schiff's base of (-)-ethyl gossypol; Schiff's base of (+)-ethyl
gossypol; Schiff's base of (.+-.)-hemigossypolone; Schiff's base of
(-)-hemigossypolone; Schiff's base of (+)-hemigossypolone,
(.+-.)-apogossypol, (-)-apogossypol, (+)-apogossypol,
(.+-.)-apogossypol acetic acid, (-)-apogossypol acetic acid,
(+)-apogossypol acetic acid, (.+-.)-ethyl apogossypol, (-)-ethyl
apogossypol, (+)-ethyl apogossypol. Acids that may be used in
combination with gossypol include, but are not limited to, formic
acid, acetic acid, propionic acid, and butyric acid.
Physiologically acceptable salts include, but are not limited to,
salts comprising sodium hydroxide, potassium hydroxide, lithium
hydroxide, barium hydroxide, sodium carbonate, potassium carbonate,
sodium acetate, potassium acetate, pyridine, triethylamine, and
quinoline.
[0125] Gossypol derivatives include any derivatives that are useful
in the present invention. One of skill in the art is familiar with
derivatization techniques. Many gossypol derivatives are known
including, but not limited to, the following compounds:
##STR00001##
wherein R=methyl, ethyl, propyl, isopropyl, butyl, s-butyl,
t-butyl, pentyl, hexyl, heptyl, dodecyl, .beta.-methyl
phenylalanine ethyl, phenylalanine methyl ester (Razakantoanina et
al. Parasitol. Res., 86:665-668 (2000));
##STR00002## ##STR00003## ##STR00004##
wherein R=methyl and R'=hydrogen, methyl (Dao et al. Bioorg. Med.
Chem., 11:2001-2006 (2003));
##STR00005##
wherein R=methyl, ethyl, propyl, isopropyl, butyl, s-butyl,
t-butyl, pentyl, hexyl, heptyl, dodecyl, .beta.-methyl
phenylalanine ethyl, phenylalanine methyl ester (Dao et al. Eur. J.
Med. Chem., 35:805-813 (2000));
##STR00006## ##STR00007##
wherein R=methyl, ethyl, propyl, butyl, pentyl, propenyl, or
t-butyl (Deck et al. J. Med. Chem., 34:3301-3305 (1991));
##STR00008##
wherein
##STR00009##
Przybylski et al. J. Mol. Structure, 611(1-3):193-201 (2002);
##STR00010##
(A. I. Meyers and J. Jeffrey Willemsen, Chem. Commun., 16:1573-1574
(1997));
##STR00011##
(R. E. Royer et al., J. Med. Chem., 38:2427-2432 (1995));
##STR00012##
(R. E. Royer et al., J. Med. Chem., 29:1799-1801 (1986));
##STR00013##
(C. M. Venuti, J. Org. Chem., 46(15):3124-3127 (1981));
##STR00014##
wherein R=Me, Bz and R'=Me, H, and Bz;
##STR00015##
wherein R=Me, and H, (I. V. Ognyanoc et al., Helv. Chim. Acta,
72:353-360 (1989));
##STR00016##
(P. C. Meltzer et al., Org. Chem., 50(17):3121-3124 (1985));
##STR00017##
(R. Adams et al., J. Am. Chem. Soc., 60:2193-2204 (1938)). Other
derivatives of gossypol are disclosed in the following references:
Le Blanc et al. Pharmacol. Res., 46:551-555 (2002); Baumgrass et
al. J. Biol. Chem., 276:47914-47921 (2001); Shelley et al.
Anticancer Drugs, 11:209-216 (2000); Sonenberg et al.
Contraception, 37:247-255, (1988); Whaley et al. Contraception,
33:605-616 (1986); Dorsett et al. J. Pharm. Sci., 64:1073-1075
(1975); Wu et al. Yao Xue Xue Bao, 24:502-511 (1989); Hoffer et al.
Contraception, 37:301-331 (1988); Guo et al. Yao Xue Xue Bao,
22:597-602 (1987); and Manmade et al. Experientia, 39:1276-1277
(1983).
[0126] As used herein, the term "gossypol acetic acid" refers to a
composition of gossypol comprising an amount of acetic acid
sufficient to detectably stabilize the gossypol composition as
compared to gossypol compositions without acetic acid. The range of
acetic acid in "gossypol acetic acid" compositions is preferably
from about 0.01% to 99% (by weight), more preferably from about
0.1% to 50%, even more preferably from about 0.5% to 20%. In one
embodiment, the gossypol acetic acid is a complex consisting of
equimolar quantities of gossypol and acetic acid (Sigma-Aldrich
Corp., St. Louis, Mo.).
[0127] As used herein, the terms "(-)-gossypol," or "(-)-gossypol
compound/composition," refer to an optically active composition of
gossypol wherein the active molecules comprising the composition
rotate plane polarized light counterclockwise (e.g., levorotatory
molecules) as measured by a polarimeter. Preferably, the
(-)-gossypol compound has an enantiomeric excess of 1% to 100%. In
one embodiment, the (-)-gossypol compound has an enantiomeric
excess of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (-)-gossypol.
In one example of a "(-)-gossypol compound", the specific rotation
([.alpha.].sub.D) of the compound is about 350.degree. to about
-390.degree., about -375.degree. to about -390.degree. , or about
-385.degree. to about -390.degree.. (See e.g., Dowd, Chirality,
15:486 (2003); Ciesielska et al., Chem. Phys. Lett. 353:69 (2992);
Freedman et al., Chirality, 15:196 (2003); and Zhou et Kexue
Tongbao, 28:1574 (1983)). Methods for resolving racemic gossypol
compounds into substantially purified (+)- or (-)-gossypol are
known (See e.g., Zhou et al., Kexue Tongbao, 28:1574 (1983)
(wherein: L-phenylalanine methyl ester was mixed with the aldehyde
groups of gossypol to form a Schiff's base with two
diastereoisomers which were then resolved on a normal silica flash
chromatography column. The filtrate was concentrated, and the
residue was purified by chromatography on silica gel eluting with
hexanes:EtOAc=3:1 to give two fractions. Acid hydrolysis of the two
fractions in 5N HCl:THF (1:5, room temperature, overnight)
regenerated the individual gossypol enantiomers, respectively. The
first fraction with a higher R.sub.f value contained (-)-gossypol,
and the second fraction with a lower R.sub.f value contained
(+)-gossypol. The crude gossypol fractions were extracted into
ether from the residue after removing THF from the reaction
mixture. The gossypol fractions were then purified by
chromatography on silica gel and eluted with hexanes:EtOAc (3:1
ratio) to give optically pure gossypol, with a yield of 30-40% in
two steps. The optical rotatory dispersion values for these
products were .alpha..sub.D=-352.degree. (c=0.65, CHCl.sub.3) for
(-)-gossypol, and .alpha..sub.D=+341.degree. (c=0.53,
CHCl.sub.3)).
[0128] As used herein, the term "gossypol Schiff's base(s)" refers
to the gossypol compound that results from the reaction of an
aldehyde or ketone form of gossypol with a primary amine to yield
an imine of gossypol. Examples of primary amines that can be used
include, but are not limited to, branched and unbranched
alkylamines (e.g., methylamine, ethylamine, propylamine,
isopropylamine, butylamine, t-butylamine), substituted and
unsubstituted arylamines (e.g., phenylamine, benzylamine), and
amino acids, such as glycine, alanine, leucine, isoleucine,
phenylalanine, tyrosine, tryptophan, lysine, arginine, histidine,
aspartate, glutamate, asparagine, glutamine, cysteine, and
methionine.
[0129] As used herein, the term "Bcl-2 family proteins," refers to
both the anti-apoptotic members of the Bcl-2 family, including, but
not limited to Bcl-2, Bcl-X.sub.L, Mcl-1, A1/BFL-1, BOO-DNA, Bcl-w,
Bcl-6, Bcl-8 and Bcl-y, and the pro-apoptotic members of the Bcl-2
family, including, but not limited to Bak, Bax, Bad, tBid,
Harakiri, Bim, Bmf, and optionally other proteins with BH3 (Bcl-2
homology 3) binding pockets that are regulated by gossypol
compounds.
[0130] As used herein, the terms "overexpression of Bcl-2," or
"overexpression of a Bcl-2 family protein" refer to an elevated
level (e.g., aberrant) of mRNAs encoding for a Bcl-2 family
protein(s), and/or to elevated levels of such Bcl-2 family
protein(s) in cells or tissues as compared to similar normal
corresponding nonpathological cells and tissues expressing basal
levels of mRNAs encoding Bcl-2 family proteins or having basal
levels of Bcl-2 family proteins. Methods for detecting the levels
of mRNAs encoding Bcl-2 family proteins, or levels of Bcl-2 family
proteins, in a cell or tissue include, but are not limited to,
Western blotting using Bcl-2 family protein antibodies,
immunohistochemical methods, and methods of nucleic acid
amplification or direct RNA detection. As important as the absolute
levels of Bcl-2 family proteins in cells, tissues, or organs are to
determining that they overexpress Bcl-2 family proteins, so also
are the relative levels of anti-apoptotic Bcl-2 family proteins to
other pro-apoptotic signalling molecules (e.g., pro-apoptotic Bcl-2
family proteins) within such cells, tissues or organs. When the
balance of these two are such that, were it not for the levels of
the anti-apoptotic Bcl-2 family proteins, the pro-apototic
signalling molecules would be sufficient to cause the cells to
execute the apoptosis program and die, said cells in such tissues
or organs would be dependent on the anti-apoptotic Bcl-2 family
proteins for their survival. In such cells, exposure to an
inhibiting effective amount of an anti-apoptotic Bcl-2 family
protein inhibitor will be sufficient to cause the cells to execute
the apoptosis program and die. Thus, the term "overexpression of
Bcl-2 family protein" also refers to cells in tissues and organs
that, due to the relative levels of pro-apoptotic signals and
anti-apoptotic signals, undergo apoptosis in response to inhibiting
effective amounts of compounds that inhibit the function of
anti-apoptotic Bcl-2 proteins.
[0131] As used herein, the terms "anticancer agent," "conventional
anticancer agent," or "cancer therapeutic drug" refer to any
therapeutic agents (e.g., chemotherapeutic compounds and/or
molecular therapeutic compounds), radiation therapies, or surgical
interventions, used in the treatment of cancer (e.g., in
mammals).
[0132] As used herein, the terms "drug" and "chemotherapeutic
agent" refer to pharmacologically active molecules that are used to
diagnose, treat, or prevent diseases or pathological conditions in
a physiological system (e.g., a subject, or in vivo, in vitro, or
ex vivo cells, tissues, and organs). Drugs act by altering the
physiology of a living organism, tissue, cell, or in vitro system
to which the drug has been administered. It is intended that the
terms "drug" and "chemotherapeutic agent" encompass
anti-hyperproliferative and antineoplastic compounds as well as
other biologically therapeutic compounds.
[0133] As used herein the term "prodrug" refers to a
pharmacologically inactive derivative of a parent "drug" molecule
that requires biotransformation (e.g., either spontaneous or
enzymatic) within the target physiological system to release, or to
convert (e.g., enzymatically, mechanically, electromagnetically,
etc.) the "prodrug" into the active "drug." "Prodrugs" are designed
to overcome problems associated with stability, toxicity, lack of
specificity, or limited bioavailability. Exemplary "prodrugs"
comprise an active "drug" molecule itself and a chemical masking
group (e.g., a group that reversibly suppresses the activity of the
"drug"). Some preferred "prodrugs" are variations or derivatives of
compounds that have groups cleavable under metabolic conditions.
Exemplary "prodrugs" become pharmaceutically active in vivo or in
vitro when they undergo solvolysis under physiological conditions
or undergo enzymatic degradation or other biochemical
transformation (e.g., phosphorylation, hydrogenation,
dehydrogenation, glycosylation, etc.). Prodrugs often offer
advantages of solubility, tissue compatibility, or delayed release
in the mammalian organism. (See e.g., Bundgard, Design of Prodrugs,
pp. 7-9, 21-24, Elsevier, Amsterdam (1985); and Silverman, The
Organic Chemistry of Drug Design and Drug Action, pp. 352-401,
Academic Press, San Diego, Calif. (1992)). Common "prodrugs"
include acid derivatives such as esters prepared by reaction of
parent acids with a suitable alcohol (e.g., a lower alkanol),
amides prepared by reaction of the parent acid compound with an
amine (e.g., as described above), or basic groups reacted to form
an acylated base derivative (e.g., a lower alkylamide).
[0134] The term "derivative" of a compound, as used herein, refers
to a chemically modified compound wherein the chemical modification
takes place either at a functional group of the compound, aromatic
ring, or carbon backbone. Such derivatives include esters of
alcohol-containing compounds, esters of carboxy-containing
compounds, amides of amine-containing compounds, amides of
carboxy-containing compounds, imines of amino-containing compounds,
acetals of aldehyde-containing compounds, ketals of
carbonyl-containing compounds, and the like.
[0135] As used herein, the term "pharmaceutically acceptable salt"
refers to any salt (e.g., obtained by reaction with an acid or a
base) of a compound of the present invention that is
physiologically tolerated in the target subject (e.g., a mammalian
subject, and/or in vivo or ex vivo, cells, tissues, or organs).
"Salts" of the compounds of the present invention may be derived
from inorganic or organic acids and bases. Examples of acids
include, but are not limited to, hydrochloric, hydrobromic,
sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,
glycolic, lactic, salicylic, succinic, toluene-p-sulfonic,
tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic,
benzoic, malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic
acid, and the like. Other acids, such as oxalic, while not in
themselves pharmaceutically acceptable, may be employed in the
preparation of salts useful as intermediates in obtaining the
compounds of the invention and their pharmaceutically acceptable
acid addition salts.
[0136] Examples of bases include, but are not limited to, alkali
metal (e.g., sodium) hydroxides, alkaline earth metal (e.g.,
magnesium) hydroxides, ammonia, and compounds of formula
NW.sub.4.sup.+, wherein W is C.sub.1-4 alkyl, and the like.
[0137] Examples of salts include, but are not limited to: acetate,
adipate, alginate, aspartate, benzoate, benzenesulfonate,
bisulfate, butyrate, citrate, camphorate, camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, chloride, bromide, iodide,
2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,
2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate,
persulfate, phenylpropionate, picrate, pivalate, propionate,
succinate, tartrate, thiocyanate, tosylate, undecanoate, and the
like. Other examples of salts include anions of the compounds of
the present invention compounded with a suitable cation such as
Na.sup.+, NH.sub.4.sup.+, and NW.sub.4.sup.+ (wherein W is a
C.sub.1-4 alkyl group), and the like. For therapeutic use, salts of
the compounds of the present invention are contemplated as being
pharmaceutically acceptable. However, salts of acids and bases that
are non-pharmaceutically acceptable may also find use, for example,
in the preparation or purification of a pharmaceutically acceptable
compound.
[0138] An "effective amount" is an amount sufficient to effect
beneficial or desired results. An effective amount can be
administered in one or more administrations.
[0139] As used herein, the term "administration" refers to the act
of giving a drug, prodrug, or other agent (e.g., a gossypol
compound), or therapeutic treatment (e.g., radiation therapy) to a
physiological system (e.g., a subject or in vivo, in vitro, or ex
vivo cells, tissues, and organs). Exemplary routes of
administration to the human body can be through the eyes
(opthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs
(inhalant), oral mucosa (buccal), ear, by injection (e.g.,
intravenously, subcutaneously, intratumorally, intraperitoneally,
etc.) and the like.
[0140] "Coadministration" refers to administration of more than one
chemical agent (e.g., a gossypol compound and/or drugs, prodrugs,
etc.) or therapeutic treatment (e.g., radiation therapy) to a
physiological system (e.g., a subject or in vivo, in vitro, or ex
vivo cells, tissues, and organs). "Coadministration" of the
respective chemical agents (e.g., a gossypol compound and/or drugs,
prodrugs, etc.) and therapeutic treatments (e.g., radiation
therapy) may be concurrent, or in any temporal order or physical
combination.
[0141] As used herein, the term "synergistic" refers to an effect
obtained when gossypol and a second agent are administered together
(e.g., at the same time or one after the other) that is greater
than the additive effect of gossypol and the second agent when
administered individually. The synergistic effect allows for lower
doses of gossypol and/or the second agent to be administered or
provides greater efficacy at the same doses. The synergistic effect
obtained can be at least 10%, at least 20%, at least 30%, at least
40%, at least 50%, at least 60%, at least 70%, at least 80%, at
least 90%, at least 100%, at least 125%, at least 150%, at least
175%, at least 200%, at least 250%, at least 300%, at least 350%,
at least 400%, or at least 500% more than the additive effect of
the gossypol compound and the second agent when administered
individually. For example, with respect to the treatment of cancer,
the synergistic effect can be a decrease in the rate of tumor
growth, a decrease in tumor mass, a decrease in the number of
metastases, an increase in time to tumor progression, or an
increase in survival time. As described herein, gossypol compounds
(e.g., (-)-gossypol) and chemotherapeutic agents, when administered
individually, often only inhibit tumor cell proliferation rather
than cause regression of the tumor mass. According to the present
invention, it is possible to cause actual regression of tumor mass
by the administration of gossypol compounds (e.g., (-)-gossypol)
and chemotherapeutic agents. The co-administration of a gossypol
compound and an anticancer agent may allow for the use of lower
doses of the gossypol compound and/or the anticancer agent such
that the cancer is effectively treated while avoiding any
substantial toxicity to the subject.
[0142] The term "sensitize," and grammatical equivalents thereof,
refers to making, through the administration of a first agent(s)
(e.g., a gossypol compound and optionally a chemotherapeutic agent
and/or radiation), a subject, cell, tissue, or organ more
susceptible, or more responsive, to the biological effects (e.g.,
promotion or retardation of an aspect of cellular function
including, but not limited to, cell growth, proliferation,
invasion, angiogenesis, apoptosis) of a second or more agent. The
"sensitizing effect" of a first agent (e.g., a gossypol compound
and optionally a chemotherapeutic agent and/or radiation) on a
target cell, tissue, or organ can be measured as the difference in
the intended biological effect (e.g., promotion or retardation of
an aspect of cellular function including, but not limited to, cell
growth, proliferation, invasion, angiogenesis, apoptosis) observed
upon the administration of a second or more agent with and without
administration of the first agent. In this regard, the second or
more agent can be exogenous to the subject, cell, tissue or organ.
Further in this regard, the second or more agent can be endogenous
to the subject, cell, tissue, or organ.
[0143] As used herein, the term "pharmacological properties" refers
to any desirable or favorable biological activities or
physicochemical characteristics of an agent (e.g., a gossypol
compound) administered to a physiological system.
[0144] As used herein, the term "pharmacokinetic properties" refers
to the action of an agent (e.g., a gossypol compound) in a subject,
cell, tissue, or organ over a period of time including, but not
limited to, the processes of absorption, distribution, localization
in tissues, biotransformation, and excretion.
[0145] As used herein, the term "bioavailability" refers to any
measure of the ability of a an agent (e.g., a gossypol compound) to
be absorbed into a biological target fluid (e.g., blood, cytoplasm,
CNS fluid, and the like), tissue, organelle or intercellular space
after administration to a physiological system (e.g., a subject or
in vivo, in vitro, or ex vivo cells, tissues, and organs).
[0146] As used herein, the term "biodistribution" refers to the
location of an agent (e.g., a gossypol compound) in organelles,
cells (e.g., in vivo or in vitro), tissues, organs, or organisms,
after administration to a physiological system.
[0147] As used herein, the term "dysregulation of the process of
cell death" refers to any aberration in the ability of (e.g.,
predisposition) a cell to undergo cell death via either necrosis or
apoptosis. Dysregulation of cell death is associated with or
induced by a variety of conditions, including for example,
autoimmune disorders (e.g., systemic lupus erythematosus,
rheumatoid arthritis, graft-versus-host disease, myasthenia gravis,
Sjogren's syndrome, etc.), chronic inflammatory conditions (e.g.,
psoriasis, asthma and Crohn's disease), hyperproliferative
disorders (e.g., tumors, B cell lymphomas, T cell lymphomas, etc.),
viral infections (e.g., herpes, papilloma, HIV), and other
conditions such as osteoarthritis and atherosclerosis. It should be
noted that when the dysregulation is induced by or associated with
a viral infection, the viral infection may or may not be detectable
at the time dysregulation occurs or is observed. That is,
viral-induced dysregulation can occur even after the disappearance
of symptoms of viral infection.
[0148] A "hyperproliferative disease," as used herein refers to any
condition in which a localized population of proliferating cells in
an animal is not governed by the usual limitations of normal
growth. Examples of hyperproliferative disorders include tumors,
neoplasms, lymphomas and the like. A neoplasm is said to be benign
if it does not undergo invasion or metastasis and malignant if it
does either of these. A "metastatic" cell or tissue means that the
cell can invade and destroy neighboring body structures.
Hyperplasia is a form of cell proliferation involving an increase
in cell number in a tissue or organ without significant alteration
in structure or function. Metaplasia is a form of controlled cell
growth in which one type of fully differentiated cell substitutes
for another type of differentiated cell. Metaplasia can occur in
epithelial or connective tissue cells. A typical metaplasia
involves a somewhat disorderly metaplastic epithelium.
[0149] The pathological growth of activated lymphoid cells often
results in an autoimmune disorder or a chronic inflammatory
condition. As used herein, the term "autoimmune disorder" refers to
any condition in which an organism produces antibodies or immune
cells which recognize the organism's own molecules, cells or
tissues. Non-limiting examples of autoimmune disorders include
autoimmune hemolytic anemia, autoimmune hepatitis, Berger's disease
or IgA nephropathy, Celiac Sprue, chronic fatigue syndrome, Crohn's
disease, dermatomyositis, fibromyalgia, graft versus host disease,
Grave's disease, Hashimoto's thyroiditis, idiopathic
thrombocytopenia purpura, lichen planus, multiple sclerosis,
myasthenia gravis, psoriasis, rheumatic fever, rheumatic arthritis,
scleroderma, Sjogren's syndrome, systemic lupus erythematosus, type
1 diabetes, ulcerative colitis, vitiligo, and the like.
[0150] As used herein, the term "neoplastic disease" refers to any
abnormal growth of cells or tissues being either benign
(non-cancerous) or malignant (cancerous).
[0151] As used herein, the term "anti-neoplastic agent" refers to
any compound that retards the proliferation, growth, or spread of a
targeted (e.g., malignant) neoplasm.
[0152] As used herein, the term "regression" refers to the return
of a diseased subject, cell, tissue, or organ to a
non-pathological, or less pathological state as compared to basal
nonpathogenic exemplary subject, cell, tissue, or organ. For
example, regression of a tumor includes a reduction of tumor mass
as well as complete disappearance of a tumor or tumors.
[0153] As used herein, the terms "prevent," "preventing," and
"prevention" refer to a decrease in the occurrence of
hyperproliferative or neoplastic cells in a subject. The prevention
may be complete, e.g., the total absence of hyperproliferative or
neoplastic cells in a subject. The prevention may also be partial,
such that the occurrence of hyperproliferative or neoplastic cells
in a subject is less than that which would have occurred without
the present invention.
[0154] As used herein the term, "in vitro" refers to an artificial
environment and to processes or reactions that occur within an
artificial environment. In vitro environments can consist of, but
are not limited to, test tubes and cell cultures. The term "in
vivo" refers to the natural environment (e.g., an animal or a cell)
and to processes or reactions that occur within a natural
environment.
[0155] As used herein, the term "host cell" refers to any
eukaryotic or prokaryotic cell (e.g., mammalian cells, avian cells,
amphibian cells, plant cells, fish cells, and insect cells),
whether located in vitro or in vivo.
[0156] As used herein, the term "cell culture" refers to any in
vitro culture of cells. Included within this term are continuous
cell lines (e.g., with an immortal phenotype), primary cell
cultures, finite cell lines (e.g., non-transformed cells), and any
other cell population maintained in vitro, including oocytes and
embryos.
[0157] As used herein, the term "subject" refers to organisms to be
treated by the methods of the present invention. Such organisms
include, but are not limited to, humans and veterinary animals
(dogs, cats, horses, pigs, cattle, sheep, goats, and the like). In
the context of the invention, the term "subject" generally refers
to an individual who will receive or who has received treatment
(e.g., administration of gossypol compound(s), and optionally one
or more anticancer agents) for a disease characterized by
overexpression of Bcl-2 family proteins (e.g., Bcl-2, Bcl-X.sub.L,
Bcl-w, Mcl-1, A-1(Bfl-1), and Boo).
[0158] The term "diagnosed," as used herein, refers to the
recognition of a disease by its signs and symptoms (e.g.,
resistance to conventional cancer therapies), or genetic analysis,
pathological analysis, histological analysis, and the like.
[0159] As used herein, the term "competes for binding" is used in
reference to a first molecule (e.g., a gossypol compound) with an
activity that binds to the same target (e.g., Bcl-2 and/or
Bcl-X.sub.L) as does a second molecule (e.g., a pro-apoptotic Bcl-2
family protein, such as Bax, Bak, Bid, and Bad, etc.). The
efficiency (e.g., kinetics or thermodynamics) of binding by the
first molecule may be the same as, or greater than, or less than,
the efficiency of the target binding by the second molecule. For
example, the equilibrium binding constant (Kd) for binding to the
target may be different for the two molecules.
[0160] As used herein, the term "antisense" is used in reference to
nucleic acid sequences (e.g., RNA, phosphorothioate DNA) that are
complementary to a specific RNA sequence (e.g., mRNA). Included
within this definition are antisense RNA ("asRNA") molecules
involved in gene regulation by bacteria. Antisense RNA may be
produced by any method, including synthesis by splicing the gene(s)
of interest in a reverse orientation to a viral promoter that
permits the synthesis of a coding strand. For example, once
introduced into an embryo, this transcribed strand combines with
natural mRNA produced by the embryo to form duplexes. These
duplexes then block either the further transcription of the mRNA or
its translation. In this manner, mutant phenotypes may be
generated. The term "antisense strand" is used in reference to a
nucleic acid strand that is complementary to the "sense" strand.
The designation (-) (i.e., "negative") is sometimes used in
reference to the antisense strand, with the designation (+)
sometimes used in reference to the sense (i.e., "positive") strand.
Regions of nucleic acid sequences that are accessible to antisense
molecules can be determined using available computer analysis
methods.
[0161] The term "sample" as used herein is used in its broadest
sense. A sample suspected of indicating a condition characterized
by the overexpression of a Bcl-2 family protein may comprise a
cell, tissue, or fluids, chromosomes isolated from a cell (e.g., a
spread of metaphase chromosomes), genomic DNA (in solution or bound
to a solid support such as for Southern blot analysis), RNA (in
solution or bound to a solid support such as for Northern blot
analysis), cDNA (in solution or bound to a solid support) and the
like. A sample suspected of containing a protein may comprise a
cell, a portion of a tissue, an extract containing one or more
proteins and the like.
[0162] The term "test compound" refers to any chemical entity,
pharmaceutical, drug, and the like, that can be used to treat or
prevent a disease, illness, sickness, or disorder of bodily
function, or otherwise alter the physiological or cellular status
of a sample (e.g., the level of Bcl-2 family proteins in a cell).
Test compounds comprise both known and potential therapeutic
compounds. A test compound can be determined to be therapeutic by
using the screening methods of the present invention. A "known
therapeutic compound" refers to a therapeutic compound that has
been shown (e.g., through animal trials or prior experience with
administration to humans) to be effective in such treatment or
prevention. In preferred embodiments, "test compounds" are
anticancer agents. In particularly preferred embodiments, "test
compounds" are anticancer agents that induce apoptosis in
cells.
[0163] As used herein, the term "purified" or "to purify" refers to
the removal of undesired components from a sample. As used herein,
the term "substantially purified" refers to molecules (e.g.,
polynucleotides, polypeptides, chemical compounds (e.g., gossypol
compounds)) that are removed from their natural environment,
isolated or separated, and are at least 60% free, preferably at
least 75% free, and most preferably at least 90% free from other
components with which they are naturally associated. For example,
an "isolated polynucleotide" is therefore a substantially purified
polynucleotide.
[0164] As used herein, the term "genome" refers to the genetic
material (e.g., chromosomes) of an organism or a host cell.
[0165] The term "nucleotide sequence of interest" refers to any
nucleotide sequence (e.g., RNA or DNA), the manipulation of which
may be deemed desirable for any reason (e.g., treat disease, confer
improved qualities, etc.), by one of ordinary skill in the art.
Such nucleotide sequences include, but are not limited to, coding
sequences, or portions thereof, of structural genes (e.g., reporter
genes, selection marker genes, oncogenes, drug resistance genes,
growth factors, etc.), and non-coding regulatory sequences which do
not encode an mRNA or protein product (e.g., promoter sequence,
polyadenylation sequence, termination sequence, enhancer sequence,
etc.).
[0166] "Nucleic acid sequence" and "nucleotide sequence" as used
herein refer to an oligonucleotide or polynucleotide, and fragments
or portions thereof, and to DNA or RNA of genomic or synthetic
origin which may be single- or double-stranded, and represent the
sense or antisense strand. As used herein, the terms "nucleic acid
molecule encoding," "DNA sequence encoding," "DNA encoding," "RNA
sequence encoding," and "RNA encoding" refer to the order or
sequence of deoxyribonucleotides or ribonucleotides along a strand
of deoxyribonucleic acid or ribonucleic acid. The order of these
deoxyribonucleotides or ribonucleotides determines the order of
amino acids along the polypeptide (protein) chain translated from
the mRNA. The DNA or RNA sequence thus codes for the amino acid
sequence.
[0167] The term "gene" refers to a nucleic acid (e.g., DNA or RNA)
sequence that comprises coding sequences necessary for the
production of a polypeptide or precursor (e.g., proinsulin). The
polypeptide can be encoded by a full length coding sequence or by
any portion of the coding sequence so long as the desired activity
or functional properties (e.g., enzymatic activity, ligand binding,
signal transduction, etc.) of the full-length or fragment are
retained. The term also encompasses the coding region of a
structural gene and includes sequences located adjacent to the
coding region on both the 5' and 3' ends for a distance of about 1
kb or more on either end such that the gene corresponds to the
length of the full-length mRNA. The sequences that are located 5'
of the coding region and which are present on the mRNA are referred
to as 5' untranslated sequences. The sequences that are located 3'
or downstream of the coding region and which are present on the
mRNA are referred to as 3' untranslated sequences. The term "gene"
encompasses both cDNA and genomic forms of a gene. A genomic form
or clone of a gene contains the coding region interrupted with
non-coding sequences termed "introns" or "intervening regions" or
"intervening sequences." Introns are segments of a gene which are
transcribed into nuclear RNA (hnRNA); introns may contain
regulatory elements such as enhancers. Introns are removed or
"spliced out" from the nuclear or primary transcript; introns
therefore are absent in the messenger RNA (mRNA) transcript. The
mRNA functions during translation to specify the sequence or order
of amino acids in a nascent polypeptide.
[0168] As used herein, the term "exogenous gene" refers to a gene
that is not naturally present in a host organism or cell, or is
artificially introduced into a host organism or cell.
[0169] As used herein, the term "vector" refers to any genetic
element, such as a plasmid, phage, transposon, cosmid, chromosome,
virus, virion, etc., which is capable of replication when
associated with the proper control elements and which can transfer
gene sequences between cells. Thus, the term includes cloning and
expression vehicles, as well as viral vectors.
[0170] As used herein, the term "gene expression" refers to the
process of converting genetic information encoded in a gene into
RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of
the gene (i.e., via the enzymatic action of an RNA polymerase), and
for protein encoding genes, into protein through "translation" of
mRNA. Gene expression can be regulated at many stages in the
process. "Up-regulation" or "activation" refers to regulation that
increases the production of gene expression products (i.e., RNA or
protein), while "down-regulation" or "repression" refers to
regulation that decreases production. Molecules (e.g.,
transcription factors) that are involved in up-regulation or
down-regulation are often called "activators" and "repressors,"
respectively.
[0171] The terms "homology" and "percent identity" when used in
relation to nucleic acids refers to a degree of complementarity.
There may be partial homology (i.e., partial identity) or complete
homology (i.e., complete identity). A partially complementary
sequence is one that at least partially inhibits a completely
complementary sequence from hybridizing to a target nucleic acid
sequence and is referred to using the functional term
"substantially homologous." The inhibition of hybridization of the
completely complementary sequence to the target sequence may be
examined using a hybridization assay (Southern or Northern blot,
solution hybridization and the like) under conditions of low
stringency. A substantially homologous sequence or probe (i.e., an
oligonucleotide which is capable of hybridizing to another
oligonucleotide of interest) will compete for and inhibit the
binding (i.e., the hybridization) of a completely homologous
sequence to a target sequence under conditions of low stringency.
This is not to say that conditions of low stringency are such that
non-specific binding is permitted; low stringency conditions
require that the binding of two sequences to one another be a
specific (i.e., selective) interaction. The absence of non-specific
binding may be tested by the use of a second target which lacks
even a partial degree of complementarity (e.g., less than about 30%
identity); in the absence of non-specific binding the probe will
not hybridize to the second non-complementary target.
[0172] When used in reference to a single-stranded nucleic acid
sequence, the term "substantially homologous" refers to any probe
that can hybridize (i.e., it is the complement of) the
single-stranded nucleic acid sequence under conditions of low
stringency as described above. A gene may produce multiple RNA
species that are generated by differential splicing of the primary
RNA transcript. cDNAs that are splice variants of the same gene
will contain regions of sequence identity or complete homology
(representing the presence of the same exon or portion of the same
exon on both cDNAs) and regions of complete non-identity (for
example, representing the presence of exon "A" on cDNA 1 wherein
cDNA 2 contains exon "B" instead). Because the two cDNAs contain
regions of sequence identity they will both hybridize to a probe
derived from the entire gene or portions of the gene containing
sequences found on both cDNAs; the two splice variants are
therefore substantially homologous to such a probe and to each
other. The present invention is not limited to the situation where
hybridization takes place only between completely homologous
sequences. In some embodiments, hybridization takes place with
substantially homologous sequences.
[0173] As used herein, the term "protein of interest" refers to a
protein encoded by a nucleic acid of interest.
[0174] As used herein, the term "native" (or wild type) when used
in reference to a protein, refers to proteins encoded by partially
homologous nucleic acids so that the amino acid sequence of the
proteins varies. As used herein, the term "variant" encompasses
proteins encoded by homologous genes having both conservative and
nonconservative amino acid substitutions that do not result in a
change in protein function, as well as proteins encoded by
homologous genes having amino acid substitutions that cause
decreased (e.g., null mutations) protein function or increased
protein function.
[0175] The term "reverse Northern blot" as used herein refers to
the analysis of DNA by electrophoresis of DNA on agarose gels to
fractionate the DNA on the basis of size followed by transfer of
the fractionated DNA from the gel to a solid support, such as
nitrocellulose or a nylon membrane. The immobilized DNA is then
probed with a labeled oligo-ribonucleotide probe or RNA probe to
detect DNA species complementary to the ribo probe used.
[0176] As used herein, the term "pathogen" refers to a biological
agent that causes a disease state (e.g., infection, cancer, etc.)
in a host. "Pathogens" include, but are not limited to, viruses,
bacteria, archaea, fungi, protozoans, mycoplasma, and parasitic
organisms.
[0177] As used herein, the term "microorganism" is used to refer to
any species or type of microorganism, including but not limited to,
bacteria, archaea, fungi, protozoans, mycoplasma, and parasitic
organisms. As used herein, the term "fungi" is used in reference to
eukaryotic organisms such as the molds and yeasts, including
dimorphic fungi.
[0178] As used herein, the term "virus" refers to minute infectious
agents, which with certain exceptions, are not observable by light
microscopy, lack independent metabolism, and are able to replicate
only within a living host cell. The individual particles (i.e.,
virions) consist of nucleic acid and a protein shell or coat; some
virions also have a lipid containing membrane. The term "virus"
encompasses all types of viruses, including animal, plant, phage,
and other viruses.
[0179] The terms "bacteria" and "bacterium" refer to all
prokaryotic organisms, including those within all of the phyla in
the kingdom Procaryotae. It is intended that the term encompass all
microorganisms considered to be bacteria including Mycoplasma,
Chlamydia, Actinomyces, Streptomyces, and Rickettsia. All forms of
bacteria are included within this definition including cocci,
bacilli, spirochetes, spheroplasts, protoplasts, etc. Also included
within this term are prokaryotic organisms which are gram negative
or gram positive. "Gram negative" and "gram positive" refer to
staining patterns with the Gram-staining process which is well
known in the art. (See e.g., Finegold and Martin, Diagnostic
Microbiology, 6th Ed., CV Mosby St. Louis, pp 13-15 (1982)). "Gram
positive bacteria" are bacteria which retain the primary dye used
in the Gram stain, causing the stained cells to appear dark blue to
purple under the microscope. "Gram negative bacteria" do not retain
the primary dye used in the Gram stain, but are stained by the
counterstain. Thus, gram negative bacteria appear red.
[0180] As used herein, the term "antigen binding protein" refers to
proteins which bind to a specific antigen. "Antigen binding
proteins" include, but are not limited to, immunoglobulins,
including polyclonal, monoclonal, chimeric, single chain, and
humanized antibodies, Fab fragments, F(ab')2 fragments, and Fab
expression libraries. Various procedures known in the art are used
for the production of polyclonal antibodies. For the production of
antibodies, various host animals can be immunized by injection with
the peptide corresponding to the desired epitope including, but not
limited to, rabbits, mice, rats, sheep, goats, etc. In a preferred
embodiment, the peptide is conjugated to an immunogenic carrier
(e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyhole
limpet hemocyanin (KLH)). Various adjuvants are used to increase
the immunological response, depending on the host species,
including, but not limited to, Freund's (complete and incomplete),
mineral gels such as aluminum hydroxide, surface active substances
such as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol, and
potentially useful human adjuvants such as BCG (Bacille
Calmette-Guerin) and Corynebacterium parvum.
[0181] For preparation of monoclonal antibodies, any technique that
provides for the production of antibody molecules by continuous
cell lines in culture may be used (See e.g., Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.). These include, but are not
limited to, the hybridoma technique originally developed by Kohler
and Milstein (Kohler and Milstein, Nature, 256:495-497 (1975)), as
well as the trioma technique, the human B-cell hybridoma technique
(See e.g., Kozbor et al., Immunol. Today, 4:72 (1983)), and the
EBV-hybridoma technique to produce human monoclonal antibodies
(Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, Inc., pp. 77-96 (1985)).
[0182] According to the invention, techniques described for the
production of single chain antibodies (U.S. Pat. No. 4,946,778;
herein incorporated by reference) can be adapted to produce
specific single chain antibodies as desired. An additional
embodiment of the invention utilizes the techniques known in the
art for the construction of Fab expression libraries (Huse et al.,
Science, 246:1275-1281 (1989)) to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity.
[0183] Antibody fragments that contain the idiotype (antigen
binding region) of the antibody molecule can be generated by known
techniques. For example, such fragments include, but are not
limited to: the F(ab')2 fragment that can be produced by pepsin
digestion of an antibody molecule; the Fab' fragments that can be
generated by reducing the disulfide bridges of an F(ab')2 fragment,
and the Fab fragments that can be generated by treating an antibody
molecule with papain and a reducing agent.
[0184] Genes encoding antigen binding proteins can be isolated by
methods known in the art. In the production of antibodies,
screening for the desired antibody can be accomplished by
techniques known in the art (e.g., radioimmunoassay, ELISA
(enzyme-linked immunosorbant assay), "sandwich" immunoassays,
immunoradiometric assays, gel diffusion precipitin reactions,
immunodiffusion assays, in situ immunoassays (using colloidal gold,
enzyme or radioisotope labels, for example), Western Blots,
precipitation reactions, agglutination assays (e.g., gel
agglutination assays, hemagglutination assays, etc.), complement
fixation assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc.) etc.
[0185] As used herein, the term "instructions for administering
said gossypol compound to a subject" includes instructions for
using the compositions contained in the kit for the treatment of
conditions characterized by the overexpression of a Bcl-2 family
protein in a cell or tissue. The term also refers to instructions
for using the compositions contained in the kit to treat cancers
characterized as being resistant to at least one conventional
anticancer therapy (e.g., chemotherapy). In some embodiments, the
instructions further comprise a statement of the recommended or
usual dosages of the compositions contained within the kit pursuant
to 21 C.F.R. .sctn.201 et seq. Additional information concerning
labeling and instruction requirements applicable to the methods and
compositions of the present are available at the Internet web page
of the U.S.F.D.A.
[0186] As used herein, the term "third party" refers to any entity
engaged in selling, warehousing, distributing, or offering for sale
a compound contemplated for co-administration with a gossypol
compound for treating conditions characterized by the
overexpression of the Bcl-2 family proteins.
[0187] As used herein, the term "modulate" refers to the activity
of a compound (e.g., gossypol compound) to affect (e.g., to promote
or retard) an aspect of the cellular function including, but not
limited to, cell growth, proliferation, invasion, angiogenesis,
apoptosis, and the like.
GENERAL DESCRIPTION OF THE INVENTION
[0188] Gossypol is a naturally occurring double biphenolic compound
derived from crude cotton seed oil (Gossypium sp.). Naturally
occurring gossypol exists in two enantiomeric forms, (+) or (-),
that when present together comprise racemic gossypol. Human trials
of racemic gossypol as a male contraceptive have demonstrated the
safety of long term administration of gossypol. Racemic gossypol is
well tolerated in humans.
[0189] Gossypol is a known inhibitor of spermatogenesis that may be
administered orally with few side effects. Some researchers have
shown, however, that hypokalemia may result from prolonged gossypol
administration. Accordingly, in some embodiments, the present
methods and compositions further comprise the co-administration of
potassium supplements to patients being treated with gossypol
compounds.
[0190] Although an understanding of the mechanism is not necessary
to practice the present invention and the present invention is not
so limited, the present invention contemplates that gossypol is a
potent inhibitor of Bcl-2 and Bcl-X.sub.L and that the anti-tumor
activity of gossypol is due, at least in part, to inhibition of the
anti-apoptotic activity of Bcl-2 and Bcl-X.sub.L and the subsequent
induction of apoptosis in cancer cells expressing Bcl-2 family
proteins. Thus, the present invention provides compositions and
methods for targeting subjects characterized as overexpressing a
Bcl-2 family protein. In some of the embodiments, the cancer cells
show elevated expression levels of Bcl-2 family proteins as
compared to nonpathological samples (e.g., non-cancerous cells or
tissues). In other embodiments, the cancer cells or supporting
cells operationally manifest elevated expression levels of Bcl-2
family proteins by virtue of executing the apoptosis program and
dying in response to an inhibiting effective amount of a gossypol
compound (e.g., (-)-gossypol), said response occurring, at least in
part, to the dependence in such cells on anti-apoptotic Bcl-2
family protein function for their survival.
[0191] In the clinical trials to date, gossypol has shown low
toxicity in patients. In some embodiments, it is contemplated that
gossypol compounds provide efficient single agent treatments for
metastatic cancers. The present invention further contemplates that
gossypol compounds represent new classes of anticancer agents that
specifically antagonize the anti-apoptotic effects of Bcl-2 and
Bcl-X.sub.L.
[0192] The present invention provides in vivo data that show
gossypol compounds significantly inhibit tumor growth, but that in
some embodiments gossypol compounds achieve even greater inhibition
of tumor growth inhibition when used in combination
(co-administration) with one or more conventional anticancer agents
(e.g., docetaxel). Accordingly, in preferred embodiments, gossypol
compounds are administered to patients suffering from diseases
characterized by the overexpression of Bcl-2 and/or Bcl-X.sub.L
(e.g., cancer). Gossypol induces apoptosis in cancer cells
expressing high levels of Bcl-2 and/or Bcl-X.sub.L, but gossypol in
general has less effect on cells with low levels Bcl-X.sub.L and/or
Bcl-2 expression. In other preferred embodiments, gossypol
compounds are administered with one or more anticancer agents
and/or radiation.
[0193] Bcl-2 is the founding member of a family of proteins that
includes both anti-apoptotic molecules (e.g., Bcl-2, Bcl-X.sub.L,
Mcl-1, A1/BFL-1, BOO-DIVA, Bcl-w, Bcl-6, Bcl-8, and Bcl-y, and the
like) and pro-apoptotic molecules (e.g., Bax, Bak, Bid, and Bad,
and the like). The bcl-2 gene is a human proto-oncogene located on
chromosome 18. The bcl-2 gene was discovered as a translocated
locus in a B-cell leukemia. This translocation is also found in
some B-cell lymphomas. In cancerous B cells, the portion of
chromosome 18 containing the bcl-2 locus undergoes a reciprocal
translocation with the portion of chromosome 14 containing antibody
heavy chains. This t(14;18) translocation places the bcl-2 gene
close to the heavy chain gene enhancer. The product of the bcl-2
gene, Bcl-2 protein, is an integral membrane protein found in the
membranes of the endoplasmic reticulum (ER), nuclear envelope, and
the outer membrane of mitochondria. It is contemplated that Bcl-2,
and Bcl-X.sub.L, function as crucial antagonists of apoptosis.
[0194] Although an understanding of the mechanism is not necessary
to practice the present invention and the present invention is not
so limited, it is contemplated that anti-apoptotic proteins Bcl-2
and Bcl-X.sub.L suppress apoptosis by forming heterodimers with
pro-apoptotic Bcl-2 family members such as Bak, Bad, Bax, Mtd
(Bok), Bim, Hrk (DP5), Blk, Bnip3, Bnip3L, and Diva. Additional
anti-apoptotic members (or related proteins) of the Bcl-2 family
are thought to include, but are not limited to, Mcl-1, A1/BFL-1,
BOO-DIVA, Bcl-w, Bcl-6, Bcl-8, and Bcl-y.
[0195] Research into the three-dimensional (3D) structures of Bcl-2
and Bcl-X.sub.L showed that both molecules have a hydrophobic
binding pocket (named BH3) that is important to their
anti-apoptotic affects. In particular, experimental 3D high
resolution structures of Bcl-X.sub.L (S. W. Muchmore et al.,
Nature, 381:335-341 (1996); and M. Aritomi et al., J. Biol. Chem.,
272:27886-27892 (1997)) alone and in complex with a Bak BH3 (Bcl-2
homology domain 3) peptide (S. Michael et al., Science, 275:983-986
(1997)) have been determined. Bcl-2 and Bcl-X.sub.L share a high
degree of homology in their amino acid sequences (45% of identity
and 56% of similarity). It has been demonstrated that when there
exists a sequence identity of more than 30% between a target
protein (Bcl-2) and a template protein (Bcl-X.sub.L), current
computational homology modeling methods, such as Modeller (A. Sali
et al., Structure, Function, and Genetics, 23:318-326 (1995)) can
provide accurate 3D structures of the target protein. (See, A.
Sali, Cum Opin. Biotech., 6:437-451 (1995)). Therefore, in
preferred embodiments of the present invention, computational
homology modeling is used to model the 3D structure of Bcl-2 (the
target protein) based upon the experimental 3D structural
coordinates of Bcl-X.sub.L (the template protein) before the
three-dimensional experimental Bcl-2 structures were
determined.
[0196] Fluorescence-polarization based assays showed that gossypol,
(-)-gossypol and (+)-gossypol bind to Bcl-2 and Bcl-X.sub.L
proteins and compete with pro-apoptotic Bid, Bad, and Bak BH3
peptides. Analysis using nuclear magnetic resonance (NMR) methods
conclusively confirmed that gossypol, (-)-gossypol, and
(+)-gossypol bind to the BH3 binding groove in Bcl-X.sub.L. Thus,
gossypol compounds bind to the surface groove in Bcl-2 and
Bcl-X.sub.L and block the binding of pro-apoptotic proteins (e.g.
Bid, Bad and Bak), therefore inhibiting the anti-apoptotic
functions of Bcl-2 and Bcl-X.sub.L.
[0197] In some embodiments, the present invention provides BH3
domain-containing proteins as targets for inhibition. It should be
understood that where the specification refers to Bcl-2 families of
proteins, the same disclosure pertains to BH3 domain-containing
proteins. Thus, in some embodiments, the present invention provides
compositions and methods for the regulation of biological
conditions related to the aberrant expression of BH3
domain-containing proteins. Likewise, in some other embodiments,
the present invention provides methods and compositions for
screening agents and compounds that modulate (e.g., inhibit or
promote) the aberrant expression of BH3 domain-containing
proteins.
[0198] Bcl-2 and Bcl-X.sub.L are highly homologous proteins. Many
forms of human cancers (e.g., myeloid leukemia and breast cancer)
overexpress Bcl-2, and/or Bcl-X.sub.L. Both Bcl-2 and Bcl-X.sub.L
have been found to be overexpressed in human breast cancers. In
particular, Bcl-2 is found to be overexpressed in 60-80% of human
breast cancers. The expression of Bcl-2 is highly correlated with
estrogen receptor (ER) positive breast cancer. Bcl-X.sub.L is
overexpressed in 40-70% of human breast cancers, 30-60% of prostate
cancers, 80% of B-cell lymphomas, 90% of colorectal
adenocarcinomas, and many other forms of cancer. The expression of
Bcl-X.sub.L is typically correlated with a poor prognosis and
shortened survival.
[0199] Several lines of evidence indicate that Bcl-2 and
Bcl-X.sub.L not only contribute to cancer progression, but also may
confer on cancer cells a resistance to apoptosis induced by
conventional anti-cancer therapies. High levels of intracellular
Bcl-2 protect cells (e.g., cancer cells) from being destroyed by
apoptosis. The majority of solid tumors are protected by at least
one of the anti-apoptotic Bcl-2 proteins. Most of the currently
available chemotherapeutic cancer agents disrupt cellular DNA
integrity or replication, and indirectly trigger apoptosis in tumor
cells. Cancers that express high levels of Bcl-2 and/or Bcl-X.sub.L
are often resistant to chemotherapeutic agents or radiation
therapy.
[0200] However, the expression patterns of Bcl-2 and Bcl-X.sub.L
are different in some cancers that overexpress Bcl-2 family
proteins. Several reports suggest that expression of either Bcl-2
or Bcl-X.sub.L proteins is sufficient for cancer cells to show
Bcl-2 family mediated resistance to chemotherapy or radiation
therapy. (See, J. C. Reed, Pharmacology, 41:501-553 (1997); J. C.
Reed et al., J. Cell Biochem., 6:23-32 (1996)). Additional research
suggests that some cancer cells are able to switch from
overexpression of Bcl-2 to Bcl-X.sub.L. (See, Z. Han et al., Cancer
Res., 56:621-628 (1996)). Accordingly, some embodiments of the
present invention provide administering a therapeutic amount of one
or more Bcl-2 antagonists (e.g., small molecules, such as gossypol
compounds) to patients having a cancer characterized by
overexpression of a Bcl-2 family member protein. Similarly, other
embodiments of the present invention provide administering a
therapeutic amount of one or more Bcl-X.sub.L antagonists (e.g.,
small molecules, such as gossypol compounds) to patients having a
cancer characterized by overexpression of Bcl-X.sub.L. In still
further embodiments, the present invention provides methods for
administering a combination of two or more anti-apoptotic Bcl-2
family protein antagonists (e.g., small molecules, such as gossypol
compounds) to a patient having a cancer characterized by the
overexpression of anti-apoptotic Bcl-2 family proteins. In some
embodiments, a Bcl-2 antagonist and/or a Bcl-X.sub.L antagonist are
administered to a subject; optionally, one or more additional
anticancer agents may also be administered. The present invention
further contemplates providing compositions and methods comprising
one or more antagonists to Bcl-2 family protein(s) (e.g., an
anti-apoptotic Bcl-2 family protein) and one or more additional
anticancer agents (e.g., TAXOL, TAXOTERE, etc.). In preferred
embodiments, the present invention comprises anticancer methods and
compositions comprising providing a subject with a therapeutically
effective amount of a gossypol compound, e.g., gossypol,
gossypolone, Schiff's bases of gossypol and gossypolone,
enantiomers (e.g., (-)-gossypol and (+)-gossypol), and
pharmaceutically acceptable salts of these compounds.
DETAILED DESCRIPTION OF THE INVENTION
[0201] The present invention relates to naturally occurring and
chemically synthesized small molecule antagonists of Bcl-2 family
proteins. In particular, the present invention provides gossypol
compounds (e.g., isomers, enantiomers, racemic compounds,
metabolites, derivatives, pharmaceutically acceptable salts, in
combination with acids or bases, and the like) and methods of using
these compounds as antagonists of the anti-apoptotic effects of
Bcl-2 family member proteins (e.g., Bcl-2, Bcl-X.sub.L, and the
like). The present invention also provides compositions comprising
gossypol compounds and optionally one or more additional
therapeutic agents (e.g., chemotherapeutic or anti-neoplastic
agents). The present invention also provides methods for treating
diseases and pathologies (e.g., neoplastic diseases) comprising
administering a composition comprising gossypol compounds and
optionally one or more additional therapeutic agents (e.g.,
chemotherapeutic or anti-neoplastic agents) and/or techniques
(e.g., radiation therapies, surgical interventions, and the like)
to a subject or in vitro cells, tissues, and organs.
[0202] Exemplary compositions and methods of the present invention
are described in more detail in the following sections: I. Binding
activity of Bcl-2 and Bcl-X.sub.L; II. Structure-based approach for
discovery of small molecule inhibitors of Bcl-2 and Bcl-X.sub.L;
III. Characterization of Bcl-2 family of proteins in cancer cell
lines; IV. Gossypol compounds inhibit cancer cell growth and
proliferation; V. Proposed mechanism of gossypol activity; VI.
Activity of gossypol in MDA-231 xenograft mice alone and in
combination with conventional anticancer agents; VII. Therapeutic
agents combined or co-administered with gossypol compounds; VIII.
Targeting agents and techniques; IX. Pharmaceutical formulations,
administration routes, and dosing considerations, and X. Exemplary
combination therapies.
I. Binding Activity of Bcl-2 and Bcl-X.sub.L
[0203] Although an understanding of the mechanism is not necessary
to practice the present invention and the present invention is not
so limited, it is contemplated that the anti-apoptotic effects of
Bcl-2 and Bcl-X.sub.L proteins can be attributed, at least in part,
to their ability to heterodimerize with pro-apoptotic Bcl-2 family
member proteins such as Bak, Bax and Bad. The experimental
structures of Bcl-2 and Bcl-X.sub.L show that BH1 (Bcl-2 Homology
Domain 1), BH2, and BH3 domains of Bcl-2 and Bcl-X.sub.L form a
hydrophobic binding pocket (the BH3 binding pocket) into which the
Bak or Bad BH3 domain can bind. (See e.g., S. W. Muchmore et al.,
Nature, 381:335-341 (1996); M. Aritomi et al., J. Biol. Chem.,
272:27886-27892 (1997); S. Michael et al., Science, 275:983-986
(1997); A. M. Petros et al., Protein Sci., 9:2528-2534 (2000); and
A. M. Petros et al., Proc. Natl. Acad. Sci. U.S.A., 98:3012-3017
(2001)). The binding site in Bcl-2/Bcl-X.sub.L is essential for its
anti-apoptotic function. (See e.g., X. M. Yin et al., Nature,
369:321-323 (1994); S. C. Cosulich et al., Curr. Biol., 7:913-920
(1997); S. Michael et al., supra; and A. M. Petros et al.,
supra).
[0204] In preferred embodiments, the present invention provides
small molecules that bind to Bcl-2 and/or Bcl-X.sub.L BH3 binding
sites thus blocking their hetero-dimerization with pro-apoptotic
Bcl-2 family member proteins (e.g., Bad, Bak, and Bax etc.) such
that their anti-apoptotic function is antagonized and apoptosis is
induced in cells with Bcl-2 and/or Bcl-X.sub.L overexpression. In
some of these embodiments, the present invention further provides
methods comprising the administration of one or more additional
therapeutic agents (e.g., anticancer agents such as TAXOL or
TAXOTERE) in combination with the disclosed small molecule
Bcl-2/Bcl-X.sub.L inhibitors (e.g., gossypol compounds).
Particularly preferred compositions and methods comprise gossypol
compounds administered in combination with at least one anticancer
agent (e.g., TAXOL, TAXOTERE, or cisplatin).
[0205] The present invention provides small molecule
Bcl-2/Bcl-X.sub.L inhibitors that have various advantages over
other available protein antagonists (e.g., antisense
oligonucleotides, antibodies, and peptides). For example, various
compositions of the present invention have improved oral
availability and lower cost among other advantages.
II. Structure-Based Approach for Discovery of Small Molecule
Inhibitors of Bcl-2 and Bcl-X.sub.L
[0206] Preferred embodiments of the present invention used a
powerful structure-based virtual screening methodology to identify
small molecule antagonists of anti-apoptotic Bcl-2 family proteins
(e.g., Bcl-2 and Bcl-X.sub.L) from large 3D chemical databases. The
methods took advantage of powerful computational docking programs
to identify small organic molecules that interact with binding
sites in the targeted proteins (e.g., the BH3 site in Bcl-2 and/or
Bcl-X.sub.L).
[0207] In one embodiment, the targeted protein, Bcl-X.sub.L, was
screened in docking studies (e.g., using the united-atom
approximation) to identify small-molecule inhibitors that bind to
targeted protein from a library of chemicals (e.g., synthetic
organic compounds and natural products). For example, in one
embodiment, polar hydrogens were added to the targeted protein, and
Kollman united-atom partial charges were assigned. All water
molecules were removed. Atomic solvation parameters and fragmental
volumes were assigned to the protein atoms using the AutoDock
utility, AddSol. (See, AutoDock Web page; G. Morris et al., J.
Comp. Chem., 19:1639-1662 (1998)).
[0208] In another embodiment, the 3D structure of Bcl-2 was modeled
using the MODELLER homology modeling program and methods based upon
the 3D structure of Bcl-X.sub.L. (A. Sali et al., Structure,
Function, and Genetics, 23:318-326 (1995); and A. Sali, Curr. Opin.
Biotech., 6:437-451 (1995)). A BLAST sequence alignment of Bcl-2
(SEQ ID NO:1) and Bcl-X.sub.L (SEQ ID NO:2) proteins is shown in
FIG. 1. In preferred embodiments, this sequence alignment was used
in the various homology modeling experiments of the present
invention. Since the Bak BH3 peptide binds to both Bcl-2 and
Bcl-X.sub.L with good affinity (See, J. L. Wang et al., Cancer
Res., 60:1498-1502 (2000); and J. L. Wang et al., Proc. Natl. Acad.
Sci. U.S.A., 97:7124-7129 (2000)), the 3D structure of Bcl-2 in
complex with the Bak BH3 peptide was modeled based upon the
experimental NMR structure of Bcl-X.sub.L complexed with Bak BH3
peptide. (S. Michael et al., Science, 275:983-986 (1997)). The
modeled 3D complex structure was further refined using molecular
dynamic (MD) simulations in explicit water using the CHARMM program
(B. R. Brooks et al., J. Comp. Chem., 4,187-217 (1983); and P. V.
R. Schleyer et al., CHARMM: The Energy Function and Its
Parameterization with an Overview of the Program, in The
Encyclopedia of Computational Chemistry, 1:271-277 eds., John Wiley
& Sons, Chichester (1998)) with the MSI CHARMM force field. The
refined structure of Bcl-2 in complex with Bak BH3 peptide is shown
in FIGS. 2A and 2B, respectively. FIG. 2A shows a ribbon
representation of the overall Bcl-2 structure complexed with Bak
BH3 peptide. FIG. 2B shows a detailed representation of the BH3
binding site. The carbon atoms in the Bak BH3 peptide are in
magenta, while the carbon atoms in the Bcl-2 protein are in green,
the oxygen atoms are in red and the nitrogen atoms are in blue. The
potential Bcl-2/Bcl-X.sub.L inhibitors were confirmed using
biochemical and biological assays.
[0209] In some embodiments, as compared to random screening
methods, the structure-based 3D-database screening methods of the
present invention are more effective and less costly. In one
embodiment, a three-dimensional structural database containing
approximately 6,000 natural products isolated from traditional
herbal medicine libraries was screened using the DOCK program to
identify inhibitors of Bcl-2 and/or Bcl-X.sub.L. In another
embodiment, the latest version of the National Cancer Institute's
(NCI) 3D-database of approximately 250,000 organic synthetic
compounds and natural products (G. W. A. Milne et al., J. Chem.
Inf. Comput. Sci., 34:1219-1224 (1994)) was screened using the DOCK
program (S. Makin and I. D. Kuntz, J. Comput. Chem. 18:1812-1825
(1997)) to identify about 250-500 potential small molecule
Bcl-2/Bcl-X.sub.L inhibitors. Of the 259 compounds initially
selected, 141 were available from the NCI chemical repository and
were thus obtained for use in in vitro binding assays.
[0210] In one embodiment, further testing of the potential
non-peptide small molecule inhibitors was done using an established
sensitive and quantitative in vitro fluorescence polarization (FP)
based binding assay. (See, I. J. Enyedy et al., J. Med. Chem.,
44:313-4324 (2001)). The 141 candidate compounds were screened
using the FP bind assay for their ability to compete with Bak BH3
peptide in binding to Bcl-2. From the 141 compounds tested, a
subset of 20 compounds was found to display IC.sub.50 values
ranging from 0.7 .mu.M to 25 .mu.M. Fifteen distinct classes of
chemicals were represented within the 20 compound subset. The
IC.sub.50 value of the natural Bak BH3 peptide was 0.3 .mu.M in the
FP binding assays. Each of the 20 small molecules inhibitors
identified blocked the binding (complexing) of Bcl-2 and Bak BH3
peptides. Several of the small molecule inhibitors also potently
induced apoptosis and thus inhibited the viability and growth of
cancer cells with Bcl-2 overexpression.
[0211] Since Bcl-X.sub.L and Bcl-2 have similar 3D structures, it
was reasoned that some of the potential Bcl-2 inhibitors would also
bind to Bcl-X.sub.L. Accordingly, additional screening efforts were
directed at discovering potential non-peptide small molecule
antagonists of Bcl-X.sub.L using the FP based binding assay
described above.
[0212] Although Bcl-2 and Bcl-X.sub.L have similar functions, the
two proteins have different expression patterns in human cancers.
Furthermore, although Bcl-2 and Bcl-X.sub.L share structurally
similar BH3 binding sites, there are differences between the two.
It was discovered that while some small molecule inhibitors of
Bcl-2 also have good binding affinity for Bcl-X.sub.L, some other
small molecule inhibitors only weakly bound Bcl-X.sub.L (although
these molecules are still effective antagonists in some
compositions and methods). Some relatively weak Bcl-2 small
molecule inhibitors had much higher potency when binding to
Bcl-X.sub.L. In particular, it was found that gossypol binds to
both Bcl-2 and Bcl-X.sub.L proteins. FIG. 3 shows direct inhibition
of the binding between Bak BH3 peptide and Bcl-2 (Bcl-X.sub.L) by
gossypol measured using a FP based binding assay. The non-FP
labeled Bak peptide has an IC.sub.50 value of 0.3 .mu.M to
Bcl-X.sub.L.
[0213] In one embodiment, it was found that racemic gossypol binds
to Bcl-2 and Bcl-X.sub.L. FIG. 3 shows racemic gossypol directly
inhibits binding between Bak BH3 peptide and Bcl-2. FIG. 3 further
shows that racemic gossypol directly inhibits binding between Bak
BH3 peptide and Bcl-X.sub.L.
[0214] In another embodiment, it was found that enantiomers of
gossypol (e.g., (-)-gossypol and (+)-gossypol) bind to Bcl-2 and
Bcl-X.sub.L proteins. FIG. 4 shows racemic gossypol, (-)-gossypol,
and (+)-gossypol directly inhibit binding between Bid BH3 peptide
and Bcl-2. FIG. 5 shows racemic gossypol, (-)-gossypol, and
(+)-gossypol directly inhibit binding between Bad BH3 peptide and
Bcl-X.sub.L.
[0215] In yet another embodiment, it was found that gossypolone
binds to Bcl-2 and Bcl-X.sub.L proteins. FIG. 6A shows racemic
gossypolone directly inhibits binding between Bak BH3 peptide and
Bcl-X.sub.L. FIG. 6B shows competitive inhibition by (-)-gossypol
ethyl Schiff's base of the binding of Bak BH3 peptide to
Bcl-X.sub.L protein.
III. Characterization of Bcl-2 Family of Proteins in Cancer Cell
Lines
[0216] To better understand the molecular mechanism of small
molecule inhibitors of Bcl-2 and Bcl-X.sub.L, the expression of
Bcl-2 family proteins was characterized in various breast and other
cancer cell lines from the National Cancer Institute's anti-cancer
drug screening program. The results from 5 representative cancer
cell lines (i.e., the MDA-MB-231, T-47D, and MDA-435 breast cancer
cell lines, the HL-60 leukemia cell line, and the HT-29 colon
cancer cell line) and 1 normal fibroblast (WI-38) cell line are
shown in FIG. 7. The HL-60 leukemia cell line had the highest level
of Bcl-2 expression, MDA-MB-231 and MDA-MB-435 breast cancer cell
lines also showed very high levels of Bcl-2. Breast cancer cell
lines MDA-MB-231 and T-47D, as well as colon cancer cell line HT-29
showed very high levels of Bcl-X.sub.L expression. The normal
fibroblast cell line showed low levels of Bcl-2 and Bcl-X.sub.L
expression.
[0217] The Bcl-2 family proteins act as arbiters of programmed cell
death. The balance between anti-apoptotic molecules (e.g., Bcl-2
and Bcl-X.sub.L) and pro-apoptotic molecules (e.g., Bid, Bax, Bak
and Bad) plays an important role in apoptosis. For this reason, the
expression status of pro-apoptotic proteins Bid, Bax, Bak and Bad
in cancer cell lines MDA-MB-231, T-47D, MDA-435, HL-60, and HT-29,
and normal fibroblast cell line WI-38 were also determined (FIG.
7). All of the cell lines tested, including the normal fibroblast
cell line WI-38, expressed high levels of Bax and most of the
cancer cell lines also expressed high levels of Bak. There is
significant variations in the expression levels of Bid and Bad
between different cell lines. Taken together, all 5 of the cancer
cell lines showed high levels of both anti-apoptotic and
pro-apoptotic Bcl-2 family proteins, while the normal fibroblast
cell line (WI-38) showed only low levels of Bcl-2 and Bcl-X.sub.L,
but high levels of pro-apoptotic Bcl-2 family members proteins Bax
and Bad.
[0218] Several other breast cancer cell lines used by the National
Cancer Institute (NCI) in their anticancer drug screening efforts
were also tested. These cell lines include BT-549, HS 578T, MCF-7
and NCI/ADR-Resistant. Of these, MCF-7 and BT-549 showed high
levels of Bcl-2 protein expression. BT-549 also showed a high level
of Bcl-X.sub.L protein expression. The cell lines HS 578T and
NCI/ADR-RES had medium levels of Bcl-X.sub.L protein. Thus, among
the 7 human breast cancer cell lines examined (i.e., MDA-MB-231,
T-47D, MDA-435, BT-549, HS 578T, MCF-7, and NCI/ADR-Resistant) 5 of
the cell lines had high levels of either, or both, Bcl-2 and
Bcl-X.sub.L expression. Two breast cancer cell lines had medium
levels of Bcl-2 or Bcl-X.sub.L expression. None of the 7 breast
cancer cell lines tested had low expression of Bcl-2 and
Bcl-X.sub.L.
IV. Gossypol Compounds Inhibit Cancer Cell Growth and
Proliferation
[0219] The fluorescence polarization assays showed that gossypol
antagonizes the binding of Bak, Bid, or Bad BH3 peptide to Bcl-2
and Bcl-X.sub.L. Thus, the present invention contemplates that
small molecule inhibitors (e.g., gossypol compounds) that bind to
the BH3 binding domain in Bcl-2 and/or Bcl-X.sub.L will block the
anti-apoptotic functions of these proteins, and in turn induce
apoptosis in cells (e.g., cancer cells) with elevated Bcl-2 and/or
Bcl-X.sub.L expression. It is further contemplated that small
molecule inhibitors (e.g., gossypol compounds) also decrease
cellular viability and proliferation in cells (e.g., cancer cells)
with high Bcl-2 and/or Bcl-X.sub.L expression. Gossypol inhibits
cell proliferation (and growth) in cancer, and more particularly,
in human breast cancers (e.g., MDA-MB-231 cells). As described
herein, the MDA-MB-231 breast cancer cell line has high levels of
expression of both Bcl-2 and Bcl-X.sub.L.
[0220] Various cancer cell inhibition studies were performed with
gossypol compounds. For example, the ability of gossypol compounds
to inhibit MDA-MB-231 cell growth was tested in a 5 day MIT assay.
Gossypol was shown to inhibit MDA-MB-231 cell growth with an
IC.sub.50 value of 2.0 .mu.M. The results in MB-231 and WI-38 cells
treated with 20 .mu.M of gossypol for 24 hours as detected by the
Hoechst Dye assay are shown in FIGS. 8A and 8B, respectively.
Treatment of MDA-MB-231 cancer cells with gossypol induces
apoptosis. FIG. 8A shows the induction of apoptosis in the MDA-231
cells. Gossypol did not induce normal WI-38 fibroblast cells to
undergo apoptosis. FIG. 8B shows that gossypol treatment did not
induce apoptosis in normal WI-38 fibroblast cells having low levels
(e.g., basal levels) of Bcl-2 and Bcl-X.sub.L expression.
[0221] In other tests, gossypol was shown to induce apoptosis in
T-47D breast cancer cells having high levels of Bcl-X.sub.L
expression, but low levels of Bcl-2 expression. It was also found
that gossypol induces apoptosis in other cancer cell lines with
high Bcl-X.sub.L expression such as HT-29, a human colon cancer
cell line, but not in cancer cell lines with low levels of Bcl-2
and Bcl-X.sub.L expression such as DU-145, a prostate cancer cell
line.
[0222] Further tests using Annexin-V flow cytometry (FACS) assays
were conducted to more quantitatively assess the ability of
gossypol to induce apoptosis in MDA-MB-231 breast cancer cells. For
example, FIG. 9 shows gossypol induced apoptosis in human
MDA-MB-231 breast cancer cells treated with gossypol for 24 hours
detected using Annexin-V flow cytometry. It was observed that 5.0
.mu.M of gossypol induced 59% of the MDA-MB-231 cells to undergo
apoptosis. While the present invention is not limited to a
particular mechanism(s), it is contemplated that induction of
apoptosis by gossypol is dose-dependent. At administrations of 10.0
and 20.0 .mu.M, respectively, gossypol induced 74% and 96% of
cancer cells, again respectively, to undergo apoptosis. MDA-MB-231
cells overexpress Bcl-2 and Bcl-X.sub.L.
[0223] Since gossypol (e.g., racemic gossypol, (-)-gossypol and
(+)-gossypol) is a potent inhibitor of Bcl-X.sub.L, the present
invention contemplates that in certain embodiments, gossypol
induces apoptosis in cancer cells with high levels of Bcl-X.sub.L
expression but low levels of Bcl-2. Indeed, gossypol induces
dose-dependent apoptosis in human T-47D breast cancer cells, which
as shown above, have high levels of Bcl-X.sub.L but low levels of
Bcl-2. FIG. 10 shows the dose dependent induction of apoptosis in
human TD-47 cancer cells treated with gossypol for 24 hours as
detected using Annexin-V flow cytometry.
[0224] Since gossypol (e.g. racemic gossypol, (-)-gossypol and
(+)-gossypol) is also a potent inhibitor of Bcl-2, the present
invention contemplates that in certain embodiments, gossypol
induces apoptosis in cancer cells with high expression levels of
Bcl-2 expression but low levels of Bcl-X.sub.L. Since gossypol
binds to both Bcl-2 and Bcl-X.sub.L proteins, the present invention
contemplates that, in certain embodiments, gossypol induces
apoptosis in cancer cells with high levels of Bcl-2 and Bcl-X.sub.L
expression.
[0225] These tests show that gossypol is a potent inhibitor of
Bcl-X.sub.L and induces cancer cells expressing high levels (e.g.,
overexpression as compared to a basal expression rate for a normal
example of the cell type) of Bcl-X.sub.L to undergo apoptosis, but
does not induce apoptosis in cells with normal levels of Bcl-2 and
Bcl-X.sub.L expression (e.g., WI-38 cells).
[0226] Preferred embodiments of the present invention provide
methods of administering one or more gossypol compounds to a
subject having a condition characterized by the overexpression of
Bcl-2 family proteins. The gossypol compounds contemplated for use
in the present inventive methods include, but are not limited to,
(.+-.)-gossypol; (-)-gossypol (Super G); (+)-gossypol;
(.+-.)-gossypolone; (-)-gossypolone; (+)-gossypolone;
(.+-.)-gossypol acetic acid; (-)-gossypol acetic acid; (+)-gossypol
acetic acid; (.+-.)-ethyl gossypol; (-)-ethyl gossypol; (+)-ethyl
gossypol; (.+-.)-hemigossypolone; (-)-hemigossypolone;
(+)-hemigossypolone; Schiff's base of (.+-.)-gossypol; Schiff's
base of (-)-gossypol; Schiff's base of (+)-gossypol; Schiff's base
of (.+-.)-gossypolone; Schiff's base of (-)-gossypolone; Schiff's
base of (+)-gossypolone; Schiff's base of (.+-.)-gossypol acetic
acid; Schiff's base of (-)-gossypol acetic acid; Schiff's base of
(+)-gossypol acetic acid; Schiff's base of (.+-.)-ethyl gossypol;
Schiff's base of (-)-ethyl gossypol; Schiff's base of (+)-ethyl
gossypol; Schiff's base of (.+-.)-hemigossypolone; Schiff's base of
(-)-hemigossypolone; Schiff's base of (+)-hemigossypolone,
(+)-apogossypol, (-)-apogossypol, (+)-apogossypol,
(.+-.)-apogossypol acetic acid, (-)-apogossypol acetic acid,
(+)-apogossypol acetic acid, (.+-.)-ethyl apogossypol, (-)-ethyl
apogossypol, (+)-ethyl apogossypol as well as derivatives,
metabolites, isomers, acids, and pharmaceutically acceptable salts
thereof.
[0227] In particularly, preferred embodiments, the (-)-gossypol
enantiomer (including derivatives, metabolites, acids, Schiff's
bases and pharmaceutically acceptable salts thereof) are
administered to a subject or to in vitro cells, tissues, or
organs.
[0228] NMR analysis of the binding of the (-)-gossypol enantiomer
shows that the (-)-gossypol specifically binds to the BH3 binding
groove in Bcl-X.sub.L (See, FIGS. 11A and 11B). FIG. 12 shows data
from growth inhibition experiments comparing (-)-gossypol and
(+)-gossypol enantiomers and racemic gossypol in the MDA-MB-231
(2-LMP) breast cancer cell line using a 5-day MTT assay.
[0229] Additional experiments showed that the T.sub.112 of
elimination of (+)-gossypol enantiomer in humans is 29 times that
of (-)-gossypol. In some embodiments, the present invention
contemplates that the longer residency of the (+)-gossypol
enantiomer is potentially beneficial in certain prolonged gossypol
treatments methods. Therefore, in some embodiments, the
(+)-gossypol enantiomer is potentially more toxic to cells. In
still some embodiments, the present invention contemplates that the
longer residency of the (+)-gossypol enantiomer is potentially
beneficial in certain prolonged gossypol treatments methods.
[0230] In other embodiments, (-)-gossypol is administered to
patients having a condition characterized by the overexpression of
a Bcl-2 family protein. Table 1 compares the inhibition of cell
growth in a number of head-neck cancer cell lines treated with
(-)-gossypol and cisplatin (CTTP) (a standard agent for the
treatment head-neck cancers). Briefly, Table 1 compares the
inhibition of cell growth demonstrated by (-)-gossypol and
cisplatin in a panel of human head-neck cancer cells and the
expression status of Bcl-2 family proteins (Bcl-2, Bcl-X.sub.L and
Bcl-X.sub.S). The IC.sub.50 value is the concentration of the drug
(e.g., (-)-gossypol and cisplatin) required to inhibit the cell
growth by 50% as compared to control cells.
TABLE-US-00001 TABLE 1 (-)- gossypol Cisplatin (IC.sub.50, .mu.M)
Bcl-X.sub.L Bcl-X.sub.S Bcl-2 (IC.sub.50, .mu.M) UM-SCC-23 1.5 +++
+ - 25 UM-SCC-1 1.5 +++ + - 30 UM-SCC-6 11 +++ +++ - >50
UM-SCC-22A 3 ++ + - 5 UM-SCC-12 4 ++ + - 22 UM-SCC-81B 4 ++ - - 22
UM-SCC-17B 5 ++ + ++ 13 UM-SCC-14A 11 + +++ - 29 UM-SCC-74B 4 - +
++ 12 UM-SCC-25 8 + - - 43 Fib-1 20 - - - >50 Fib-2 20 - - -
>50 Fib-3 18 - - - >50
V. Proposed Mechanism of Gossypol Activity
[0231] Although an understanding of any particular proposed
mechanism is not necessary to make and use the compositions and
methods of the present invention and the present invention is not
limited to any particular mechanism(s), it is contemplated that one
of the key molecules in the Bcl-2/Bcl-X.sub.L mediated apoptosis
pathway is cytochrome-c (Cyt-c). It is further contemplated that
one of the key functions of Bcl-2/Bcl-X.sub.L is to heterodimerize
with Bax, Bak, or Bad and to block release of Cyt-c from
mitochondria. Thus, the ability of gossypol to induce Cyt-c release
from mitochondria to the cytosol in cancer cells was tested. Breast
cancer cell lines MDA-231 and T47D were treated with either 5 or 20
.mu.M of gossypol for 24 hours. FIG. 13 shows that Cyt-c was
released from mitochondria into the cytosol after treatment with 20
.mu.M of gossypol in both the MDA-231 and T47D breast cancer cell
lines (HM, Cyt-c found in the heavy membrane; Cytosol, Cyt-c found
in the cytosol).
[0232] It is also contemplated that Bcl-2 mediated apoptosis
involves caspase (e.g., caspase-3 and -9) activation once Cyt-c is
released from the mitochondria. Therefore, tests were conducted to
determine whether gossypol activates caspase-3. In one embodiment,
the amount of caspase-3 cleavage in lysates of MDA-231 breast
cancer cells was measured after 12 or 24 hours following treatment
with gossypol. FIG. 14 shows that caspase-3 was cleaved after
treatment with gossypol into 17 and 21 kD fragments in a dose
dependent manner. Similar results were obtained in T-47D human
breast cancer cells and HT-29 colon cancer cells treated with
gossypol, both of which have high levels of Bcl-X.sub.L expression
and relatively low levels of Bcl-2 expression following treatment
with gossypol.
[0233] In contrast, treatment of human DU-145 prostate cancer
cells, having low Bcl-2 and Bcl-X.sub.L expression, with 5, 10, or
20 .mu.M of gossypol for 24 hours had no effect on caspase-3
activation. Therefore, activation of caspase-3 by gossypol is
specific and correlative to expression levels of Bcl-2 and
Bcl-X.sub.L in cancer cells.
VI. Activity of Gossypol in MDA-231 Xenograft Mice Alone and in
Combination with Conventional Anticancer Agents
[0234] The potential of gossypol compounds as anticancer
therapeutics was further evaluated in MDA-231 xenograft mice. A
gossypol treatment regime was started at day 7 after tumors had
grown to 8-10 mm in diameter. Each treatment group had five mice
bearing two tumors each (one tumor on each side). A control group
of five mice received no gossypol. In the treated mice, gossypol
was administered daily in two different oral doses, a 30 and a 90
mg/kg dose, for three weeks. It was found that at both 30 and 90
mg/kg daily doses, there is more than 70% inhibition of tumor
growth by gossypol with more than a 95% confidence level at day 29.
No weight loss or deaths were seen in the mice treated with
gossypol. There did not appear to be any significant difference in
the anticancer activity of gossypol in the 30 mg/kg and 90 mg/kg
doses. These results suggest that a 30 mg/kg daily dose of gossypol
successfully inhibits tumor growth without supplementing the
gossypol therapy with adjuvants or additional anticancer compounds
or therapies.
[0235] Overexpression of Bcl-2 and/or Bcl-X.sub.L proteins appears
to protect cancer cells from apoptosis induced by some conventional
anticancer therapies (e.g., docetaxel). Some embodiments of the
present invention, therefore, provide methods for administering an
effective dose(s) of gossypol (and enantiomers, derivatives, acids
(e.g., acetic acid) and pharmaceutically acceptable salts thereof)
in combination with at least one conventional anticancer therapy
(e.g., chemotherapeutic agents, such as docetaxel and/or radiation
therapy). In preferred embodiments, gossypol is administered in
combination with one or more conventional anticancer therapies to
treat diseases (e.g., cancer) characterized by overexpression of
Bcl-2 family proteins (e.g., Bcl-2 and/or Bcl-X.sub.L). In one
embodiment of the present invention, when gossypol is administered,
it is not co-administered with radiation and heat.
[0236] Although an understanding of any mechanism is not necessary
to practice the present invention and the present invention is not
so limited, it is contemplated that administration of at least one
gossypol compound sensitizes cancer cells having high levels of
expression of Bcl-2 family proteins (e.g., Bcl-2 and/or
Bcl-X.sub.L) which are resistant to conventional anticancer
therapies, to treatment with additional anticancer agents (e.g.,
docetaxel). The present invention is, however, not limited to the
administration of any particular combination of gossypol compounds
and anticancer therapeutic agents, nor is the invention limited to
any particular sequence or level of agents being administered.
[0237] In one, embodiment of the present invention, the
co-administration of a gossypol compound and one or more anticancer
agents produces a synergistic effect, i.e., an effect that is more
than the additive effect of each compound administered
individually. In a further embodiment of the present invention, the
co-administration of a gossypol compound and one or more anticancer
agents allows lower doses of the gossypol compound and/or the one
or more anticancer agents to be used. The ability to achieve
efficacy using lower doses allows the administration of doses that
do not induce any substantial toxicity in the subject. In another
embodiment of the present invention, the co-administration of a
gossypol compound and one or more anticancer agents may lead to
complete regression of a tumor whereas either compound alone would
provide only a partial regression. In a further embodiment of the
present invention, the administration of a gossypol compound
sensitizes neoplastic cells to the therapeutic effect of anticancer
agents. Thus, a lower dose of the anticancer agent is sufficient to
kill the neoplastic cells when co-administered with a gossypol
compound.
[0238] Examples of lower dose ranges of gossypol compounds and some
anticancer agents that can be used in combination with gossypol
compounds for the treatment of particular cancers are presented in
Tables 2-4 below. These examples are not intended to limit the
present invention in any way.
TABLE-US-00002 TABLE 2 Racemic Gossypol Cisplatin Docetaxel
Radiation Breast 1-200 mg/d; 5-30 mg/m.sup.2 10-40 mg/m.sup.2 2-65
Gy total Cancer 1, 5, 10, 15, 20, every wk; every wk; dose; 25, 30,
35, 40, 5, 10, 15, 20, 25, 10, 15, 20, 25, 2, 5, 10, 15, 20, 45,
50, 55, 60, 30 mg/m.sup.2 every 30, 35, 40 mg/m.sup.2 25, 30, 35,
40, 65, 70, 75, 80, wk; every wk; 45, 50, 55, 60, 65 85, 90, 95,
100, 5-120 mg/m.sup.2 10-60 mg/m.sup.2 Gy total dose 105, 110, 115,
every 3 wk; every 2 wk; 120, 125, 130, 5, 10, 15, 20, 25, 10, 15,
20, 25, 135, 140, 145, 30, 35, 40, 45, 30, 35, 40, 45, 150, 155,
160, 50, 55, 60, 65, 50, 55, 60 mg/m.sup.2 165, 170, 175, 70, 75,
80, 85, every 2 wk; 180, 185, 190, 90, 95, 100, 105, 20-100
mg/m.sup.2 195, 200 mg/d; 110, 115, 120 mg/m.sup.2 every 3 wk;
40-400 mg every every 3 wk 20, 25, 30, 35, wk; 40, 45, 50, 55,
40-400 mg every 60, 65, 70, 75, wk; 80, 85, 90, 95, 40, 50, 60, 70,
100 mg/m.sup.2 every 80, 90, 100, 120, 3 wk 140, 160, 180, 200,
220, 240, 260, 280, 300, 320, 340, 360, 380, 400 mg every week
Prostate 1-200 mg/d; 5-20 mg/m.sup.2/d for 5-35 mg/m.sup.2/d for
2-78 Gy total Cancer 1, 5, 10, 15, 20, 3 d; 2 d; dose; 25, 30, 35,
40, 5, 10, 15, 20 mg/m.sup.2/d 5, 10, 15, 20, 25, 2, 5, 10, 15, 20,
45, 50, 55, 60, for 3 d; 30, 35 mg/m.sup.2/d 25, 30, 35, 40, 65,
70, 75, 80, 5-20 mg/m.sup.2 for 2 d; 45, 50, 55, 60, 85, 90, 95,
100, every other d; 5, 10, 15, 20, 25 mg/m.sup.2/d 65, 70, 75, 78
Gy 105, 110, 115, 5, 10, 15, 20 mg/m.sup.2 for 4 d total dose 120,
125, 130, every 135, 140, 145, other d; 150, 155, 160, 10-70
mg/m.sup.2 165, 170, 175, every 4 wk; 180, 185, 190, 10, 15, 20,
25, 195, 200 mg/d; 30, 35, 40, 45, 40-400 mg every 50, 55, 60, 65,
70 mg/m.sup.2 wk; every 4 wk 40-400 mg every wk; 40-400 mg every
wk; 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240,
260, 280, 300, 320, 340, 360, 380, 400 mg every week Colon 1-200
mg/d; 5-30 mg/m.sup.2/d for 10-185 mg/m.sup.2 2-60 Gy total Cancer
1, 5, 10, 15, 20, 3 d; every 3 wk; dose; 25, 30, 35, 40, 5, 10, 15,
20, 25, 10, 15, 25, 35, 2, 5, 10, 15, 20, 45, 50, 55, 60, 30
mg/m.sup.2/d for 45, 55, 65, 75, 25, 30, 35, 40, 65, 70, 75, 80, 3
d 85, 95, 105, 115, 45, 50, 55, 60 Gy 85, 90, 95, 100, 125, 135,
145,, total dose 105, 110, 115, 155, 165, 175, 120, 125, 130, 185
mg/m.sup.2 every 135, 140, 145, 3 wk 150, 155, 160, 165, 170, 175,
180, 185, 190, 195, 200 mg/d; 40-400 mg every wk; 40-400 mg every
wk; 40-400 mg every wk; 40, 50, 60, 70, 80, 90, 100, 120, 140, 160,
180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 mg every
week Pancreatic 1-200 mg/d; 25-50 mg/m.sup.2 5-35 mg/m.sup.2 2-65
Gy total Cancer 1, 5, 10, 15, 20, every wk; every wk; dose; 25, 30,
35, 40, 25, 30, 35, 40, 5, 10, 15, 20, 25, 2, 5, 10, 15, 20, 45,
50, 55, 60, 45, 50 mg/m.sup.2 30, 35 mg/m.sup.2 25, 30, 35, 40, 65,
70, 75, 80, every wk; every wk; 45, 50, 55, 60, 65 85, 90, 95, 100,
5-15 mg/m.sup.2 6-100 mg/m.sup.2 Gy total dose 105, 110, 115, every
3 wk; every 3 wk; 120, 125, 130, 5, 10, 15 mg/m.sup.2 6, 10, 15,
20, 25, 135, 140, 145, every 3 wk; 30, 35, 40, 45, 150, 155, 160,
10-100 mg/m.sup.2 50, 55, 60, 65, 165, 170, 175, every 4 wk; 70,
75, 80, 85, 180, 185, 190, 10, 15, 20, 25, 90, 95, 100 mg/m.sup.2
195, 200 mg/d; 30, 35, 40, 45, every 3 wk; 40-400 mg every 50, 55,
60, 65, 6-60 mg/m.sup.2 wk; 70, 75, 80, 85, every 4 wk; 40-400 mg
every 90, 95, 100 mg/m.sup.2 6, 10, 15, 20, 25, wk; every 4 wk 30,
35, 40, 45, 40-400 mg every 50, 55, 60 mg/m.sup.2 wk; every 4 wk
40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240,
260, 280, 300, 320, 340, 360, 380, 400 mg every week Head/Neck
1-200 mg/d; 5-20 mg/m.sup.2/d for 2-40 mg/m.sup.2 2-66 Gy total
Cancer 1, 5, 10, 15, 20, 3 d; every wk; dose; 25, 30, 35, 40, 5,
10, 15, 20 mg/m.sup.2/d 2, 5, 10, 15, 20, 2, 5, 10, 15, 20, 45, 50,
55, 60, for 3 d; 25, 30, 35, 40 mg/m.sup.2 25, 30, 35, 40, 65, 70,
75, 80, 5-10 mg/m.sup.2 every wk; 45, 50, 55, 60, 66 85, 90, 95,
100, every wk; 6-60 mg/m.sup.2 Gy total dose 105, 110, 115, 5, 7.5,
10 mg/m.sup.2 every 3 wk; 120, 125, 130, every wk; 6, 10, 15, 20,
25, 135, 140, 145, 10-65 mg/m.sup.2 30, 35, 40, 45, 150, 155, 160,
every 2 wk; 50, 55, 60 mg/m.sup.2 165, 170, 175, 10, 15, 20, 25,
every 3 wk; 180, 185, 190, 30, 35, 40, 45, 6-80 mg/m.sup.2 195, 200
mg/d; 50, 55, 60, 65 mg/m.sup.2 every 4 wk; 40-400 mg every every 2
wk; 6, 10, 15, 20, 25, wk; 10-100 mg/m.sup.2 30, 35, 40, 45, 40-400
mg every every 3 wk; 50, 55, 60, 65, wk; 10, 15, 20, 25, 70, 75, 80
mg/m.sup.2 40-400 mg every 30, 35, 40, 45, every 4 wk wk; 50, 55,
60, 65, 40, 50, 60, 70, 70, 75, 80, 85, 80, 90, 100, 120, 90, 95,
100 mg/m.sup.2 140, 160, 180, every 3 wk; 200, 220, 240, 5-20
mg/m.sup.2/d for 260, 280, 300, 5 d every 4 wk; 320, 340, 360, 5,
10, 15, 20 mg/m.sup.2/d 380, 400 mg for 5 d every week every 4 wk
Non-Small 1-200 mg/d; 5-30 mg/m.sup.2/d for 5-40 mg/m.sup.2 2-86 Gy
total Cell Lung 1, 5, 10, 15, 20, 2 d every 3 wk; every wk; dose;
Cancer 25, 30, 35, 40, 5, 10, 15, 20, 25, 5, 10, 15, 20, 25, 2, 5,
10, 15, 20, 45, 50, 55, 60, 30 mg/m.sup.2/d for 2 d 30, 35, 40
mg/m.sup.2 25, 30, 35, 40, 65, 70, 75, 80, every 3 wk; every wk;
45, 50, 55, 60, 85, 90, 95, 100, 10-100 mg/m.sup.2 6-175 mg/m.sup.2
65, 70, 75, 80, 86 105, 110, 115, every 3 wk; every 3 wk; Gy total
dose 120, 125, 130, 10, 15, 20, 25, 6, 10, 15, 25, 35, 135, 140,
145, 30, 35, 40, 45, 45, 55, 65, 75, 150, 155, 160, 50, 55, 60, 65,
85, 95, 105, 115, 165, 170, 175, 70, 75, 80, 85, 125, 135, 145,
180, 185, 190, 90, 95, 100 mg/m.sup.2 155, 165, 175 mg/m.sup.2 195,
200 mg/d; every 3 wk; 8-80 mg/m.sup.2 40-400 mg every 10-100
mg/m.sup.2/d every 4 wk; wk; every 4 wk; 8, 15, 20, 25, 30, 40-400
mg every 10, 15, 20, 25, 35, 40, 45, 50, wk; 30, 35, 40, 45, 55,
60, 65, 70, 40-400 mg every 50, 55, 60, 65, 75, 80 mg/m.sup.2 wk;
70, 75, 80, 85, every 4 wk 40, 50, 60, 70, 90, 95, 100 mg/m.sup.2/d
80, 90, 100, 120, every 4 wk 140, 160, 180, 200, 220, 240, 260,
280, 300, 320, 340, 360, 380, 400 mg every week Melanoma 1-200
mg/d; 10-80 mg/m.sup.2 5-100 mg/m.sup.2 2-60 Gy total 1, 5, 10, 15,
20, every wk; every 3 wk; dose; 25, 30, 35, 40, 10, 15, 20, 25, 5,
10, 15, 20, 25, 2, 5, 10, 15, 20, 45, 50, 55, 60, 30, 35, 40, 45,
30, 35, 40, 45, 25, 30, 35, 40, 65, 70, 75, 80, 50, 55, 60, 65, 50,
55, 60, 65, 45, 50, 55, 60 Gy 85, 90, 95, 100, 70, 75, 80
mg/m.sup.2 70, 75, 80, 85, total dose 105, 110, 115, every wk; 90,
95, 100 mg/m.sup.2 120, 125, 130, 5-20 mg/m.sup.2/d for every 3 wk;
135, 140, 145, 4 d every 2 wk; 8-80 mg/m.sup.2 150, 155, 160, 5,
10, 15, 20 mg/m.sup.2/d every 4 wk; 165, 170, 175, for 4 d 8, 10,
15, 20, 25, 180, 185, 190, every 2 wk; 30, 35, 40, 45, 195, 200
mg/d; 5-25 mg/m.sup.2/d for 50, 55, 60, 65, 40-400 mg every 2 d
every 3 wk; 70, 75, 80 mg/m.sup.2 wk; 5, 10, 15, 20, 25
mg/m.sup.2/d every 4 wk 40-400 mg every for 2 d wk; every 3 wk;
40-400 mg every 5-30 mg/m.sup.2/d for wk; 3 d every 3 wk; 40, 50,
60, 70, 5, 10, 15, 20, 25, 80, 90, 100, 120, 30 mg/m.sup.2/d for 3
d 140, 160, 180, every 3 wk; 200, 220, 240, 10-100 mg/m.sup.2 260,
280, 300, every 3 wk; 320, 340, 360, 10, 15, 20, 25, 380, 400 mg
30, 35, 40, 45, every week 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100 mg/m.sup.2 every 3 wk Ovarian 1-200 mg/d; 10-100 mg/m.sup.2
5-30 mg/m.sup.2 2-52 Gy total Cancer 1, 5, 10, 15, 20, every 3 wk;
every wk; dose; 25, 30, 35, 40, 10, 15, 20, 25, 5, 10, 15, 20, 25,
2, 5, 10, 15, 20, 45, 50, 55, 60, 30, 35, 40, 45, 30 mg/m.sup.2
every 25, 30, 35, 40, 65, 70, 75, 80, 50, 55, 60, 65, wk; 45, 50,
52 Gy 85, 90, 95, 100, 70, 75, 80, 85, 5-60 mg/m.sup.2 total dose
105, 110, 115, 90, 95, 100 mg/m.sup.2 every 2 wk; 120, 125, 130,
every 3 wk; 5, 10, 15, 20, 25, 135, 140, 145, 10-100 mg/m.sup.2 30,
35, 40, 45, 150, 155, 160, every 4 wk; 50, 55, 60 mg/m.sup.2 165,
170, 175, 10, 15, 20, 25, every 2 wk; 180, 185, 190, 30, 35, 40,
45, 10-100 mg/m.sup.2 195, 200 mg/d; 50, 55, 60, 65, every 3 wk;
40-400 mg every 70, 75, 80, 85, 10, 15, 20, 25, wk; 90, 95, 100
mg/m.sup.2 30, 35, 40, 45, 40-400 mg every every 4 wk 50, 55, 60,
65, wk; 70, 75, 80, 85, 40, 50, 60, 70, 90, 95, 100 mg/m.sup.2 80,
90, 100, 120, every 3 wk; 140, 160, 180, 6-60 mg/m.sup.2 200, 220,
240, every 4 wk; 260, 280, 300, 6, 10, 15, 20, 25, 320, 340, 360,
30, 35, 40, 45, 380, 400 mg 50, 55, 60 mg/m.sup.2 every week every
4 wk Lymphoma 1-200 mg/d; 5-25 mg/m.sup.2/d for 10-100 mg/m.sup.2
2-55 Gy total 1, 5, 10, 15, 20, 4 d; every 3 wk; dose; 25, 30, 35,
40, 5, 10, 15, 20, 25 mg/m.sup.2/d 10, 15, 20, 25, 2, 5, 10, 15,
20, 45, 50, 55, 60, for 4 d; 30, 35, 40, 45, 25, 30, 35, 40, 65,
70, 75, 80, 10-75 mg/m.sup.2 50, 55, 60, 65, 45, 50, 55 Gy 85, 90,
95, 100, every 3 wk; 70, 75, 80, 85, total dose 105, 110, 115, 10,
15, 20, 25, 90, 95, 100 mg/m.sup.2 120, 125, 130, 30, 35, 40, 45,
every 3 wk 135, 140, 145, 50, 55, 60, 65, 150, 155, 160, 70, 75
mg/m.sup.2 165, 170, 175, every 3 wk 180, 185, 190, 195, 200 mg/d;
40-400 mg every wk; 40-400 mg every wk; 40, 50, 60, 70, 80, 90,
100, 120, 140, 160, 180,
200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 mg every week
Hepatoma 1-200 mg/d; 5-30 mg/m.sup.2 5-36 mg/m.sup.2 2-70 Gy total
1, 5, 10, 15, 20, every wk; every wk; dose; 25, 30, 35, 40, 5, 10,
15, 20, 25, 5, 10, 15, 20, 25, 2, 5, 10, 15, 20, 45, 50, 55, 60, 30
mg/m.sup.2 every 30, 36 mg/m.sup.2 25, 30, 35, 40, 65, 70, 75, 80,
wk; every wk; 45, 50, 55, 60, 85, 90, 95, 100, 10-80 mg/m.sup.2;
5-40 mg/m.sup.2 65, 70 Gy total 105, 110, 115, 10, 15, 20, 25,
every 3 wk; dose 120, 125, 130, 30, 35, 40, 45, 5, 10, 15, 20, 25,
135, 140, 145, 50, 55, 60, 65, 30, 35, 40 mg/m.sup.2 150, 155, 160,
70, 75, 80 mg/m.sup.2 every 3 wk; 165, 170, 175, 10-60 mg/m.sup.2
180, 185, 190, every 4 wk; 195, 200 mg/d; 10, 15, 20, 25, 40-400 mg
every 30, 35, 40, 45, wk; 50, 55, 60 mg/m.sup.2 40-400 mg every
every 4 wk wk; 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,
200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 mg every week
Sarcoma 1-200 mg/d; 5-20 mg/m.sup.2/d for 4-100 mg/m.sup.2 2-66 Gy
total 1, 5, 10, 15, 20, 5 d every 3 wk; every 3 wk; dose; 25, 30,
35, 40, 5, 10, 15, 20 mg/m.sup.2/d 4, 10, 15, 20, 25, 2, 5, 10, 15,
20, 45, 50, 55, 60, for 5 d 30, 35, 40, 45, 25, 30, 35, 40, 65, 70,
75, 80, every 3 wk; 50, 55, 60, 65, 45, 50, 55, 60, 66 85, 90, 95,
100, 5-20 mg/m.sup.2/d for 70, 75, 80, 85, Gy total dose 105, 110,
115, 5 d every 4 wk; 90, 95, 100 mg/m.sup.2 120, 125, 130, 5, 10,
15, 20 mg/m.sup.2/d every 3 wk 135, 140, 145, for 5 d 150, 155,
160, every 4 wk; 165, 170, 175, 5-30 mg/m.sup.2; 180, 185, 190, 5,
10, 15, 20, 25, 195, 200 mg/d; 30 mg/m.sup.2 40-400 mg every wk;
40-400 mg every wk; 40, 50, 60, 70, 80, 90, 100, 120, 140, 160,
180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 mg every
week Chronic 1-200 mg/d; 5-35 mg/m.sup.2/d for 2-8 Gy total
Lymphocytic 1, 5, 10, 15, 20, 2 d; dose; Leukemia 25, 30, 35, 40,
5, 10, 15, 20, 25, 2, 4, 6, 8 Gy total 45, 50, 55, 60, 30, 35
mg/m.sup.2/d dose 65, 70, 75, 80, for 2 d; 85, 90, 95, 100, 5-25
mg/m.sup.2/d for 105, 110, 115, 4 d; 120, 125, 130, 5, 10, 15, 20,
25 mg/m.sup.2/d 135, 140, 145, for 4 d 150, 155, 160, 165, 170,
175, 180, 185, 190, 195, 200 mg/d; 40-400 mg every wk; 40-400 mg
every wk; 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200,
220, 240, 260, 280, 300, 320, 340, 360, 380, 400 mg every week
Acute 1-200 mg/d; 2-45 Gy total Myelogenous 1, 5, 10, 15, 20, dose;
Leukemia 25, 30, 35, 40, 2, 5, 10, 15, 20, 45, 50, 55, 60, 25, 30,
35, 40, 45 65, 70, 75, 80, Gy total dose 85, 90, 95, 100, 105, 110,
115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175,
180, 185, 190, 195, 200 mg/d; 40-400 mg every wk; 40-400 mg every
wk; 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240,
260, 280, 300, 320, 340, 360, 380, 400 mg every week Multiple 1-200
mg/d; 10-75 mg/m.sup.2 2-40 Gy total Myeloma 1, 5, 10, 15, 20,
every 3 wk; dose; 25, 30, 35, 40, 10, 15, 20, 25, 2, 5, 10, 15, 20,
45, 50, 55, 60, 30, 35, 40, 45, 25, 30, 35, 40 Gy 65, 70, 75, 80,
50, 55, 60, 65, total dose 85, 90, 95, 100, 70, 75 mg/m.sup.2 105,
110, 115, every 3 wk 120, 125, 130, 135, 140, 145, 150, 155, 160,
165, 170, 175, 180, 185, 190, 195, 200 mg/d; 40-400 mg every wk;
40-400 mg every wk; 40, 50, 60, 70, 80, 90, 100, 120, 140, 160,
180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 mg every
week
TABLE-US-00003 TABLE 3 Gemcitabine CHOP Carboplatin Doxorubicin
Breast 100-1200 mg/m.sup.2 C-100-1000 mg/m.sup.2; 40-265
mg/m.sup.2/d 2-20 mg/m.sup.2 Cancer every wk; H-10-50 mg/m.sup.2;
for 4 d; every wk; 100, 200, 300, O-1-2 mg/m.sup.2; 40, 80, 120,
160, 2, 4, 6, 8, 10, 12, 400, 500, 600, P-10-40 mg; 200, 265
mg/m.sup.2/d 14, 16, 18, 20 mg/m.sup.2 700, 800, 900, C-100, 200,
300, for 4 d; every wk; 100, 1100, 1200 mg/m.sup.2 400, 500, 600,
5-20 mg/m.sup.2/d for 10-75 mg/m.sup.2 every wk; 700, 800, 900, 20
d; every 2 wk; 100-2,000 mg/m.sup.2 1000 mg/m.sup.2; 5, 10, 15, 20
mg/m.sup.2/d 10, 20, 30, 40, every 2 wk; H-10, 20, 30, 40, for 20
d; 50, 60, 75 mg/m.sup.2 100, 200, 400, 50 mg/m.sup.2; 50-300
mg/m.sup.2 every 2 wk; 600, 800, 1000, O-1, 1.2, 1.4, every 4 wk;
10-75 mg/m.sup.2 1200, 1400, 1.6, 1.8, 2 mg/m.sup.2; 50, 100, 150,
every 3 wk; 1600, 1800, 2000 mg/m.sup.2 P-10, 20, 30, 40 mg 200,
250, 300 mg/m.sup.2 10, 20, 30, 40, every 2 wk; every 4 wk; 50, 60,
75 mg/m.sup.2 150-1500 mg/m.sup.2 500-1600 mg/m.sup.2; every 3 wk;
every 3 wk; 500, 750, 1000, 10-50 mg/m.sup.2 150, 300, 600, 1250,
1600 mg/m.sup.2 every 4 wk; 900, 1200, 1500 mg/m.sup.2 10, 15, 20,
25, every 3 wk 30, 35, 40, 45, 50 mg/m.sup.2 every 4 wk; 5-30
mg/m.sup.2/d for 3 d every 4 wk; 5, 10, 15, 20, 25, 30 mg/m.sup.2/d
for 3 d every 4 wk Prostate 60-1200 mg/m.sup.2 40-800 mg/m.sup.2
2-20 mg/m.sup.2 Cancer every 2 wk; every 4 wk; every wk; 60, 120,
200, 40, 100, 200, 2, 4, 6, 8, 10, 12, 400, 600, 800, 300, 400,
500, 14, 16, 18, 20 mg/m.sup.2 100, 1200 mg/m.sup.2 600, 700, 800
mg/m.sup.2 every wk; every 2 wk every 4 wk; 4-50 mg/m.sup.2 2-20
mg/m.sup.2/d for every 3 wk; 21 d every 6 wk; 4, 10, 15, 20, 25, 2,
4, 8, 12, 16, 20 mg/m.sup.2/d 30, 35, 40, 45, 50 mg/m.sup.2 for 21
d every 3 wk; every 6 wk 3-50 mg/m.sup.2 every 4 wk; 3, 6, 10, 15,
20, 25, 30, 35, 40, 45, 50 mg/m.sup.2 every 4 wk Colon 60-2200
mg/m.sup.2 2-20 mg/m.sup.2/d for 3-30 mg/m.sup.2 Cancer every wk;
20 d; every wk; 60, 120, 200, 2, 4, 8, 12, 16, 20 mg/m.sup.2/d 3,
6, 10, 15, 20, 400, 600, 800, for 20 d 25, 30 mg/m.sup.2 1000,
1200, every wk; 1400, 1600, 1-15 mg/m.sup.2/d for 1800, 2000, 2200
mg/m.sup.2 4 d; every wk 1, 3, 5, 7, 9, 11, 13, 15 mg/m.sup.2/d for
4 d Pancreatic 100-1500 mg/m.sup.2 10-100 mg/m.sup.2 4-40
mg/m.sup.2 Cancer every wk; every wk; every 3 wk; 100, 300, 500,
10, 20, 30, 40, 4, 8, 12, 16, 20, 700, 900, 1100, 50, 60, 70, 80,
24, 28, 32, 36, 40 mg/m.sup.2 1300, 1500 mg/m.sup.2 90, 100
mg/m.sup.2 every 3 wk; every wk every wk; 2-40 mg/m.sup.2 30-300
mg/m.sup.2 every 4 wk; every 3 wk; 2, 4, 8, 12, 16, 30, 60, 90,
120, 20, 24, 28, 32, 150, 180, 210, 36, 40 mg/m.sup.2 240, 270, 300
mg/m.sup.2 every 4 wk every 3 wk; 20-200 mg/m.sup.2 every 8 wk; 20,
40, 60, 80, 100, 120, 140, 160, 180, 200 mg/m.sup.2 every 8 wk
Head/Neck 50-1800 mg/m.sup.2 10-90 mg/m.sup.2 2-20 mg/m.sup.2
Cancer every wk; every wk; every wk; 50, 150, 300, 10, 20, 30, 40,
2, 4, 6, 8, 10, 12, 600, 900, 1200, 50, 60, 70, 80, 90 mg/m.sup.2
14, 16, 18, 20 mg/m.sup.2 1500, 1800 mg/m.sup.2 every wk; every wk;
every wk 10-70 mg/m.sup.2/d 5-75 mg/m.sup.2 for 5 d every 4 wk;
every 3 wk; 10, 20, 30, 40, 5, 15, 25, 35, 45, 50, 60, 70
mg/m.sup.2/d 55, 65, 75 mg/m.sup.2 for 5 d every 3 wk; every 4 wk
5-30 mg/m.sup.2 every 4 wk; 5, 10, 15, 20, 25, 30 mg/m.sup.2 every
4 wk; 5-30 mg/m.sup.2/d for 3 d every 4 wk 5, 10, 15, 20, 25, 30
mg/m.sup.2/d for 3 d every 4 wk Non-Small 75-1500 mg/m.sup.2 4-40
mg/m.sup.2/d for 5-55 mg/m.sup.2 Cell Lung every wk; 33 d; every 2
wk; Cancer 75, 150, 300, 4, 10, 15, 20, 25, 5, 10, 15, 20, 25, 600,
900, 1200, 30, 35, 40 mg/m.sup.2/d 30, 35, 40, 45, 1500 mg/m.sup.2
for 33 d 50, 55 mg/m.sup.2 every wk every 2 wk; 5-50 mg/m.sup.2
every 3 wk; 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 mg/m.sup.2 every
3 wk; 5-30 mg/m.sup.2 every 4 wk; 5, 10, 15, 20, 25, 30 mg/m.sup.2
every 4 wk Melanoma 80-1000 mg/m.sup.2 40-400 mg/m.sup.2 5-50
mg/m.sup.2 every wk; every 3 wk; every 3 wk; 80, 150, 300, 40, 80,
120, 160, 5, 10, 15, 20, 25, 500, 750, 1000 mg/m.sup.2 200, 240,
280, 30, 35, 40, 45, 50 mg/m.sup.2 every wk 320, 360, 400
mg/m.sup.2 every 3 wk every 3 wk; 30-400 mg/m.sup.2 every 4 wk; 30,
50, 100, 150, 200, 250, 300, 350, 400 mg/m.sup.2 every 4 wk Ovarian
60-1250 mg/m.sup.2 30-360 mg/m.sup.2 4-50 mg/m.sup.2 Cancer every
wk; every 4 wk; every wk; 60, 120, 250, 30, 60, 90, 120, 4, 10, 15,
20, 25, 500, 750, 1000, 150, 180, 210, 30, 35, 40, 45, 50
mg/m.sup.2 1250 mg/m.sup.2 240, 270, 300, every wk; every wk; 330,
360 mg/m.sup.2 5-75 mg/m.sup.2 80-2000 mg/m.sup.2 every 4 wk every
3 wk; every 2 wk; 5, 15, 25, 35, 45, 80, 2090, 400, 55, 65, 75
mg/m.sup.2 600, 800, 1000, every 3 wk; 1200, 1400, 5-30
mg/m.sup.2/d for 1600, 1800, 2000 mg/m.sup.2 3 d every 4 wk; every
2 wk 5, 10, 15, 20, 25, 30 mg/m.sup.2/d for 3 d every 4 wk; 5-40
mg/m.sup.2/d for 4 d; 5, 10, 15, 20, 25, 30, 35, 40 mg/m.sup.2/d
for 4 d Lymphoma 80-1250 mg/m.sup.2 C-100-1500 mg/m.sup.2; 30-300
mg/m.sup.2 2-20 mg/m.sup.2 every wk; H-10-70 mg/m.sup.2; every 3
wk; every wk; 80, 150, 250, O-1-2 mg/m.sup.2; 30, 60, 90, 120, 2,
4, 6, 8, 10, 12, 500, 750, 1000, P-10-100 mg; 150, 180, 210, 14,
16, 18, 20 mg/m.sup.2 1250 mg/m.sup.2 C-100, 300, 500, 240, 270,
300 mg/m.sup.2 every wk; every wk; 700, 900, 1100, every 3 wk; 5-75
mg/m.sup.2 1500-2000 mg/m.sup.2 1300, 1500 mg/m.sup.2; 30-400
mg/m.sup.2 every 3 wk; every 2 wk; H-10, 30, 50, 70 mg/m.sup.2;
every 4 wk; 5, 15, 25, 35, 45, 1500, 1600, O-1, 1.2, 1.4, 30, 50,
100, 150, 55, 65, 75 mg/m.sup.2 1700, 1800, 1.6, 1.8, 2 mg/m.sup.2;
200, 250, 300, every 3 wk; 1900, 2000 mg/m.sup.2 P-10, 20, 30, 40,
350, 400 mg/m.sup.2 5-80 mg/m.sup.2 every 2 wk; 50, 60, 70, 80,
every 4 wk every 4 wk; 2-10 mg/m.sup.2/min 90, 100 mg 5, 10, 15,
20, 25, for 12 hr; 30, 35, 40, 45, 2, 4, 6, 8, 10 mg/m.sup.2/min
50, 55, 60, 65, for 70, 75, 80 mg/m.sup.2 12 hr every 4 wk; 3-30
mg/m.sup.2/d for 3 d every 4 wk; 3, 6, 10, 15, 20, 25, 30
mg/m.sup.2/d for 3 d every 4 wk Hepatoma 70-700 mg/m.sup.2 2-20
mg/m.sup.2 every 3 wk; every wk; 70, 150, 300, 2, 4, 6, 8, 10, 12,
450, 600, 700 mg/m.sup.2 14, 16, 18, 20 mg/m.sup.2 every 3 wk;
every wk; 25-560 mg/m.sup.2 4-60 mg/m.sup.2 every 4 wk; every 3 wk;
25, 50, 100, 150, 4, 10, 15, 20, 25, 200, 250, 300, 30, 35, 40, 45,
350, 400, 450, 50, 55, 60 mg/m.sup.2 500, 560 mg/m.sup.2 every 3
wk; every 4 wk 3-50 mg/m.sup.2 every 4 wk; 3, 5, 10, 15, 20, 25,
30, 35, 40, 45, 50 mg/m.sup.2 every 4 wk Sarcoma 50-500 mg/m.sup.2
3-60 mg/m.sup.2 every 3 wk; every 3 wk; 50, 100, 150, 3, 5, 10, 15,
20, 200, 250, 300, 25, 30, 35, 40, 350, 400, 450, 45, 50, 55, 60
mg/m.sup.2 500 mg/m.sup.2 every every 3 wk; 3 wk; 5-75 mg/m.sup.2
30-300 mg/m.sup.2 every 4 wk; every 4 wk; 5, 15, 25, 35, 45, 30,
60, 90, 120, 55, 65, 75 mg/m.sup.2 150, 180, 210, every 4 wk; 240,
270, 300 mg/m.sup.2 2-20 mg/m.sup.2/d for every 4 wk; 3 d; 30-300
mg/m.sup.2/d 2, 4, 6, 8, 10, 12, for 4 d; 14, 16, 18, 20
mg/m.sup.2/d 30, 60, 90, 120, for 3 d; 150, 180, 210, 1-4
mg/m.sup.2/d for 240, 270, 300 mg/m.sup.2/d 4 d; for 4 d 1, 1.5, 2,
2.5, 3, 3.5, 4 mg/m.sup.2/d for 4 d Chronic C-100-1500 mg/m.sup.2;
5-50 mg/m.sup.2; Lymphocytic H-10-70 mg/m.sup.2; 5, 10, 15, 20, 25,
Leukemia O-1-2 mg/m.sup.2; 30, 35, 40, 45, 50 mg/m.sup.2; P-10-100
mg; 3-36 mg/m.sup.2/d for C-100, 300, 500, 4 d every 3 wk 700, 900,
1100, 3, 6, 9, 12, 15, 1300, 1500 mg/m.sup.2; 18, 21, 24, 27, H-10,
30, 50, 70 mg/m.sup.2; 30, 33, 36 mg/m.sup.2/d O-1, 1.2, 1.4, for 4
d 1.6, 1.8, 2 mg/m.sup.2; every 3 wk P-10, 20, 30, 40, 50, 60, 70,
80, 90, 100 mg Acute 15-150 mg/m.sup.2/d 2-25 mg/m.sup.2
Myelogenous for 3 d every wk; every wk; Leukemia 15, 30, 45, 60, 2,
5, 10, 15, 20, 75, 90, 105, 120, 25 mg/m.sup.2 every 135, 150
mg/m.sup.2/d wk; for 3 d 2-25 mg/m.sup.2/d for every wk; 3 d;
30-315 mg/m.sup.2/d 2, 5, 10, 15, 20, for 5 d every 2 wk; 25
mg/m.sup.2/d for 3 d; 30, 60, 90, 120, 5-50 mg/m.sup.2; 150, 180,
210, 5, 10, 15, 20, 25, 240, 270, 315 mg/m.sup.2/d 30, 35, 40, 45,
50 mg/m.sup.2 for 5 d every 2 wk; 20-216 mg/m.sup.2/d for 5 d; 20,
50, 80, 110, 140, 170, 216 mg/m.sup.2/d for 5 d Multiple 10-200
mg/m.sup.2/d 3-30 mg/m.sup.2 Myeloma for 4 d; every 3 wk; 10, 25,
50, 75, 3, 6, 10, 15, 20, 100, 125, 150, 25, 30 mg/m.sup.2 175, 200
mg/m.sup.2/d every 3 wk; for 4 d; 3-50 mg/m.sup.2; 40-400
mg/m.sup.2 3, 6, 10, 15, 20, every 4 wk; 25, 30, 35, 40,
40, 80, 120, 160, 45, 50 mg/m.sup.2 200, 240, 280, 320, 360, 400
mg/m.sup.2 every 4 wk
TABLE-US-00004 TABLE 4 Oxaliplatin Bortezomib Gefitinib Bevacizumab
Colon 10-85 mg/m.sup.2 5-10 mg/kg Cancer every 2 wk; every 2 wk;
10, 15, 20, 25, 5, 6, 7, 8, 9, 10 mg/kg 30, 35, 40, 45, every 2 wk
50, 55, 60, 65, 70, 75, 80, 85 mg/m.sup.2 every 2 wk; 10-130
mg/m.sup.2 every 3 wk; 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,
110, 120, 130 mg/m.sup.2 every 3 wk Pancreatic 10-100 mg/m.sup.2
Cancer every wk; 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/m.sup.2
every wk; 8, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85 mg/m.sup.2 every 2 wk; 8-85 mg/m.sup.2 every 2 wk Head/Neck
5-60 mg/m.sup.2 25-500 mg/d; Cancer every wk; 25, 50, 75, 100, 5,
10, 15, 20, 25, 150, 200, 250, 30, 35, 40, 45, 300, 350, 400, 50,
55, 60 mg/m.sup.2 450, 500 mg/d every wk Non-Small 5-65 mg/m.sup.2
25-500 mg/d; Cell Lung every wk; 25, 50, 75, 100, Cancer 5, 10, 15,
20, 25, 150, 200, 250, 30, 35, 40, 45, 300, 350, 400, 50, 55, 60,
65 mg/m.sup.2 450, 500 mg/d every wk; 10-130 mg/m.sup.2 every 3 wk;
10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 mg/m.sup.2
every 3 wk Ovarian 10-130 mg/m.sup.2 Cancer every wk; 10, 20, 30,
40, 50, 60, 70, 80, 90, 100, 110, 120, 130 mg/m.sup.2 every wk
Lymphoma 0.2-1.04 mg/m.sup.2 2x wk; 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1.04 mg/m.sup.2 2x wk Multiple 0.1-1.3 mg/m.sup.2 2x
Myeloma wk; 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1,
1.2, 1.3 mg/m.sup.2 2x wk
[0239] Since racemic gossypol achieves significant inhibition of
tumor growth at a daily dose of about 30 mg/kg, this dose level was
initially selected for testing gossypol in combination with
conventional anticancer therapeutics. A group of 10 mice received
an orally administered daily dose of 30 mg/kg gossypol starting at
day 7 and lasting for 4 weeks. Also starting at day 7, the same 10
mice were administered a weekly i.p. dose (7.5 mg/kg) of docetaxel
for 3 weeks. The results of this experiment are shown in FIG. 15.
In particular, FIG. 15 shows the inhibition of tumor growth by
gossypol, or by gossypol and docetaxel in human breast cancer
xenograft MDA-MB-231 nude mice. Each experimental group had 10
animals. FIG. 15 shows that administration of gossypol alone (30
mg/kg daily), or docetaxel alone in a sub-optimal dose (7.5 mg/kg
weekly), significantly inhibited tumor growth in the test animals,
however, test animals that received a combination therapy of
gossypol and docetaxel showed even greater tumor growth inhibition.
Importantly, 3 out of 10 mice (6 tumors) treated with a combination
gossypol and docetaxel showed complete tumor regression. Overall,
there was more than 90% inhibition in tumor growth in the
combination therapy group as compared to the control group.
Statistical analyses were performed using the SAS (See, G. Verbeke
and G. Molenberghs, Linear mixed models in practice: An
SAS-orientated approach, Springer-Verlag, vol. 126 (1997)) program.
Results of these experiments are provided in Table 5.
TABLE-US-00005 TABLE 5 Control Gossypol Docetaxel Racemic Gossypol
0.008* (0.06.sup..dagger-dbl.) Docetaxel 0.003 (0.01) Racemic
Gossypol + 0.00001 0.005 0.01 Docetaxel (0.0000004) (0.009) (0.002)
*day 41 .sup..dagger-dbl.day 47
[0240] The results show that the anticancer activity of gossypol
and docetaxel is statistically significant as compared to controls
at both day 41 and day 47 of the study. Furthermore, the anticancer
activity of the combination of gossypol and docetaxel is
significant as compared to the control (untreated) groups, and to
the groups treated with gossypol or docetaxel alone. Taken
together, these data indicate that gossypol has a significant
anticancer activity alone, but in some embodiments achieves even
greater activity when administered in combination with a
conventional anticancer agent (e.g., chemotherapeutic such as
docetaxel).
[0241] Although an understanding of any mechanism is not necessary
to practice the present invention and the present invention is not
so limited, it is contemplated that the synergistic effects
observed in some combinations of conventional anticancer agents and
gossypol compounds are due to a similarity of the compounds'
mechanisms of action (e.g., induction of apoptosis). For example,
in one embodiment (-)-gossypol enantiomer co-administered with the
conventional anticancer therapeutic agent TAXOL provides a
synergistic benefit. FIG. 16 shows the synergistic effects of
co-administration of (-)-gossypol enantiomer and TAXOL. Briefly,
this experiment used (-)-gossypol or (+)-gossypol in a fixed ratio
to TAXOL in a MCF-7 breast cancer cell line ((-)-gossypol IC.sub.50
8.71 .mu.M; (+)-gossypol IC.sub.50 22.88 .mu.M; TAXOL+(-)-gossypol
IC.sub.50 2.755 .mu.M; and TAXOL+(+)-gossypol IC.sub.50 10.57
.mu.M). FIGS. 17A and 17B show that there is a strong synergy
between (-)-gossypol and TAXOL as well as between (+)-gossypol and
TAXOL.
VII. Therapeutic Agents Combined or Co-Administered with Gossypol
Compounds
[0242] A wide range of therapeutic agents find use with the present
invention. Any therapeutic agent that can be co-administered with
gossypol compounds, or associated with gossypol compounds is
suitable for use in the methods of the present invention.
[0243] Some embodiments of the present invention provide methods
for administering an effective amount of gossypol (acids,
enantiomers, isomers, metabolites, derivatives, and
pharmaceutically acceptable salts thereof) and at least one
additional non-gossypol therapeutic agent (e.g., including, but not
limited to, chemotherapeutic antineoplastics, antimicrobials,
antivirals, antifungals, and anti-inflammatory agents) and/or
therapeutic technique (e.g., surgical intervention,
radiotherapies). In some of these embodiments, the subject has a
disease characterized by the intracellular overexpression of Bcl-2
family proteins (e.g., Bcl-2 and/or Bcl-X.sub.L).
[0244] Various classes of antineoplastic (e.g., anticancer) agents
are contemplated for use in certain embodiments of the present
invention. Anticancer agents suitable for use with the present
invention include, but are not limited to, agents that induce
apoptosis, agents that inhibit adenosine deaminase function,
inhibit pyrimidine biosynthesis, inhibit purine ring biosynthesis,
inhibit nucleotide interconversions, inhibit ribonucleotide
reductase, inhibit thymidine monophosphate (TMP) synthesis, inhibit
dihydrofolate reduction, inhibit DNA synthesis, form adducts with
DNA, damage DNA, inhibit DNA repair, intercalate with DNA,
deaminate asparagines, inhibit RNA synthesis, inhibit protein
synthesis or stability, inhibit microtubule synthesis or function,
and the like.
[0245] In some embodiments, exemplary anticancer agents suitable
for use in compositions and methods of the present invention
include, but are not limited to: 1) alkaloids, including
microtubule inhibitors (e.g., vincristine, vinblastine, and
vindesine, etc.), microtubule stabilizers (e.g., paclitaxel
(TAXOL), and docetaxel, etc.), and chromatin function inhibitors,
including topoisomerase inhibitors, such as epipodophyllotoxins
(e.g., etoposide (VP-16), and teniposide (VM-26), etc.), and agents
that target topoisomerase I (e.g., camptothecin and isirinotecan
(CPT-11), etc.); 2) covalent DNA-binding agents (alkylating
agents), including nitrogen mustards (e.g., mechlorethamine,
chlorambucil, cyclophosphamide, ifosphamide, and busulfan
(MYLERAN), etc.), nitrosoureas (e.g., carmustine, lomustine, and
semustine, etc.), and other alkylating agents (e.g., dacarbazine,
hydroxymethylmelamine, thiotepa, and mitomycin, etc.); 3)
noncovalent DNA-binding agents (antitumor antibiotics), including
nucleic acid inhibitors (e.g., dactinomycin (actinomycin D), etc.),
anthracyclines (e.g., daunorubicin (daunomycin, and cerubidine),
doxorubicin (adriamycin), and idarubicin (idamycin), etc.),
anthracenediones (e.g., anthracycline analogues, such as
mitoxantrone, etc.), bleomycins (BLENOXANE), etc., and plicamycin
(mithramycin), etc.; 4) antimetabolites, including antifolates
(e.g., methotrexate, FOLEX, and MEXATE, etc.), purine
antimetabolites (e.g., 6-mercaptopurine (6-MP, PURINETHOL),
6-thioguanine (6-TG), azathioprine, acyclovir, ganciclovir,
chlorodeoxyadenosine, 2-chlorodeoxyadenosine (CdA), and
2'-deoxycoformycin (pentostatin), etc.), pyrimidine antagonists
(e.g., fluoropyrimidines (e.g., 5-fluorouracil (ADRUCIL),
5-fluorodeoxyuridine (FdUrd) (floxuridine)) etc.), and cytosine
arabinosides (e.g., CYTOSAR (ara-C) and fludarabine, etc.); 5)
enzymes, including L-asparaginase, and hydroxyurea, etc.; 6)
hormones, including glucocorticoids, antiestrogens (e.g.,
tamoxifen, etc.), nonsteroidal antiandrogens (e.g., flutamide,
etc.), and aromatase inhibitors (e.g., anastrozole (ARIMIDEX),
etc.); 7) platinum compounds (e.g., cisplatin and carboplatin,
etc.); 8) monoclonal antibodies conjugated with anticancer drugs,
toxins, and/or radionuclides, etc.; 9) biological response
modifiers (e.g., interferons (e.g., IFN-.alpha., etc.) and
interleukins (e.g., IL-2, etc.), etc.); 10) adoptive immunotherapy;
11) hematopoietic growth factors; 12) agents that induce tumor cell
differentiation (e.g., all-trans-retinoic acid, etc.); 13) gene
therapy techniques; 14) antisense therapy techniques; 15) tumor
vaccines; 16) therapies directed against tumor metastases (e.g.,
batimastat, etc.); 17) angiogenesis inhibitors; 18) proteosome
inhibitors (e.g., VELCADE); 19) inhibitors of acetylation and/or
methylation (e.g., HDAC inhibitors); 20) modulators of NF kappa B;
21) inhibitors of cell cycle regulation (e.g., CDK inhibitors); 22)
modulators of p53 protein function; and 23) radiation.
[0246] Any oncolytic agent that is routinely used in a cancer
therapy context finds use in the compositions and methods of the
present invention. For example, the U.S. Food and Drug
Administration maintains a formulary of oncolytic agents approved
for use in the United States. International counterpart agencies to
the U.S.F.D.A. maintain similar formularies. Table 6 provides a
list of exemplary antineoplastic agents approved for use in the
U.S. Those skilled in the art will appreciate that the "product
labels" required on all U.S. approved chemotherapeutics describe
approved indications, closing information, toxicity data, and the
like, for the exemplary agents.
TABLE-US-00006 TABLE 6 Aldesleukin Proleukin Chiron Corp.,
(des-alanyl-1, serine-125 human interleukin-2) Emeryville, CA
Alemtuzumab Campath Millennium and (IgG1.kappa. anti CD52 antibody)
ILEX Partners, LP, Cambridge, MA Alitretinoin Panretin Ligand
(9-cis-retinoic acid) Pharmaceuticals, Inc., San Diego CA
Allopurinol Zyloprim GlaxoSmithKline,
(1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one Research Triangle
monosodium salt) Park, NC Altretamine Hexalen US Bioscience, West
(N,N,N',N',N'',N'',-hexamethyl-1,3,5-triazine-2,4, Conshohocken, PA
6-triamine) Amifostine Ethyol US Bioscience (ethanethiol,
2-[(3-aminopropyl)amino]-, dihydrogen phosphate (ester))
Anastrozole Arimidex AstraZeneca (1,3-Benzenediacetonitrile,
a,a,a',a'-tetramethyl- Pharmaceuticals, LP,
5-(1H-1,2,4-triazol-1-ylmethyl)) Wilmington, DE Arsenic trioxide
Trisenox Cell Therapeutic, Inc., Seattle, WA Asparaginase Elspar
Merck & Co., Inc., (L-asparagine amidohydrolase, type EC-2)
Whitehouse Station, NJ BCG Live TICE BCG Organon Teknika,
(lyophilized preparation of an attenuated strain of Corp., Durham,
NC Mycobacterium bovis (Bacillus Calmette-Gukin [BCG], substrain
Montreal) bexarotene capsules Targretin Ligand
(4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2- Pharmaceuticals
napthalenyl) ethenyl] benzoic acid) bexarotene gel Targretin Ligand
Pharmaceuticals Bleomycin Blenoxane Bristol-Myers Squibb (cytotoxic
glycopeptide antibiotics produced by Co., NY, NY Streptomyces
verticillus; bleomycin A.sub.2 and bleomycin B.sub.2) Capecitabine
Xeloda Roche (5'-deoxy-5-fluoro-N-[(pentyloxy)carbonyl]- cytidine)
Carboplatin Paraplatin Bristol-Myers Squibb (platinum, diammine
[1,1- cyclobutanedicarboxylato(2-)-0,0']-,(SP-4-2)) Carmustine
BCNU, BiCNU Bristol-Myers Squibb
(1,3-bis(2-chloroethyl)-1-nitrosourea) Carmustine with Polifeprosan
20 Implant Gliadel Wafer Guilford Pharmaceuticals, Inc., Baltimore,
MD Celecoxib Celebrex Searle (as
4-[5-(4-methylphenyl)-3-(trifluoromethyl)- Pharmaceuticals,
1H-pyrazol-1-yl] England benzenesulfonamide) Chlorambucil Leukeran
GlaxoSmithKline (4-[bis(2chlorethyl)amino]benzenebutanoic acid)
Cisplatin Platinol Bristol-Myers Squibb (PtCl.sub.2H.sub.6N.sub.2)
Cladribine Leustatin, 2-CdA R. W. Johnson
(2-chloro-2'-deoxy-b-D-adenosine) Pharmaceutical Research
Institute, Raritan, NJ Cyclophosphamide Cytoxan, Neosar
Bristol-Myers Squibb (2-[bis(2-chloroethyl)amino]
tetrahydro-2H-13,2- oxazaphosphorine 2-oxide monohydrate)
Cytarabine Cytosar-U Pharmacia & Upjohn
(1-b-D-Arabinofuranosylcytosine, C.sub.9H.sub.13N.sub.3O.sub.5)
Company cytarabine liposomal DepoCyt Skye Pharmaceuticals, Inc.,
San Diego, CA Dacarbazine DTIC-Dome Bayer AG,
(5-(3,3-dimethyl-1-triazeno)-imidazole-4- Leverkusen, carboxamide
(DTIC)) Germany Dactinomycin, actinomycin D Cosmegen Merck
(actinomycin produced by Streptomyces parvullus,
C.sub.62H.sub.86N.sub.12O.sub.16) Darbepoetin alfa Aranesp Amgen,
Inc., (recombinant peptide) Thousand Oaks, CA daunorubicin
liposomal DanuoXome Nexstar
((8S-cis)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-a- Pharmaceuticals,
Inc., L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro- Boulder, CO
6,8,11-trihydroxy-1-methoxy-5,12- naphthacenedione hydrochloride)
Daunorubicin HCl, daunomycin Cerubidine Wyeth Ayerst,
((1S,3S)-3-Acetyl-1,2,3,4,6,11-hexahydro- Madison, NJ
3,5,12-trihydroxy-10-methoxy-6,11-dioxo-1- naphthacenyl
3-amino-2,3,6-trideoxy-(alpha)-L- lyxo-hexopyranoside
hydrochloride) Denileukin diftitox Ontak Seragen, Inc.,
(recombinant peptide) Hopkinton, MA Dexrazoxane Zinecard Pharmacia
& Upjohn ((S)-4,4'-(1-methyl-1,2-ethanediyl)bis-2,6- Company
piperazinedione) Docetaxel Taxotere Aventis
((2R,3S)--N-carboxy-3-phenylisoserine, N-tert- Pharmaceuticals,
Inc., butyl ester, 13-ester with 5b-20-epoxy- Bridgewater, NJ
12a,4,7b,10b,13a-hexahydroxytax-11-en-9-one 4- acetate 2-benzoate,
trihydrate) Doxorubicin HCl Adriamycin, Pharmacia & Upjohn
(8S,10S)-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo- Rubex Company
hexopyranosyl)oxy]-8-glycolyl-7,8,9,10-
tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12- naphthacenedione
hydrochloride) doxorubicin Adriamycin PFS Pharmacia & Upjohn
Intravenous Company injection doxorubicin liposomal Doxil Sequus
Pharmaceuticals, Inc., Menlo park, CA dromostanolone propionate
Dromostanolone Eli Lilly & Company,
(17b-Hydroxy-2a-methyl-5a-androstan-3-one Indianapolis, IN
propionate) dromostanolone propionate Masterone Syntex, Corp., Palo
injection Alto, CA Elliott's B Solution Elliott's B Orphan Medical,
Inc Solution Epirubicin Ellence Pharmacia & Upjohn
((8S-cis)-10-[(3-amino-2,3,6-trideoxy-a-L- Company
arabino-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-
6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-
5,12-naphthacenedione hydrochloride) Epoetin alfa Epogen Amgen, Inc
(recombinant peptide) Estramustine Emcyt Pharmacia & Upjohn
(estra-1,3,5(10)-triene-3,17-diol(17(beta))-,3- Company
[bis(2-chloroethyl)carbamate] 17-(dihydrogen phosphate), disodium
salt, monohydrate, or estradiol 3-[bis(2-chloroethyl)carbamate] 17-
(dihydrogen phosphate), disodium salt, monohydrate) Etoposide
phosphate Etopophos Bristol-Myers Squibb
(4'-Demethylepipodophyllotoxin 9-[4,6-O--(R)-
ethylidene-(beta)-D-glucopyranoside], 4'- (dihydrogen phosphate))
etoposide, VP-16 Vepesid Bristol-Myers Squibb
(4'-demethylepipodophyllotoxin 9-[4,6-0-(R)-
ethylidene-(beta)-D-glucopyranoside]) Exemestane Aromasin Pharmacia
& Upjohn (6-methylenandrosta-1,4-diene-3,17-dione) Company
Filgrastim Neupogen Amgen, Inc (r-metHuG-CSF) floxuridine
(intraarterial) FUDR Roche (2'-deoxy-5-fluorouridine) Fludarabine
Fludara Berlex Laboratories, (fluorinated nucleotide analog of the
antiviral Inc., Cedar Knolls, agent vidarabine,
9-b-D-arabinofuranosyladenine NJ (ara-A)) Fluorouracil, 5-FU
Adrucil ICN Pharmaceuticals, (5-fluoro-2,4(1H,3H)-pyrimidinedione)
Inc., Humacao, Puerto Rico Fulvestrant Faslodex IPR
Pharmaceuticals, (7-alpha-[9-(4,4,5,5,5-penta
fluoropentylsulphinyl) Guayama, Puerto
nonyl]estra-1,3,5-(10)-triene-3,17-beta-diol) Rico Gemcitabine
Gemzar Eli Lilly (2'-deoxy-2',2'-difluorocytidine monohydrochloride
(b-isomer)) Gemtuzumab Ozogamicin Mylotarg Wyeth Ayerst (anti-CD33
hP67.6) Goserelin acetate Zoladex Implant AstraZeneca (acetate salt
of [D-Ser(But).sup.6,Azgly.sup.10]LHRH; pyro- Pharmaceuticals
Glu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro- Azgly-NH2 acetate
[C.sub.59H.sub.84N.sub.18O.sub.14.cndot.(C.sub.2H.sub.4O.sub.2).sub.x
Hydroxyurea Hydrea Bristol-Myers Squibb Ibritumomab Tiuxetan
Zevalin Biogen IDEC, Inc., (immunoconjugate resulting from a
thiourea Cambridge MA covalent bond between the monoclonal antibody
Ibritumomab and the linker-chelator tiuxetan [N-
[2-bis(carboxymethyl)amino]-3-(p-
isothiocyanatophenyl)-propyl]-[N-[2-
bis(carboxymethyl)amino]-2-(methyl)- ethyl]glycine) Idarubicin
Idamycin Pharmacia & Upjohn (5,12-Naphthacenedione,
9-acetyl-7-[(3-amino- Company 2,3,6-trideoxy-(alpha)-L-lyxo-
hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,9,11-
trihydroxyhydrochloride, (7S-cis)) Ifosfamide IFEX Bristol-Myers
Squibb (3-(2-chloroethyl)-2-[(2-
chloroethyl)amino]tetrahydro-2H-1,3,2- oxazaphosphorine 2-oxide)
Imatinib Mesilate Gleevec Novartis AG, Basel,
(4-[(4-Methyl-1-piperazinyl)methyl]-N-[4-methyl- Switzerland
3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]- phenyl]benzamide
methanesulfonate) Interferon alfa-2a Roferon-A Hoffmann-La Roche,
(recombinant peptide) Inc., Nutley, NJ Interferon alfa-2b Intron A
Schering AG, Berlin, (recombinant peptide) (Lyophilized Germany
Betaseron) Irinotecan HCl Camptosar Pharmacia & Upjohn
((4S)-4,11-diethyl-4-hydroxy-9-[(4-piperidinopiperidino)carbonyloxy]-
Company 1H-pyrano[3',4':6,7] indolizino[1,2-b]
quinoline-3,14(4H,12H) dione hydrochloride trihydrate) Letrozole
Femara Novartis (4,4'-(1H-1,2,4-Triazol-1-ylmethylene)
dibenzonitrile) Leucovorin Wellcovorin, Immunex, Corp., (L-Glutamic
acid, N[4[[(2amino-5-formyl- Leucovorin Seattle, WA 1,4,5,6,7,8
hexahydro4oxo6- pteridinyl)methyl]amino]benzoyl], calcium salt
(1:1)) Levamisole HCl Ergamisol Janssen Research
((-)-(S)-2,3,5,6-tetrahydro-6-phenylimidazo [2,1- Foundation, b]
thiazole monohydrochloride C.sub.11H.sub.12N.sub.2S.cndot.HCl)
Titusville, NJ Lomustine CeeNU Bristol-Myers Squibb
(1-(2-chloro-ethyl)-3-cyclohexyl-1-nitrosourea) Meclorethamine,
nitrogen mustard Mustargen Merck
(2-chloro-N-(2-chloroethyl)-N-methylethanamine hydrochloride)
Megestrol acetate Megace Bristol-Myers Squibb
17.alpha.(acetyloxy)-6-methylpregna-4,6-diene- 3,20-dione
Melphalan, L-PAM Alkeran GlaxoSmithKline (4-[bis(2-chloroethyl)
amino]-L-phenylalanine) Mercaptopurine, 6-MP Purinethol
GlaxoSmithKline (1,7-dihydro-6H-purine-6-thione monohydrate) Mesna
Mesnex Asta Medica (sodium 2-mercaptoethane sulfonate) Methotrexate
Methotrexate Lederle Laboratories (N-[4-[[(2,4-diamino-6-
pteridinyl)methyl]methylamino]benzoyl]-L- glutamic acid)
Methoxsalen Uvadex Therakos, Inc., Way
(9-methoxy-7H-furo[3,2-g][1]-benzopyran-7-one) Exton, Pa Mitomycin
C Mutamycin Bristol-Myers Squibb mitomycin C Mitozytrex SuperGen,
Inc., Dublin, CA Mitotane Lysodren Bristol-Myers Squibb
(1,1-dichloro-2-(o-chlorophenyl)-2-(p- chlorophenyl) ethane)
Mitoxantrone Novantrone Immunex (1,4-dihydroxy-5,8-bis[[2-[(2-
Corporation hydroxyethyl)amino]ethyl]amino]-9,10- anthracenedione
dihydrochloride) Nandrolone phenpropionate Durabolin-50 Organon,
Inc., West Orange, NJ Nofetumomab Verluma Boehringer Ingelheim
Pharma KG, Germany Oprelvekin Neumega Genetics Institute, (IL-11)
Inc., Alexandria, VA Oxaliplatin Eloxatin Sanofi Synthelabo,
(cis-[(1R,2R)-1,2-cyclohexanediamine-N,N'] Inc., NY, NY
[oxalato(2-)-O,O'] platinum)
Paclitaxel TAXOL Bristol-Myers Squibb
(5.beta.,20-Epoxy-1,2a,4,7.beta.,10.beta.,13a-
hexahydroxytax-11-en-9-one 4,10-diacetate 2- benzoate 13-ester with
(2R,3S)-N-benzoyl-3- phenylisoserine) Pamidronate Aredia Novartis
(phosphonic acid (3-amino-1-hydroxypropylidene) bis-, disodium
salt, pentahydrate, (APD)) Pegademase Adagen Enzon
((monomethoxypolyethylene glycol succinimidyl) (Pegademase
Pharmaceuticals, Inc., 11-17-adenosine deaminase) Bovine)
Bridgewater, NJ Pegaspargase Oncaspar Enzon
(monomethoxypolyethylene glycol succinimidyl L-asparaginase)
Pegfilgrastim Neulasta Amgen, Inc (covalent conjugate of
recombinant methionyl human G-CSF (Filgrastim) and
monomethoxypolyethylene glycol) Pentostatin Nipent Parke-Davis
Pharmaceutical Co., Rockville, MD Pipobroman Vercyte Abbott
Laboratories, Abbott Park, IL Plicamycin, Mithramycin Mithracin
Pfizer, Inc., NY, NY (antibiotic produced by Streptomyces plicatus)
Porfimer sodium Photofrin QLT Phototherapeutics, Inc., Vancouver,
Canada Procarbazine Matulane Sigma Tau
(N-isopropyl-.mu.-(2-methylhydrazino)-p-toluamide Pharmaceuticals,
Inc., monohydrochloride) Gaithersburg, MD Quinacrine Atabrine
Abbott Labs (6-chloro-9-(1-methyl-4-diethyl-amine)butylamino-
2-methoxyacridine) Rasburicase Elitek Sanofi-Synthelabo,
(recombinant peptide) Inc., Rituximab Rituxan Genentech, Inc.,
(recombinant anti-CD20 antibody) South San Francisco, CA
Sargramostim Prokine Immunex Corp (recombinant peptide)
Streptozocin Zanosar Pharmacia & Upjohn (streptozocin
2-deoxy-2- Company [[(methylnitrosoamino)carbonyl]amino]-a(and b)-
D-glucopyranose and 220 mg citric acid anhydrous) Talc Sclerosol
Bryan, Corp., (Mg.sub.3Si.sub.4O.sub.10(OH).sub.2) Woburn, MA
Tamoxifen Nolvadex AstraZeneca ((Z)2-[4-(1,2-diphenyl-1-butenyl)
phenoxy]-N,N- Pharmaceuticals dimethylethanamine 2-hydroxy-1,2,3-
propanetricarboxylate (1:1)) Temozolomide Temodar Schering
(3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-as-
tetrazine-8-carboxamide) teniposide, VM-26 Vumon Bristol-Myers
Squibb (4'-demethylepipodophyllotoxin 9-[4,6-0-(R)-2-
thenylidene-(beta)-D-glucopyranoside]) Testolactone Teslac
Bristol-Myers Squibb (13-hydroxy-3-oxo-13,17-secoandrosta-1,4-dien-
17-oic acid [dgr]-lactone) Thioguanine, 6-TG Thioguanine
GlaxoSmithKline (2-amino-1,7-dihydro-6H-purine-6-thione) Thiotepa
Thioplex Immunex (Aziridine, 1,1',1''-phosphinothioylidynetris-, or
Corporation Tris (1-aziridinyl) phosphine sulfide) Topotecan HCl
Hycamtin GlaxoSmithKline ((S)-10-[(dimethylamino)
methyl]-4-ethyl-4,9- dihydroxy-1H-pyrano[3',4':6,7] indolizino
[1,2-b] quinoline-3,14-(4H,12H)-dione monohydrochloride) Toremifene
Fareston Roberts (2-(p-[(Z)-4-chloro-1,2-diphenyl-1-butenyl]-
Pharmaceutical phenoxy)-N,N-dimethylethylamine citrate (1:1))
Corp., Eatontown, NJ Tositumomab, I 131 Tositumomab Bexxar Corixa
Corp., Seattle, (recombinant murine immunotherapeutic WA monoclonal
IgG.sub.2a lambda anti-CD20 antibody (I 131 is a
radioimmunotherapeutic antibody)) Trastuzumab Herceptin Genentech,
Inc (recombinant monoclonal IgG.sub.1 kappa anti-HER2 antibody)
Tretinoin, ATRA Vesanoid Roche (all-trans retinoic acid) Uracil
Mustard Uracil Mustard Roberts Labs Capsules Valrubicin,
N-trifluoroacetyladriamycin-14- Valstar Anthra --> Medeva
valerate ((2S-cis)-2-[1,2,3,4,6,11-hexahydro-2,5,12- trihydroxy-7
methoxy-6,11-dioxo-[[4 2,3,6-
trideoxy-3-[(trifluoroacetyl)-amino-.alpha.-L-lyxo-
hexopyranosyl]oxyl]-2-naphthacenyl]-2-oxoethyl pentanoate)
Vinblastine, Leurocristine Velban Eli Lilly
(C.sub.46H.sub.56N.sub.4O.sub.10.cndot.H.sub.2SO.sub.4) Vincristine
Oncovin Eli Lilly
(C.sub.46H.sub.56N.sub.4O.sub.10.cndot.H.sub.2SO.sub.4) Vinorelbine
Navelbine GlaxoSmithKline (3',4'-didehydro-4'-deoxy-C'-
norvincaleukoblastine [R--(R*,R*)-2,3- dihydroxybutanedioate
(1:2)(salt)]) Zoledronate, Zoledronic acid Zometa Novartis
((1-Hydroxy-2-imidazol-1-yl-phosphonoethyl) phosphonic acid
monohydrate)
[0247] Preferred conventional anticancer agents for use in
administration with the disclosed gossypol compounds include, but
are not limited to, adriamycin, 5-fluorouracil, etoposide,
camptothecin, actinomycin-D, mitomycin C, cisplatin, docetaxel,
gemcitabine, carboplatin, oxaliplatin, bortezomib, gefitinib, and
bevacizumab. These agents can be prepared and used singularly, in
combined therapeutic compositions, in kits, or in combination with
immunotherapeutic agents, and the like.
[0248] In preferred embodiments, the present invention provides
methods for the administration of effective amounts of gossypol
compounds and at least one conventional anticancer agent (e.g., an
agent that induces apoptosis). In some preferred embodiments, the
subject has a disease characterized by the overexpression of Bcl-2
family protein(s) (e.g., Bcl-2 and/or Bcl-X.sub.L). In yet other
preferred embodiments, the present invention provides methods for
the administration of effective amounts of gossypol compounds and a
taxane (e.g., docetaxel) compound to subjects having diseases
characterized by the overexpression of Bcl-2 family protein(s)
(e.g., Bcl-2 and/or Bcl-X.sub.L).
[0249] Generally, taxanes (e.g., docetaxel) are an effective class
of anticancer chemotherapeutic agents. (See e.g., K. D. Miller and
G. W. Sledge, Jr. Cancer Investigation, 17:121-136 (1999)). While
the present invention is not limited to any particular
mechanism(s), it is contemplated that taxane-mediated cell death
occurs through intracellular microtubule stabilization and
subsequent induction of the apoptotic pathway. (See e.g., S. Haldar
et al., Cancer Research, 57:229-233 (1997)). In many systems,
Bcl-X.sub.L functions as a negative control on this pathway.
[0250] In some other embodiments, cisplatin and TAXOL are
specifically contemplated for administration with gossypol
compounds. Cisplatin and TAXOL induce apoptosis in tumor cells.
(See e.g., Lanni et al., Proc. Natl. Acad. Sci., 94:9679 (1997);
Tortora et al., Cancer Research 57:5107 (1997); and Zaffaroni et
al., Brit. J. Cancer 77:1378 (1998)). However, treatment with these
and other chemotherapeutic agents is difficult to accomplish
without subjecting the patient to significant toxicity. Many
anticancer chemotherapeutic agents currently in use are generally
poorly water soluble, toxic, and when given at efficacious levels
affect normal cells as well as diseased cells.
[0251] For example, paclitaxel (TAXOL), is a very promising
anticancer compound, and has shown excellent antitumor activity in
a wide variety of tumor models such as the B16 melanoma, L1210
leukemias, MX-1 mammary tumors, and CS-1 colon tumor xenografts.
However, is has poor aqueous solubility which presents a problem in
human administration. Accordingly, paclitaxel formulations
typically require the use of a cremaphor to solubilize the drug.
The human clinical dose range of paclitaxel is about 110-500
mg/m.sup.2. For administration, paclitaxel is usually dissolved in
a solution of ethanol:cremaphor (1:1) then diluted into one liter
of water or other aqueous mixture. Polyethoxylated castor oil is
the most often used cremaphor. The cremaphor mixture is
administered by infusion. Direct administration (e.g.,
subcutaneous) of the cremaphor mixture results in local toxicity
and low levels of activity.
[0252] In still further embodiments, the present invention provides
methods for monitoring the therapeutic success of cisplatin and/or
TAXOL administration in a subject. Measuring the ability of these
drugs to induce apoptosis in vitro is reported to be a marker for
in vivo efficacy. (Gibb, Gynecologic Oncology, 65:13 (1997)). The
effectiveness of cisplatin and/or TAXOL as anticancer
chemotherapeutics can be measured using techniques of the present
invention for monitoring induction of apoptosis. Cisplatin and/or
TAXOL are active against a wide-range of tumor types including, but
not limited to, breast cancer and colon cancer. (Akutsu et al.,
Eur. J. Cancer 31A:2341 (1995)).
[0253] In some embodiments of the present invention, therapeutic
gossypol compound treatments further comprise one or more agents
that directly cross-link nucleic acids (e.g., DNA) to facilitate
DNA damage leading to a synergistic effect. Agents such as
cisplatin and other DNA alkylating agents are preferred. Cisplatin
has been widely used in cancer treatments. Efficacious doses used
in clinical applications include, but are not limited to, about 20
mg/m.sup.2 for 5 days every three weeks for a total of three
courses, and 50-120 mg/m.sup.2 every 3 weeks.
[0254] Agents that damage DNA also include compounds that interfere
with DNA replication, mitosis, and chromosomal segregation. Such
chemotherapeutic compounds include, but are not limited to,
adriamycin, also known as doxorubicin, etoposide, verapamil,
podophyllotoxin, and the like. These compounds are widely used in
clinical settings for the treatment of neoplasms, and are typically
administered as a bolus intravenous injection at doses ranging from
about 25-75 mg/m.sup.2 at 21 day intervals, 20-30 mg/m.sup.2 every
week, and similar doses for adriamycin, and 100-200 mg/m.sup.2 for
etoposide for three days every 3-4 weeks intravenously or double
the intravenous dose when administered orally.
[0255] Agents that disrupt the synthesis and fidelity of nucleic
acid precursors and subunits also lead to DNA damage and find use
as chemotherapeutic agents in the present invention. A number of
nucleic acid precursors have been developed. Particularly useful
are agents that have undergone extensive testing and are readily
available. 5-Fluorouracil (5-FU) is preferentially used by
neoplastic tissues, making this agent particularly useful for
targeting to neoplastic cells. The dose of 5-fluorouracil may range
from about 3 to 15 mg/kg/day, although other doses may vary
considerably according to various factors including stage of
disease, amenability of the cells to the therapy, amount of
resistance to the agent and the like.
[0256] In preferred embodiments, the anticancer agents used in the
present invention are those that are amenable to co-administration
with the disclosed gossypol compounds or are otherwise associated
with the disclosed gossypol compounds such that they can be
delivered into a subject, tissue, or cell without loss of fidelity
of anticancer effect. For a more detailed description of cancer
therapeutic agents such as a platinum complex, verapamil,
podophyllotoxin, carboplatin, procarbazine, mechlorethamine,
cyclophosphamide, camptothecin, ifosfamide, melphalan,
chlorambucil, busulfan, nitrosourea, dactinomycin, daunorubicin,
doxorubicin (adriamycin), bleomycin, plicomycin, mitomycin,
etoposide (VP16), tamoxifen, TAXOL, transplatinum, 5-fluorouracil,
vincristin, vinblastin and methotrexate and other similar
anti-cancer agents, those skilled in the art are referred to any
number of instructive manuals including, but not limited to, the
Physician's Desk Reference and to Goodman and Gilman's
"Pharmaceutical Basis of Therapeutics" ninth edition, Eds. Hardman
et al., 1996.
[0257] In some embodiments, the drugs are attached to the gossypol
compounds with photocleavable linkers. For example, several
heterobifunctional, photocleavable linkers that find use with the
present invention are described by Ottl et al. (Ottl et al.,
Bioconjugate Chem., 9:143 (1998)). These linkers can be either
water or organic soluble. They contain an activated ester that can
react with amines or alcohols and an epoxide that can react with a
thiol group. In between the two groups is
3,4-dimethoxy-6-nitrophenyl photoisomerization group, which, when
exposed to near-ultraviolet light (365 nm), releases the amine or
alcohol in intact form. Thus, the therapeutic agent, when linked to
the compositions of the present invention using such linkers, may
be released in biologically active or activatable form through
exposure of the target area to near-ultraviolet light.
[0258] In an exemplary embodiment, the alcohol group of TAXOL is
reacted with the activated ester of the organic-soluble linker.
This product in turn is reacted with the partially-thiolated
surface of appropriate dendrimers (the primary amines of the
dendrimers can be partially converted to thiol-containing groups by
reaction with a sub-stoichiometric amount of 2-iminothiolano). In
the case of cisplatin, the amino groups of the drug are reacted
with the water-soluble form of the linker. If the amino groups are
not reactive enough, a primary amino-containing active analog of
cisplatin, such as Pt(II) sulfadiazine dichloride (Pasani et al.,
Inorg. Chim. Acta 80:99 (1983) and Abel et al., Eur. J. Cancer 9:4
(1973)) can be used. Thus conjugated, the drug is inactive and will
not harm normal cells. When the conjugate is localized within tumor
cells, it is exposed to laser light of the appropriate near-UV
wavelength, causing the active drug to be released into the
cell.
[0259] Similarly, in other embodiments of the present invention,
the amino groups of cisplatin (or an analog thereof) are linked
with a very hydrophobic photocleavable protecting group, such as
the 2-nitrobenzyloxycarbonyl group (See e.g., Pillai, V. N. R.
Synthesis: 1-26 (1980)). When exposed to near-UV light (about 365
nm), the hydrophobic group is cleaved, leaving the intact drug. A
number of photocleavable linkers have been demonstrated as
effective anti-tumor conjugates and can be prepared by attaching
cancer therapeutics, such as doxorubicin, to water-soluble polymers
with appropriate short peptide linkers (See e.g., Vasey et al.,
Clin. Cancer Res., 5:83 (1999)). The linkers are stable outside of
the cell, but are cleaved by thiolproteases once within the cell.
In a preferred embodiment, the conjugate PK1 is used. As an
alternative to the photocleavable linker strategy,
enzyme-degradable linkers, such as Gly-Phe-Leu-Gly (SEQ ID NO: 3)
may be used. An alternative to photocleavable linkers are enzyme
cleavable linkers.
[0260] The present invention is not limited by the nature of the
therapeutic technique. For example, other conjugates that find use
with the present invention include, but are not limited to, using
conjugated boron dusters for BNCT (Capala et al., Bioconjugate
Chem., 7:7 (1996)), the use of radioisotopes, and conjugation of
toxins such as ricin.
[0261] Antimicrobial therapeutic agents may also be used as
therapeutic agents in the present invention. Any agent that can
kill, inhibit, or otherwise attenuate the function of microbial
organisms may be used, as well as any agent contemplated to have
such activities. Antimicrobial agents include, but are not limited
to, natural and synthetic antibiotics, antibodies, inhibitory
proteins (e.g., defensins), antisense nucleic acids, membrane
disruptive agents and the like, used alone or in combination.
Indeed, any type of antibiotic may be used including, but not
limited to, antibacterial agents, antiviral agents, antifungal
agents, and the like.
VIII. Targeting Agents and Techniques
[0262] In still further embodiments, the present invention provides
gossypol compounds (and any other chemotherapeutic agents)
associated with targeting agents (gossypol compound-targeting agent
complexes) that are able to specifically target particular cell
types (e.g., tumor cells). Generally, the gossypol compound that is
associated with a targeting agent, targets neoplastic cells through
interaction of the targeting agent with a cell surface moiety that
is taken into the cell through receptor mediated endocytosis.
[0263] Any moiety known to be located on the surface of target
cells (e.g., tumor cells) finds use with the present invention. For
example, an antibody directed against such a moiety targets the
compositions of the present invention to cell surfaces containing
the moiety. Alternatively, the targeting moiety may be a ligand
directed to a receptor present on the cell surface or vice versa.
Similarly, vitamins also may be used to target the therapeutics of
the present invention, to a particular cell.
[0264] As used herein, the term "targeting molecules" refers to
chemical moieties, and portions thereof useful for targeting
chemical compounds (e.g., gossypol compounds, drugs, prodrugs,
small molecules, therapeutic agents) to cells, tissues, and organs
of interest. Various types of targeting molecules are contemplated
for use with the present invention including, but not limited to,
signal peptides, antibodies, nucleic acids, toxins and the like.
Targeting moieties may additionally promote the binding of the
associated chemical compounds (e.g., small molecules) or the entry
of the compounds into the targeted cells, tissues, and organs.
Preferably, targeting moieties are selected according to their
specificity, affinity, and efficacy in selectively delivering
attached compounds to targeted sites within a subject, tissue, or a
cell, including specific subcellular locations and organelles.
[0265] In some preferred embodiments, the targeting molecules of
the present invention are associated with a therapeutic or other
small molecule (e.g., gossypol compound, drugs, prodrugs, small
molecules, therapeutic agents, etc.). Targeting molecules can be
associated to the therapeutic small molecules of the present
invention using a variety of linking (e.g., cleavable linkers),
spacer, and protecting groups. For example, in certain embodiments,
targeting moieties are associated (e.g., covalently or
noncovalently bound) to the small molecule therapeutic agents by
short (e.g., direct coupling), medium (e.g., using small-molecule
bifunctional linkers such as SPDP (Pierce Biotechnology, Inc.,
Rockford, Ill.)), or long (e.g., PEG bifunctional linkers (Nektar
Therapeutics, Inc., San Carlos, Calif.)) chemical linkages.
[0266] Preferably, the various targeting molecules and therapeutic
agents of the present invention are attached, associated, fixed, or
conjugated such that each entity therein is sufficiently free of
steric hindrance (e.g., via connection through a suitable linker)
such that its chemical or biological activity is, at least
partially, retained.
[0267] The small molecules of the present invention can be targeted
to a wide range biological targets including, but not limited to,
diseased cells (e.g., tumor cells) and tissues, healthy cells and
tissues, nucleic acids (e.g., DNA, cDNA, RNA, mRNA, and siRNA),
polypeptides (e.g., enzymes, cell surface proteins, etc.), cell
surface proteins, cell surface receptors, cell surface
polysaccharides, extracellular matrix proteins, intracellular
proteins, and to microorganisms and other pathogens (e.g.,
bacteria, fungi, mycoplasma, prions, viruses, and the like).
[0268] A variety of targeting molecules are contemplated for use in
association with the present compositions, including nucleic acids
(e.g., RNA and DNA), polypeptides (e.g., receptor ligands, signal
peptides, avidin, Protein A, antigen binding proteins, etc.),
polysaccharides, biotin, hydrophobic groups, hydrophilic groups,
drugs, and any organic molecules that bind to receptors. In some
embodiments, the small molecules of the present invention are
associated with multiple targeting molecules. In some of these
embodiments, the various targeting molecules are similar (e.g.,
monoclonal antibodies). In other embodiments, the targeting
molecules are dissimilar (e.g., antibodies with distinct idiotypes
or isotypes, or antibodies and nucleic acids, etc.).
[0269] In some embodiments of the present invention, any number of
cancer cell targeting groups are associated with the gossypol
compounds. Thus, the gossypol compounds associated with targeting
groups are specific for targeting cancer cells (i.e., much more
likely to attach to cancer cells and not to healthy cells).
[0270] Utilization of more than one targeting molecule in a
composition allows multiple biological targets to be targeted
and/or provides the ability to increase affinity for specific
targets. Multiple targeting molecules allow the compositions to be
"stacked," wherein a first composition is targeted to a first
biological target, and a second composition is targeted to the
first composition or to the first biological target. A number of
exemplary targeting molecules and targeting methods are describe in
more detail below.
[0271] A. General Targeting Molecules and Targeting
Considerations
[0272] Various efficiency issues affect the administration of all
drugs--and of highly cytotoxic drugs (e.g., anticancer drugs) in
particular. One issue of particular importance is ensuring that the
administered agents affect only targeted cells (e.g., cancer
cells), tissues, or organs. The nonspecific or unintended delivery
of highly cytotoxic agents to nontargeted cells can cause serious
toxicity issues.
[0273] Numerous attempts have been made to devise drug targeting
schemes to address the problems associated with nonspecific drug
delivery. (See e.g., K. N. Syrigos and A. A. Epenetos Anticancer
Res., 19:606-614 (1999); Y. J. Park et al., J. Controlled Release,
78:67-79 (2002); R. V. J. Chari, Adv. Drug Deliv. Rev., 31:89-104
(1998); and D. Putnam and J. Kopecek, Adv. Polymer Sci., 122:55-123
(1995)). Conjugating targeting moieties such as antibodies and
ligand peptides (e.g., RDG for endothelium cells) to drug molecules
has been used to alleviate some collateral toxicity issues
associated with particular drugs. However, conjugating drugs to
targeting moieties alone does not completely negate potential side
effects to nontargeted cells, since the drugs are usually bioactive
on their way to target cells. Advances in targeting moiety-prodrug
conjugates, which are inactive while traveling to specific targeted
tissues, have diminished some of these concerns. A
biotransformation, such as enzymatic cleavage, typically converts
the prodrug into a biologically active molecule at the target
site.
[0274] Accordingly, in some preferred embodiments, the present
invention provides prodrug conjugates that are inactive until they
reach their target site, where they are subsequently converted into
an active therapeutic drug molecule. ADEPT and ATTEMPTS are two
exemplary prodrug delivery systems compatible with certain
embodiments of the present invention. (See K. N. Syrigos and A. A.
Epenetos, Anticancer Res., 19:606-614 (1999); K. D. Bagshawe, Brit.
J. Cancer, 56:531-532 (1987); Y. J. Park et al., J. Controlled
Release, 72:145-156 (2001); and Y. J. Park et al., J. Controlled
Release, 78:67-79 (2002)).
[0275] The rapid clearance of some types of therapeutic agents,
especially water-soluble low-molecular weight agents, from the
subject's bloodstream provides yet another obstacle to effective
small molecule administration. Still other obstacles come from the
rapid clearance (e.g., proteolytic degradation) or potential
immunogenicity of the administered agents.
[0276] In natural systems, clearance and other pharmacokinetic
behaviors of small molecules (e.g., drugs) in a subject are
regulated by a series of transport proteins. (See e.g., H. T.
Nguyen, Clin. Chem. Lab. Anim., (2nd Ed.) pp. 309-335 (1999); and
G. J. Russell-Jones and D. H. Alpers, Pharm. Biotechnol.,
12:493-520 (1999)). Thus, in preferred embodiments, the
pharmacokinetics of agents are considered when testing and
developing potential therapeutics.
[0277] The rate of agent clearance in a subject is typically
manageable. For instance, attaching (e.g., binding) the agent to a
macromolecular carrier normally prolongs circulation and retention
times. Accordingly, some embodiments of the present invention
provide small molecules (e.g., gossypol compounds, drugs, or
prodrugs) conjugated to polyethylene glycol (PEG), or similar
biopolymers, to decrease (prevent) the molecules' degradation and
to improve its retention in the subject's bloodstream. (See e.g.,
R. B. Greenwald et al., Critical Rev. Therapeutic Drug Carrier
Syst., 17:101-161 (2000)). The ability of PEG to discourage
protein-protein interactions can reduce the immunogenicity of many
drugs.
[0278] Another issue affecting the administration of some
therapeutic agents, and especially hydrophilic and macromolecular
drugs such as peptides and nucleic acids, is that these agents have
difficulty crossing into targeted cellular membranes. Small
(typically less than 1,000 Daltons) hydrophobic molecules are less
susceptible to having difficulties entering target cell membranes.
Moreover, low molecular weight cytotoxic drugs often localize more
efficiently in normal tissues rather than in target tissues such as
tumors (K. Bosslet et al., Cancer Res., 58:1195-1201 (1998)) due to
the high interstitial pressure and unfavorable blood flow
properties within rapidly growing tumors (R. K. Jain, Int. J.
Radiat. Biol., 60:85-100 (1991); and R. K. Jain and L. T. Baxter,
Cancer Res., 48:7022-7032 (1998)).
[0279] Certain embodiments, especially those directed to delivering
cytotoxic agents, utilize one or more of the following methods or
compositions to aid delivery of the therapeutic compositions of the
present invention: microinjection (See e.g., M. Foldvari and M.
Mezei, J. Pharm. Sci., 80:1020-1028, (1991)); scrape loading (See
e.g., P. L. McNeil et al., J. Cell Biol., 98:1556-1564 (1984));
electroporation (See e.g., R. Chakrabarti et al., J. Biol. Chem.,
26:15494-15500 (1989)); liposomes (See e.g., M. Foldvari et al., J.
Pharm. Sci., 80:1020-1028 (1991); and J. N. Moreira et al., Biochim
Biophys Acta., 515:167-176 (2001)); nanocarriers such as
water-soluble polymers (e.g., enhanced permeation and retention
"EPR", See e.g., H. Maeda et al., J. Controlled Release, 65:271-284
(2000); H. Maeda et al., supra; and L. W. Seymour, Crit. Rev.
Therapeu. Drug Carrier Systems, 9:135-187 (1992)); bacterial toxins
(See e.g., T. I. Prior et al., Biochemistry, 31:3555-3559 (1992);
and H. Stenmark et al., J. Cell Biol.; 113:1025-1032 (1991));
receptor-mediated endocytosis and phagocytosis, including the
tumor-activated prodrug (TAP) system (See e.g., R. V. J. Chari,
Adv. Drug Deliv. Rev., 31:89-104 (1998); I. Mellman, Annu. Rev.
Cell Dev. Biol., 12:575-625 (1996); C. P. Leamon and P. S. Low, J.
Biol. Chem., 267 (35):24966-24971 (1992); H. Ishihara et al.,
Pharm. Res., 7:542-546 (1990); S. K. Basu, Biochem. Pharmacol.,
40:1941-1946 (1990); and G. Y. Wu and C. H. Wu, Biochemistry,
27:887-892 (1988)); other suitable compositions and methods are
known in the art.
[0280] B. Antibodies as Targeting Molecules
[0281] In some embodiments of the present invention, targeting
molecules comprise antigen binding proteins or immunoglobulins
(antibodies). Immunoglobulins can be generated to allow for the
targeting of antigens or immunogens (e.g., tumor, tissue, or
pathogen specific antigens) on various biological targets (e.g.,
pathogens, tumor cells, normal tissue). Such immunoglobulins
include, but are not limited to, polyclonal, monoclonal, chimeric,
single chain, Fab fragments, and Fab expression libraries.
[0282] Immunoglobulins (antibodies) are proteins generated by the
immune system to provide a specific molecule capable of complexing
with an invading molecule commonly referred to as an antigen.
Natural antibodies have two identical antigen-binding sites, both
of which are specific to a particular antigen. The antibody
molecule recognizes the antigen by complexing its antigen-binding
sites with areas of the antigen termed epitopes. The epitopes fit
into the conformational architecture of the antigen-binding sites
of the antibody, enabling the antibody to bind to the antigen.
[0283] The immunoglobulin molecule is composed of two identical
heavy and two identical light polypeptide chains, held together by
interchain disulfide bonds. Each individual light and heavy chain
folds into regions of about 110 amino acids, assuming a conserved
three-dimensional conformation. The light chain comprises one
variable region (termed V.sub.L) and one constant region (CL),
while the heavy chain comprises one variable region (V.sub.H) and
three constant regions (CH1, CH2 and CH3). Pairs of regions
associate to form discrete structures. In particular, the light and
heavy chain variable regions, V.sub.L and V.sub.H, associate to
form an "FV" area which contains the antigen-binding site.
[0284] The variable regions of both heavy and light chains show
considerable variability in structure and amino acid composition
from one antibody molecule to another, whereas the constant regions
show little variability. Each antibody recognizes and binds an
antigen through the binding site defined by the association of the
heavy and light chain variable regions into an FV area. The
light-chain variable region V.sub.L and the heavy-chain variable
region V.sub.H of a particular antibody molecule have specific
amino acid sequences that allow the antigen-binding site to assume
a conformation that binds to the antigen epitope recognized by that
particular antibody.
[0285] Within the variable regions are "sub-regions" in which the
amino acid sequence is extremely variable from one antibody to
another. Three of these so-called "hypervariable" regions or
"complementarity-determining regions" (CDRs) are found in each of
the light and heavy chains. The three CDRs from a light chain and
the three CDRs from a corresponding heavy chain form the
antigen-binding site.
[0286] Cleavage of naturally occurring antibody molecules with the
proteolytic enzyme papain generates fragments that retain their
antigen-binding site. These fragments, commonly known as Fabs (for
Fragment, antigen binding site) are composed of the C.sub.L,
V.sub.L, C.sub.H1 and V.sub.H regions of the antibody. In the Fab
the light chain and the fragment of the heavy chain are covalently
linked by a disulfide linkage.
[0287] Antibody fragments that contain the idiotype (antigen
binding region) of the antibody molecule can be generated by known
techniques. For example, such fragments include but are not limited
to: the F(ab')2 fragment that can be produced by pepsin digestion
of the antibody molecule; the Fab' fragments that can be generated
by reducing the disulfide bridges of the F(ab')2 fragment, and the
Fab fragments that can be generated by treating the antibody
molecule with papain and a reducing agent.
[0288] Various procedures known in the art are used for the
production of polyclonal antibodies. For the production of
antibody, various host animals can be immunized by injection with
the peptide corresponding to the desired epitope including, but not
limited to, rabbits, mice, rats, sheep, goats, etc. In a preferred
embodiment, the peptide is conjugated to an immunogenic carrier
(e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyhole
limpet hemocyanin (KLH)). Various adjuvants are used to increase
the immunological response, depending on the host species,
including but not limited to Freund's (complete and incomplete),
mineral gels such as aluminum hydroxide, surface active substances
such as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol, and
potentially useful human adjuvants such as BCG (Bacille
Calmette-Guerin) and Corynebacterium parvum.
[0289] Monoclonal antibodies against target antigens (e.g., a cell
surface protein such as a receptor) are produced by a variety of
techniques including conventional monoclonal antibody methodologies
such as the somatic cell hybridization techniques of Kohler and
Milstein, Nature, 256:495 (1975). Although in some embodiments,
somatic cell hybridization procedures are preferred, other
techniques for producing monoclonal antibodies are contemplated as
well (e.g., viral or oncogenic transformation of B
lymphocytes).
[0290] For preparation of monoclonal antibodies, any technique that
provides for the production of antibody molecules by continuous
cell lines in culture may be used (See e.g., Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.). These include, but are not
limited to, the hybridoma technique originally developed by Kohler
and Milstein (Kohler and Milstein, Nature 256:495-497 (1975)), as
well as the trioma technique, the human B-cell hybridoma technique
(See e.g., Kozbor et al., Immunol. Today 4:72 (1983)), and the
EBV-hybridoma technique to produce human monoclonal antibodies
(Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, Inc., pp. 77-96 (1985)).
[0291] In an additional embodiment of the invention, monoclonal
antibodies can be produced in germ-free animals utilizing recent
technology (See e.g., PCT/US90/02545). According to the invention,
human antibodies may be used and can be obtained by using human
hybridomas (Cote et at, Proc. Natl. Acad. Sci. U.S.A., 80:2026-2030
(1983)) or by transforming human B cells with EBV virus in vitro
(Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, pp. 77-96 (1985)).
[0292] In one embodiment, the preferred animal for preparing
hybridomas is the mouse. Hybridoma production in the mouse is a
well-established procedure. Immunization protocols and techniques
for isolation of immunized splenocytes for fusion are known in the
art. Fusion partners (e.g., murine myeloma cells) and fusion
procedures are also known. In other preferred embodiments, avian
(e.g., chickens) species are preferred for antibody production.
[0293] Human monoclonal antibodies (mAbs) directed against human
proteins can be generated using transgenic mice carrying the
complete human immune system rather than the mouse system.
Splenocytes from the transgenic mice are immunized with the antigen
of interest which are used to produce hybridomas that secrete human
mAbs with specific affinities for epitopes from a human protein.
(See e.g., Wood et al., WO 91/00906, Kucherlapati et al., WO
91/10741; Lonberg et al., WO 92/03918; Kay et al., WO 92/03917
(each of which is herein incorporated by reference in its
entirety); N. Lonberg et al., Nature, 368:856-859 (1994); L. L.
Green et al., Nature Genet., 7:13-21 (1994); S. L. Morrison et al.,
Proc. Nat. Acad. Sci. U.S.A., 81:6851-6855 (1994); Bruggeman et
al., Immunol., 7:33-40 (1993); Tuaillon et al., Proc. Nat. Acad.
Sci. U.S.A., 90:3720-3724 (1993); and Bruggeman et al. Eur. J.
Immunol., 21:1323-1326 (1991)).
[0294] Monoclonal antibodies can also be generated by other methods
known to those skilled in the art of recombinant DNA technology. An
alternative method, referred to as the "combinatorial antibody
display" method, has been developed to identify and isolate
antibody fragments having a particular antigen specificity, and can
be utilized to produce monoclonal antibodies. (See e.g., Sastry et
al., Proc. Nat. Acad. Sci. U.S.A., 86:5728 (1989); Huse et al.,
Science, 246:1275 (1989); and Orlandi et al., Proc. Nat. Acad. Sci.
U.S.A., 86:3833 (1989)). After immunizing an animal with an
immunogen as described above, the antibody repertoire of the
resulting B-cell pool is cloned. Methods are available for
obtaining DNA sequences from the variable regions of a diverse
population of immunoglobulin molecules using a mixture of oligomer
primers and PCR. For instance, mixed oligonucleotide primers
corresponding to the 5' leader (signal peptide) sequences or
framework 1 (FR1) sequences, as well as primer to a conserved 3'
constant region primer can be used for PCR amplification of the
heavy and light chain variable regions from a number of murine
antibodies. (See e.g., Larrick et al., Biotechniques, 11:152-156
(1991)). A similar strategy can also been used to amplify human
heavy and light chain variable regions from human antibodies (See
e.g., Larrick et al., Methods: Companion to Methods in Enzymology,
2:106-110 (1991)).
[0295] In one embodiment, RNA is isolated from B lymphocytes, for
example, peripheral blood cells, bone marrow, or spleen
preparations, using standard protocols (e.g., U.S. Pat. No.
4,683,292 (incorporated herein by reference in its entirety);
Orlandi, et al., Proc. Nat. Acad. Sci. U.S.A., 86:3833-3837 (1989);
Sastry et al., Proc. Nat. Acad. Sci. U.S.A., 86:5728-5732 (1989);
and Huse et al., Science, 246:1275 (1989). First strand cDNA is
synthesized using primers specific for the constant region of the
heavy chain(s) and each of the .kappa. and .lamda. light chains, as
well as primers for the signal sequence. Using variable region PCR
primers, the variable regions of both heavy and light chains are
amplified, each alone or in combination, and ligated into
appropriate vectors for further manipulation in generating the
display packages. Oligonucleotide primers useful in amplification
protocols may be unique or degenerate or incorporate inosine at
degenerate positions. Restriction endonuclease recognition
sequences may also be incorporated into the primers to allow for
the cloning of the amplified fragment into a vector in a
predetermined reading frame for expression.
[0296] The V-gene library cloned from the immunization-derived
antibody repertoire can be expressed by a population of display
packages, preferably derived from filamentous phage, to form an
antibody display library. Ideally, the display package comprises a
system that allows the sampling of very large variegated antibody
display libraries, rapid sorting after each affinity separation
round, and easy isolation of the antibody gene from purified
display packages. In addition to commercially available kits for
generating phage display libraries, examples of methods and
reagents particularly amenable for use in generating a variegated
antibody display library can be found in, for example, U.S. Pat.
No. 5,223,409; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679;
WO 93/01288; WO 92/01047; WO 92/09690; WO 90/02809 (each of which
is herein incorporated by reference in its entirety); Fuchs et al.,
Biol. Technology, 9:1370-1372 (1991); Hay et al., Hum. Antibod.
Hybridomas, 3:81-85 (1992); Huse et al., Science, 46:1275-1281
(1989); Hawkins et al., J. Mol. Biol., 226:889-896 (1992); Clackson
et al., Nature, 352:624-628 (1991); Gram et al., Proc. Nat. Acad.
Sci. U.S.A., 89:3576-3580 (1992); Garrad et al., Bio/Technolog,
2:1373-1377 (1991); Hoogenboom et al., Nuc. Acid Res., 19:4133-4137
(1991); and Barbas et al., Proc. Nat. Acad. Sci. U.S.A., 88:7978
(1991). In certain embodiments, the V region domains of heavy and
light chains can be expressed on the same polypeptide, joined by a
flexible linker to form a single-chain Fv fragment, and the scFV
gene subsequently cloned into the desired expression vector or
phage genome.
[0297] As generally described in McCafferty et al., Nature,
348:552-554 (1990), complete V.sub.H and V.sub.L domains of an
antibody, joined by a flexible linker (e.g., (Gly.sub.4-Ser).sub.3)
can be used to produce a single chain antibody which can render the
display package separable based on antigen affinity. Isolated scFV
antibodies immunoreactive with the antigen can subsequently be
formulated into a pharmaceutical preparation for use in the subject
method.
[0298] According to the invention, techniques described for the
production of single chain antibodies (U.S. Pat. No. 4,946,778;
herein incorporated by reference) can be adapted to produce
specific single chain antibodies. An additional embodiment of the
invention utilizes the techniques described for the construction of
Fab expression libraries (Huse et al., Science, 246:1275-1281
(1989)) to allow rapid and easy identification of monoclonal Fab
fragments with the desired specificity.
[0299] Once displayed on the surface of a display package (e.g.,
filamentous phage), the antibody library is screened with the
target antigen, or peptide fragment thereof, to identify and
isolate packages that express an antibody having specificity for
the target antigen. Nucleic acid encoding the selected antibody can
be recovered from the display package (e.g., from the phage genome)
and subcloned into other expression vectors by standard recombinant
DNA techniques.
[0300] Specific antibody molecules with high affinities for a
surface protein can be made according to methods known to those in
the art, e.g., methods involving screening of libraries. (See,
e.g., U.S. Pat, No. 5,233,409 and U.S. Pat. No. 5,403,484 (both
incorporated herein by reference in their entireties)). Further,
the methods of these libraries can be used in screens to obtain
binding determinants that are mimetics of the structural
determinants of antibodies.
[0301] Generally, in the production of antibodies, screening for
the desired antibody can be accomplished by techniques known in the
art (e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant
assay), "sandwich" immunoassays, immunoradiometric assays, gel
diffusion precipitin reactions, immunodiffusion assays, in situ
immunoassays (using colloidal gold, enzyme or radioisotope labels,
for example), Western Blots, precipitation reactions, agglutination
assays (e.g., gel agglutination assays, hemagglutination assays,
etc.), complement fixation assays, immunofluorescence assays,
protein A assays, and immunoelectrophoresis assays, etc.).
[0302] In particular, the Fv binding surface of a particular
antibody molecule interacts with its target ligand according to
principles of protein-protein interactions, hence sequence data for
V.sub.H and V.sub.L (the latter of which may be of the .kappa. or
.lamda. chain type) is the basis for protein engineering techniques
known to those with skill in the art. Details of the protein
surface that comprises the binding determinants can be obtained
from antibody sequence in formation, by a modeling procedure using
previously determined three-dimensional structures from other
antibodies obtained from NMR studies or crystallographic data.
[0303] In one embodiment, a variegated peptide library is expressed
by a population of display packages to form a peptide display
library. Ideally, the display package comprises a system that
allows the sampling of very large variegated peptide display
libraries, rapid sorting after each affinity separation round, and
easy isolation of the peptide-encoding gene from purified display
packages. Peptide display libraries can be in, e.g., prokaryotic
organisms and viruses, which can be amplified quickly, are
relatively easy to manipulate, and which allows the creation of a
large number of clones. Preferred display packages include, for
example, vegetative bacterial cells, bacterial spores, and most
preferably, bacterial viruses (especially DNA viruses). However,
the present invention also contemplates the use of eukaryotic
cells, including yeast and their spores, as potential display
packages. Phage display libraries are known in the art.
[0304] Other techniques include affinity chromatography with an
appropriate "receptor," e.g., a target antigen, followed by
identification of the isolated binding agents or ligands by
conventional techniques (e.g., mass spectrometry and NMR).
Preferably, the soluble receptor is conjugated to a label (e.g.,
fluorophores, colorimetric enzymes, radioisotopes, or luminescent
compounds) that can be detected to indicate ligand binding.
Alternatively, immobilized compounds can be selectively released
and allowed to diffuse through a membrane to interact with a
receptor.
[0305] Combinatorial libraries of compounds can also be synthesized
with "tags" to encode the identity of each member of the library.
(See e.g., W. C. Still et al., WO 94/08051, incorporated herein by
reference in its entirety). In general, this method features the
use of inert but readily detectable tags that are attached to the
solid support or to the compounds. When an active compound is
detected, the identity of the compound is determined by
identification of the unique accompanying tag. This tagging method
permits the synthesis of large libraries of compounds that can be
identified at very low levels among the total set of all compounds
in the library.
[0306] The term modified antibody is also intended to include
antibodies, such as monoclonal antibodies, chimeric antibodies, and
humanized antibodies, which have been modified by, for example,
deleting, adding, or substituting portions of the antibody. For
example, an antibody can be modified by deleting the hinge region,
thus generating a monovalent antibody. Any modification is within
the scope of the invention so long as the antibody has at least one
antigen binding region specific.
[0307] Chimeric mouse-human monoclonal antibodies can be produced
by recombinant DNA techniques known in the art. For example, a gene
encoding the Fc constant region of a murine (or other species)
monoclonal antibody molecule is digested with restriction enzymes
to remove the region encoding the murine Fc, and the equivalent
portion of a gene encoding a human Fc constant region is
substituted. (See e.g., Robinson et al., PCT/US86/02269; European
Patent Application 184,187; European Patent Application 171,496;
European Patent Application 173,494; WO 86/01533; U.S. Pat. No.
4,816,567; European Patent Application 125,023 (each of which is
herein incorporated by reference in its entirety) Better et al.,
Science, 240:1041-1043 (1988); Liu et al., Proc. Nat. Acad. Sci.
U.S.A., 84:3439-3443 (1987); Liu et al., J. Immunol., 139:3521-3526
(1987); Sun et al., Proc. Nat. Acad. Sci. U.S.A., 84:214-218
(1987); Nishimura et al., Canc. Res., 47:999-1005 (1987); Wood et
al., Nature, 314:446-449 (1985); and Shaw et al., J. Natl. Cancer
Inst., 80:1553-1559 (1988)).
[0308] The chimeric antibody can be further humanized by replacing
sequences of the Fv variable region which are not directly involved
in antigen binding with equivalent sequences from human Fv variable
regions. General, reviews of humanized chimeric antibodies are
provided by S. L. Morrison, Science, 229:1202-1207 (1985) and by Oi
et al., Bio. Techniques, 4:214 (1986). Those methods include
isolating, manipulating, and expressing the nucleic acid sequences
that encode all or part of immunoglobulin Fv variable regions from
at least one of a heavy or light chain. Sources of such nucleic
acids are known and, for example, may be obtained from 7E3, an
anti-GPIIbIIIa antibody producing hybridoma. The recombinant DNA
encoding the chimeric antibody, or fragment thereof, is then cloned
into an appropriate expression vector.
[0309] Suitable humanized antibodies can alternatively be produced
by CDR substitution (e.g., U.S. Pat. No. 5,225,539 (incorporated
herein by reference in its entirety); Jones et al., Nature,
321:552-525 (1986); Verhoeyan et al., Science, 239:1534 (1988); and
Beidler et al., J. Immunol., 141:4053 (1988)). All of the CDRs of a
particular human antibody may be replaced with at least a portion
of a non-human CDR or only some of the CDRs may be replaced with
non-human CDRs. It is only necessary to replace the number of CDRs
required for binding of the humanized antibody to the Fc
receptor.
[0310] An antibody is humanized by any method that is capable of
replacing at least a portion of a CDR of a human antibody with a
CDR derived from a non-human antibody. The human CDRs may be
replaced with non-human CDRs using oligonucleotide site-directed
mutagenesis.
[0311] Also within the scope of the invention are chimeric and
humanized antibodies in which specific amino acids have been
substituted, deleted or added. In particular, preferred humanized
antibodies have amino acid substitutions in the framework region,
such as to improve binding to the antigen. For example, in a
humanized antibody having mouse CDRs, amino acids located in the
human framework region can be replaced with the amino acids located
at the corresponding positions in the mouse antibody. Such
substitutions are known to improve binding of humanized antibodies
to the antigen in some instances.
[0312] In preferred embodiments, the fusion proteins include a
monoclonal antibody subunit (e.g., a human, murine, or bovine), or
a fragment thereof, (e.g., an antigen binding fragment thereof).
The monoclonal antibody subunit or antigen binding fragment thereof
can be a single chain polypeptide, a dimer of a heavy chain and a
light chain, a tetramer of two heavy and two light chains, or a
pentamer (e.g., IgM). IgM is a pentamer of five monomer units held
together by disulfide bonds linking their carboxyl-terminal
(C.mu.4/C.mu.4) domains and C.mu.3/C.mu.3 domains. The pentameric
structure of IgM provides 10 antigen-binding sites, thus serum IgM
has a higher valency than other types of antibody isotypes. With
its high valency, pentameric IgM is more efficient than other
antibody isotypes at binding multidimensional antigens (e.g., viral
particles and red blood cells. However, due to its large pentameric
structure, IgM does not diffuse well and is usually found in low
concentrations in intercellular tissue fluids. The J chain of IgM
allows the molecule to bind to receptors on secretory cells, which
transport the molecule across epithelial linings to the external
secretions that bathe the mucosal surfaces. In some embodiments,
the present invention takes advantage of the low diffusion rate of
pentameric IgM to help concentrate the fusion proteins of the
present invention at a site of interest.
[0313] In some preferred embodiments, the monoclonal antibody is a
murine antibody or a fragment thereof. In other preferred
embodiments, the monoclonal antibody is a bovine antibody or a
fragment thereof. For example, the murine antibody can be produced
by a hybridoma that includes a B cell obtained from a transgenic
mouse having a genome comprising a heavy chain transgene and a
light chain transgene fused to an immortalized cell.
[0314] The antibodies can be of the various isotypes, including,
IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgAsec, IgD,
or IgE. In some preferred embodiments, the antibody is an IgG
isotype. In other preferred embodiments, the antibody is an IgM
isotype. The antibodies can be full-length (e.g., an IgG1, IgG2,
IgG3, or IgG4 antibody) or can include only an antigen-binding
portion (e.g., a Fab, F(ab')2, Fv or a single chain Fv
fragment).
[0315] In preferred embodiments, the immunoglobulin subunit of the
fusion proteins is a recombinant antibody (e.g., a chimeric or a
humanized antibody), a subunit or an antigen binding fragment
thereof (e.g., has a variable region, or at least a complementarity
determining region (CDR)).
[0316] In preferred embodiments, the immunoglobulin subunit of the
fusion protein is monovalent (e.g., includes one pair of heavy and
light chains, or antigen binding portions thereof). In other
embodiments, the immunoglobulin subunit of the fusion protein is
divalent (e.g., includes two pairs of heavy and light chains, or
antigen binding portions thereof). In preferred embodiments, the
transgenic fusion proteins include an immunoglobulin heavy chain or
a fragment thereof (e.g., an antigen binding fragment thereof).
[0317] In preferred embodiments of the present invention, the
targeting agent is an antibody or antigen binding fragment of an
antibody (e.g., Fab units). For example, a well-studied antigen
found on the surface of many cancers (including breast HER2 tumors)
is glycoprotein p185, which is exclusively expressed in malignant
cells (Press et al., Oncogene 5:953 (1990)). Recombinant humanized
anti-HER2 monoclonal antibodies (rhuMabHER2) have even been shown
to inhibit the growth of HER2 overexpressing breast cancer cells,
and are being evaluated (in conjunction with conventional
chemotherapeutics) in phase III clinical trials for the treatment
of advanced breast cancer (Pegrarn et al., Proc. Am. Soc. Clin.
Oncol., 14:106 (1995)). Park et al. have attached Fab fragments of
rhuMabHER2 to small unilamellar liposomes, which then can be loaded
with the chemotherapeutic doxorubicin (dox) and targeted to HER2
overexpressing tumor xenografts (Park et al., Cancer Lett., 118:153
(1997) and Kirpotin et al., Biochem., 36:66 (1997)). These
dox-loaded "immunoliposomes" showed increased cytotoxicity against
tumors compared to corresponding non-targeted dox-loaded liposomes
or free dox, and decreased systemic toxicity compared to free
dox.
[0318] In some preferred embodiments, the antibodies recognize
tumor specific epitopes (e.g., TAG-72 (Kjeldsen et al., Cancer
Res., 48:2214-2220 (1988); U.S. Pat. Nos. 5,892,020; 5,892,019; and
5,512,443); human carcinoma antigen (U.S. Pat. Nos. 5,693,763;
5,545,530; and 5,808,005); TP1 and TP3 antigens from osteocarcinoma
cells (U.S. Pat. No. 5,855,866); Thomsen-Friedenreich (TF) antigen
from adenocarcinoma cells (U.S. Pat. No. 5,110,911); "KC-4 antigen"
from human prostrate adenocarcinoma (U.S. Pat. Nos. 4,708,930 and
4,743,543); a human colorectal cancer antigen (U.S. Pat. No.
4,921,789); CA125 antigen from cystadenocarcinoma (U.S. Pat. No.
4,921,790); DF3 antigen from human breast carcinoma (U.S. Pat. Nos.
4,963,484 and 5,053,489); a human breast tumor antigen (U.S. Pat.
No. 4,939,240); p97 antigen of human melanoma (U.S. Pat. No.
4,918,164); carcinoma or orosomucoid-related antigen (CORA)(U.S.
Pat. No. 4,914,021); a human pulmonary carcinoma antigen that
reacts with human squamous cell lung carcinoma but not with human
small cell lung carcinoma (U.S. Pat. No. 4,892,935); T and Tn
haptens in glycoproteins of human breast carcinoma (Springer et
al., Carbohydr. Res., 178:271-292 (1988)), MSA breast carcinoma
glycoprotein (Tjandra et al., Br. J. Surg., 75:811-817 (1988));
MFGM breast carcinoma antigen (Ishida et al., Tumor Biol., 10:12-22
(1989)); DU-PAN-2 pancreatic carcinoma antigen (Lan et al., Cancer
Res., 45:305-310 (1985)); CA125 ovarian carcinoma antigen (Hanisch
et al., Carbohydr. Res., 178:29-47 (1988)); YH206 lung carcinoma
antigen (Hinoda et al., Cancer J., 42:653-658 (1988)). Each of the
foregoing references is specifically incorporated herein by
reference.
[0319] For breast cancer, the cell surface may be targeted with
mammastatin, folic acid, EGF, FGF, and antibodies (or antibody
fragments) to the tumor-associated antigens MUC1, cMet receptor and
CD56 (NCAM).
[0320] A very flexible method to identify and select appropriate
peptide targeting groups is the phage display technique (See e.g.,
Cortese et al., Curr. Opin. Biotechol., 6:73 (1995)), which can be
conveniently carried out using commercially available kits. The
phage display procedure produces a large and diverse combinatorial
library of peptides attached to the surface of phage, which are
screened against immobilized surface receptors for tight binding.
After the tight-binding, viral constructs are isolated and
sequenced to identify the peptide sequences. The cycle is repeated
using the best peptides as starting points for the next peptide
library. Eventually, suitably high-affinity peptides are identified
and then screened for biocompatibility and target specificity. In
this way, it is possible to produce peptides conjugated to
dendrimers, producing multivalent conjugates with high specificity
and affinity for the target cell receptors (e.g., tumor cell
receptors) or other desired targets. In some embodiments, the
gossypol compounds or other therapeutic agents are associated with
dendrimers (e.g., PAMAM), or liposomes, or other carriers. Those
skilled in the art will be able to readily design dendrimer
gossypol compound molecules that take advantage of the multivalent
structure of dendrimers.
[0321] Related to the targeting approaches described above is the
"pretargeting" approach (See e.g., Goodwin and Meares, Cancer
(suppl.), 80:2675 (1997)). An example of this strategy involves
initial treatment of the patient with conjugates of tumor-specific
monoclonal antibodies and streptavidin. Remaining soluble conjugate
is removed from the bloodstream with an appropriate biotinylated
clearing agent. When the tumor-localized conjugate is all that
remains, a gossypol-linked, biotinylated agent is introduced, which
in turn localizes at the tumor sites by the strong and specific
biotin-streptavidin interaction.
[0322] In other preferred embodiments, the antibodies recognize
specific pathogens (e.g., Legionella peomophilia, Mycobacterium
tuberculosis, Clostridium tetani, Hemophilus influenzae, Neisseria
gonorrhoeae, Treponema pallidum, Bacillus anthracis, Vibrio
cholerae, Borrelia burgdorferi, Cornebacterium diphtheria,
Staphylococcus aureus, human papilloma virus, human
immunodeficiency virus, rubella virus, polio virus, and the
like).
[0323] C. Peptides as Targeting Molecules
[0324] In some preferred embodiments, targeting molecules comprise
peptides that bind specifically to tumor blood vessels. (See e.g.,
Arap et al., Science, 279:377-80 (1998)). These peptides include,
but are not limited to, peptides containing the RGD (Arg-Gly-Asp)
motif (e.g., CDCRGDCFC; SEQ ID NO:4), the NGR (Asn-Gly-Arg) motif
(e.g., CNGRCVSGCAGRC; SEQ ID NO:5), and the GSL (Gly-Ser-Leu; SEQ
ID NO:6) motif. These peptides and conjugates containing these
peptides selectively bind to various tumors, including, but not
limited to, breast carcinomas, Kaposi's sarcoma, and melanoma. It
is not intended that the present invention be limited to any
particular mechanism of action. Indeed, an understanding of the
mechanism is not necessary to make and use the present invention.
However, it is believed that these peptides are ligands for
integrins and growth factor receptors that are absent or barely
detectable in established blood vessels. In some preferred
embodiments, the peptide is preferably produced using chemical
synthesis methods. For example, peptides can be synthesized by
solid phase techniques, cleaved from the resin, and purified by
preparative high performance liquid chromatography. (See e.g.,
Creighton (1983) Proteins Structures and Molecular Principles, W.H.
Freeman and Co, New York, N.Y.). In other embodiments, the
composition of the synthetic peptides is confirmed by amino acid
analysis or sequencing.
[0325] In some preferred embodiments, targeting molecules comprise
peptides that specifically bind to glioma cells. (See e.g.,
Debinski et al., Nature Biotech., 16:449-53 (1998); Debinski et
al., J. Biol. Chem., 270(28):16775-80 (1995); and Debinski et al.,
J. Biol. Chem., 271(37):22428-33 (1996)). In some embodiments, the
present invention contemplates using drug delivery compositions
comprising IL13, or one of its variants, so that the drug delivery
compositions bind to IL13 binding sites in glioma cells.
[0326] Human high-grade gliomas are uniquely enriched in IL13
binding sites. Many of the established brain tumor cell lines,
primarily malignant gliomas, over-express hIL13 binding sites.
Human malignant glioma cell lines express a high number, up to
30,000, of binding sites for hIL13 per cell. Of interest,
glioblastoma multiforme (GBM) explant cells showed an
extraordinarily high number of hIL13 binding sites, up to 500,000
per cell. The binding of hIL13 is not neutralized by hIL4 on an
array of established human glioma cell lines that includes U-251
MG, U-373 MG, DBTRG MG, Hs-683, U-87 MG, SNB-19, and A-172 cells.
hIL13 can be engineered to increase its specific targeting of
high-grade gliomas. The pattern for IL13 and [L4R sharing on normal
cells requires IL13 to bind hIL4R. This is confirmed by the fact
that hIL13 binding is always fully competed by hIL4. The recently
proposed model for this hIL13R suggests that the shared hIL13/4R is
heterodimeric. This scenario would imply that hIL13 may contain at
least two receptor-binding sites, each recognizing a respective
subunit of the receptor. The engineered hIL13 variants (e.g.,
hIL13.E13K or hIL13.E13Y) are deprived of cell signaling abilities.
This is desirable because interaction with physiological systems
contributes prominently to the dose-limiting toxicity of some
biological therapeutics (e.g., cytokines). Significantly, the
molecule of hIL13 appears not to be sensitive to a variety of
genetically engineered modifications and these variants can be
produced in large quantities. It is thus possible to divert the
molecule of hIL13 from its physiological receptor and make it a
non-signaling compound, while the discovery of the expression of
IL13 receptors on the surface of all of the malignancies of glial
origin provides a novel strategy for the accumulation and retention
of drug delivery compositions within CNS cancers. The high-grade
glioma (HGG)-associated receptor for IL13 used in the present
affinity toward the HGG-associated receptor remains intact or is
increased. Such forms of IL13 can serve as rationally designed
vectors for variety of imaging and therapeutic approaches of
HGG.
[0327] Given the typically grim prognosis following the
identification of an intracranial malignancy, any strategy for the
pre-, intra- or post-operative identification and removal of cancer
cells is a significant improvement. In some embodiments, nucleic
acids encoding IL13 fragments, fusion proteins or functional
equivalents or variants (e.g., hIL13.E13K or hIL13.E13Y) thereof
are cloned into an appropriate expression vector, expressed and
purified (e.g., preferably as described in Debinski et al., Nature
Biotech., 16:449-53 (1998); Debinski et al., J. Biol. Chem.,
270(28):16775-80 (1995); and Debinski et al., J. Biol. Chem.,
271(37):22428-33 (1996)). In other embodiments of the present
invention, vectors include, but are not limited to, chromosomal,
nonchromosomal and synthetic DNA sequences, e.g., derivatives of
SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids;
vectors derived from combinations of plasmids and phage DNA, viral
DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
Large numbers of suitable vectors are known to those of skill in
the art, and are commercially available. Such vectors include, but
are not limited to, the following vectors: 1) Bacterial--pQE70:
pQE60; pQE-9 (Qiagen, Inc., Valencia, Calif.); pBS; pD10;
phagescript; psiX174; pbluescript SK; pBSKS; pNH8A; pNH16a; pNH18A;
pNH46A (Stratagene, Inc., La Jolla, Calif.); ptrc99a; pKK223-3;
pKK233-3; pDR540; pRIT5 (Pharmacia, Peapack, N.J.); and 2)
Eukaryotic--pWLNEO; pSV2CAT; pOG44; PXT1; pSG (Stratagene); pSVK3;
pBPV; pMSG; and pSVL (Pharmacia). Any other plasmid or vector can
be used as long as they are replicable and viable in the host. In
some preferred embodiments of the present invention, mammalian
expression vectors comprise an origin of replication, a suitable
promoter and enhancer, and any necessary ribosome binding sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
nontranscribed sequences. In other embodiments, DNA sequences
derived from the SV40 splice, and polyadenylation sites are used to
provide the required nontranscribed genetic elements.
[0328] In other embodiments, the IL13 peptide or variant thereof is
expressed in a host cell. In some embodiments of the present
invention, the host cell is a higher eukaryotic cell (e.g., a
mammalian or insect cell). In other embodiments of the present
invention, the host cell is a lower eukaryotic cell (e.g., a yeast
cell). In still other embodiments of the present invention, the
host cell can be a prokaryotic cell (e.g., a bacterial cell).
Specific examples of host cells include, but are not limited to,
Escherichia coli, Salmonella typhimurium, Bacillus subtilis, and
various species within the genera Pseudomonas, Streptomyces, and
Staphylococcus, as well as Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Drosophila S2 cells, Spodoptera Sf9
cells, Chinese Hamster Ovary (CHO) cells, COS-7 lines of monkey
kidney fibroblasts, (Gluzman, Cell, 23:175 (1981)), C127, 3T3, HeLa
and BHK cell lines.
[0329] In some embodiments of the present invention, IL13 or
variants thereof are recovered or purified from recombinant cell
cultures by methods including, but not limited to, ammonium sulfate
or ethanol precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. In other
embodiments of the present invention, protein refolding steps are
used, as necessary, in completing configuration of the mature
protein. In still other embodiments of the present invention, high
performance liquid chromatography (HPLC) is employed for final
purification steps.
[0330] Some embodiments of the present invention provide
polynucleotides having the coding sequence fused in frame to a
marker sequence that allows for purification of the polypeptide of
the present invention. A non-limiting example of a marker sequence
is a hexahistidine tag that is supplied by a vector, preferably a
pQE-9 vector, that provides for purification of the polypeptide
fused to the marker in the case of a bacterial host, or, for
example, the marker sequence may be a hemagglutinin (HA) tag when a
mammalian host (e.g., COS-7 cells) is used. The HA tag corresponds
to an epitope derived from the influenza hemagglutinin protein
(Wilson, et al., Cell, 37:767 (1984)).
[0331] D. Signal Peptide as Targeting Molecules
[0332] In some embodiments of the present invention, the targeting
molecules comprise signal peptides. These peptides are chemically
synthesized or cloned, expressed and purified as described above.
Signal peptides can assist the chemical address tags of the present
invention target the drug delivery composition (or a portion
thereof) to discrete regions within a cell. In some of these
embodiments, the signal peptide is preferably:
NH-Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Pro-Ala-Thr-Arg-Thr-Le-
u-COOH (SEQ ID NO:7). The present invention is not limited to any
particular mechanism, and an understanding of mechanisms is not
necessary to make and use the present invention, however, it is
contemplated that the peptide of SEQ ID NO:7 forms an amphipathic
helix that associates with mitochondrial membrane protein import
sites. This association allows peptide complexes to attach to
mitochondrial membranes. It is unlikely that the complex is
internalized, since there are few pores of nanometer size on intact
mitochondria. In still other embodiments, the following nuclear
localization signal is utilized:
NH-Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val-COOH (SEQ ID NO:8).
[0333] E. Nucleic Acids as Targeting Molecules
[0334] In some embodiments of the present invention, the targeting
molecules comprise nucleic acids (e.g., RNA or DNA). In some
embodiments, these nucleic acid moieties are designed to hybridize
(by base pairing) to a particular nucleic acid (e.g., chromosomal
DNA, mRNA, or ribosomal RNA) sequence in target cells and tissues.
Exemplary nucleic acids include, but are not limited to, those
coding for reverse transcriptase, REV and TAT proteins of HIV
(Tuerk et al., Gene, 137(1):33-9 (1993)); human nerve growth factor
(Binkley et al., Nuc. Acids Res., 23(16):3198-205 (1995)); and
vascular endothelial growth factor (Jellinek et al., Biochem.,
83(34):10450-6 (1994)). In other embodiments, the targeting
molecules bind ligands or biological targets directly. In some
embodiments, suitable nucleic acids that bind ligands are
identified using the SELEX procedure (U.S. Pat. Nos. 5,475,096;
5,270,163; WO 97/38134; WO 98/33941; and WO 99/07724; all of which
are herein incorporated by reference), although many additional
methods are known in the art and are suitable in certain
embodiments of the present invention.
[0335] F. Other Cellular Targeting Molecules
[0336] The targeting molecules of the present compositions may
recognize a variety of epitopes on biological targets (e.g.,
pathogens, tumor cells, normal tissues). In some embodiments,
cellular level targeting moieties are incorporated to recognize,
target, or detect a variety of pathogenic organisms including, but
not limited to, tumor specific antigens (e.g., carcinoembryonic
antigen, prostate specific antigen, tyrosinase, ras, a sialyl lewis
antigen, erb, MAGE-1, MAGE-3, BAGE, MN, gp100, gp75, p97,
proteinase 3, a mucin, CD81, CID9, CD63, CD53, CD38, CO-029, CA125,
GD2, GM2 and 0-acetyl GD3, M-TAA, M-fetal or M-urinary).
Alternatively, the targeting molecules may be a tumor suppressor, a
cytokine, a chemokine, a tumor specific receptor ligand, a
receptor, an inducer of apoptosis, or a differentiating agent.
[0337] Tumor suppressor proteins contemplated for targeting
include, but are not limited to, p16, p21, p27, p53, p73, Rb, Wilms
tumor (WT-1), DCC, neurofibromatosis type 1 (NF-1), von
Hippel-Lindau (VHL) disease tumor suppressor, Maspin, Brush-1,
BRCA-1, BRCA-2, the multiple tumor suppressor (MTS), gp95/p97
antigen of human melanoma, renal cell carcinoma-associated G250
antigen, KS 1/4 pan-carcinoma antigen, ovarian carcinoma antigen
(CA125), prostate specific antigen, melanoma antigen gp75, CD9,
CD63, CD53, CD37, R2, CD81, C0029, TI-1, L6 and SAS. Of course,
these are merely exemplary tumor suppressors. It is envisioned that
the present invention may be used in conjunction with any other
agents that are or become known to those of skill in the art as a
tumor suppressor or tumor marker.
[0338] In preferred embodiments of the present invention, the
compositions are targeted to factors expressed by oncogenes. These
include, but are not limited to, tyrosine kinases, both
membrane-associated and cytoplasmic forms, such as members of the
Src family, serine/threonine kinases, such as Mos, growth factor
receptors, such as platelet derived growth factor (PDGF), SMALL
GTPases (G proteins) including the ras family, cyclin-dependent
protein kinases (cdk), members of the myc family, including c-myc,
N-myc, and L-myc and bcl-2 and family members.
[0339] Receptors and their related ligands that find use in the
context of certain embodiments of the present invention include,
but are not limited to, the folate receptor, adrenergic receptor,
growth hormone receptor, luteinizing hormone receptor, estrogen
receptor, epidermal growth factor receptor, fibroblast growth
factor receptor, and the like.
[0340] Hormones and their receptors that find use in the cellular
level targeting aspects of the present invention include, but are
not limited to, growth hormone, prolactin, placental lactogen,
luteinizing hormone, follicle-stimulating hormone, chorionic
gonadotropin, thyroid-stimulating hormone, leptin,
adrenocorticotropin (ACTH), angiotensin I, angiotensin II,
.alpha.-endorphin, .alpha.-melanocyte stimulating hormone
(.alpha.-MSH), cholecystokinin, endothelin I, galanin, gastric
inhibitory peptide (GIP), glucagon, insulin, amylin, lipotropins,
GLP-1 (7-37) neurophysins, mammastatin, and somatostatin.
[0341] In addition, the present invention contemplates that
vitamins (both fat soluble and non-fat soluble vitamins) can be
used as targeting molecules to target biological targets (e.g.,
cells) that have receptors for, or otherwise take up, these
vitamins. Particularly preferred for this aspect of the invention
are the fat soluble vitamins D, E, and A, and analogues thereof,
and the water soluble vitamin C.
IX. Pharmaceutical Formulations, Administration Routes, and Dosing
Considerations
[0342] The present invention provides pharmaceutical compositions
which may comprise at least one gossypol compound, and in preferred
embodiments, at least one conventional anticancer agent. The
gossypol compounds and anticancer agents may be administered in any
sterile, biocompatible pharmaceutical carrier, including, but not
limited to, saline, buffered saline, dextrose, and water. In some
embodiments, the pharmaceutical compositions of the present
invention may contain one agent (e.g., a gossypol compound). In
other embodiments, the pharmaceutical compositions contain a
mixture of at least two agents (e.g., a gossypol compound and one
or more conventional anticancer agents). In still further
embodiments, the pharmaceutical compositions of the present
invention contain at least two agents (e.g., gossypol compounds and
one or more conventional anticancer agents) that are administered
to a patient under one or more of the following conditions: at
different periodicities, at different durations, at different
concentrations, by different administration routes, etc. In some
embodiments, the gossypol compound is administered prior to the
anticancer agent, e.g., 0.5, 1, 2 3, 4, 5, 10, 12, or 18 hours, 1,
2, 3, 4, 5, or 6 days, 1, 2, 3, or 4 weeks prior to the
administration of the anticancer agent. In some embodiments, the
gossypol compound is administered after the anticancer agent, e.g.,
0.5, 1, 2 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days,
1, 2, 3, or 4 weeks after the administration of the anticancer
agent. In some embodiments, the gossypol compound and the
anticancer agent are administered concurrently but on different
schedules, e.g., the gossypol compound is administered daily while
the anticancer agent is administered once a week, once every two
weeks, once every three weeks, or once every four weeks. In other
embodiments, the gossypol compound is administered once a week
while the anticancer agent is administered daily, once a week, once
every two weeks, once every three weeks, or once every four
weeks.
[0343] The compositions and methods of the present invention find
use in treating diseases or in altering physiological states that
are characterized by the overexpression of one or more Bcl-2 family
proteins (e.g., Bcl-2, Bcl-X.sub.L, Mcl-1, A1/BFL-1, and BOO-DIVA,
etc.). The invention further provides methods for inducing
apoptosis in cells by antagonizing the anti-apoptotic affects of
some Bcl-2 family proteins including, but not limited to, Bcl-2,
Bcl-X.sub.L, Mcl-1, A1/BFL-1, and BOO-DIVA.
[0344] Depending on the condition being treated, preferred
embodiments of the present pharmaceutical compositions are
formulated and administered systemically or locally. Techniques for
formulation and administration can be found in the latest edition
of "Remington's Pharmaceutical Sciences" (Mack Publishing Co,
Easton Pa.). Exemplary pharmaceutical formulations and methods of
producing pharmaceuticals are described in U.S. 20030211046A1; U.S.
20030004182A1; U.S. 2002060356384; U.S. 20020015728A1; U.S. Pat.
No. 6,511,660; U.S. Pat. No. 6,406,745; U.S. Pat. No. 6,346,269;
U.S. Pat. No. 6,039,977; U.S. Pat. No. 5,858,408; U.S. Pat. No.
5,631,023; U.S. Pat. No. 5,476,667; U.S. Pat. No. 5,044,091; U.S.
Pat. No. 4,867,970; and WO 0028969A2 each of which is incorporated
herein by reference in its entirety). Suitable routes may, for
example, include oral or transmucosal administration as well as
parenteral delivery (e.g., intramuscular, subcutaneous,
intramedullary, intrathecal, intraventricular, intravenous,
intraperitoneal, or intranasal administration).
[0345] The present invention contemplates administering gossypol
compounds and, in some embodiments, one or more conventional
anticancer agents, in accordance with acceptable pharmaceutical
delivery methods and preparation techniques. For example, gossypol
compounds and suitable anticancer agents can be administered to a
subject intravenously in a pharmaceutically acceptable carrier such
as physiological saline. Standard methods for intracellular
delivery of pharmaceutical agents are contemplated (e.g., delivery
via liposome). Such methods are well known to those of ordinary
skill in the art.
[0346] In some embodiments, the formulations of the present
invention are useful for parenteral administration (e.g.,
intravenous, subcutaneous, intramuscular, intramedullary, and
intraperitoneal). Therapeutic co-administration of some
contemplated anticancer agents (e.g., therapeutic polypeptides) can
also be accomplished using gene therapy reagents and
techniques.
[0347] In some embodiments of the present invention, gossypol
compounds are administered to a subject alone, or in combination
with one or more conventional anticancer agents (e.g., nucleotide
sequences, drugs, hormones, etc.) or in pharmaceutical compositions
where the components are optionally mixed with excipient(s) or
other pharmaceutically acceptable carriers. In preferred
embodiments of the present invention, pharmaceutically acceptable
carriers are biologically inert. In preferred embodiments, the
pharmaceutical compositions of the present invention are formulated
using pharmaceutically acceptable carriers well known in the art in
dosages suitable for oral administration. Such carriers enable the
pharmaceutical compositions to be formulated as tablets, pills,
capsules, dragees, liquids, gels, syrups, slurries, solutions,
suspensions and the like, for respective oral or nasal ingestion by
a subject. In preferred embodiments, the gossypol compounds are
administered orally to a subject.
[0348] Pharmaceutical preparations for oral use can be obtained by
combining the active compounds (e.g., gossypol compounds) with
solid excipients, optionally grinding the resulting mixture, and
processing the mixture into granules, after adding suitable
auxiliaries, if desired, to obtain tablets or dragee cores.
Suitable excipients are carbohydrate or protein fillers such as
sugars, including lactose, sucrose, mannitol, or sorbitol; starch
from corn, wheat, rice, potato, etc.; cellulose such as methyl
cellulose, hydroxypropylmethyl-cellulose, or sodium
carboxymethylcellulose; gums including arabic and tragacanth; and
proteins such as gelatin and collagen. If desired, disintegrating
or solubilizing agents may be added, such as the cross-linked
polyvinyl pyrrolidone, agar, alginic acid or a salt thereof such as
sodium alginate.
[0349] For injection, the pharmaceutical compositions of the
invention may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiologically buffered saline. For tissue
or cellular administration, penetrants appropriate to the
particular barrier to be permeated are used. Such penetrants are
known to those skilled in the art.
[0350] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
For example, an effective amount of a gossypol compound may be that
amount that induces apoptosis in a cell or tissue having elevated
levels of a Bcl-2 family protein as compared to normal
nonpathological cells or tissues. The determination of an effective
amount of an agent is well within the skills of those in the
pharmacological arts, especially in view of the disclosure provided
herein.
[0351] In addition to the active ingredients, preferred
pharmaceutical compositions may contain suitable pharmaceutically
acceptable carriers comprising excipients and auxiliaries that
facilitate processing of the active compounds into pharmaceutically
useful forms.
[0352] The pharmaceutical compositions of the present invention may
be manufactured using any acceptable techniques for preparing
pharmaceutical compositions including, but not limited to, by means
of conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes, and the like.
[0353] Ingestible formulations of the present compositions may
further include any material approved by the United States
Department of Agriculture for inclusion in foodstuffs and
substances that are generally recognized as safe (GRAS), such as
food additives, flavorings, colorings, vitamins, minerals, and
phytonutrients. The term phytonutrients, as used herein, refers to
organic compounds isolated from plants that have a biological
effect, and includes, but is not limited to, compounds of the
following classes: isoflavonoids, oligomeric proanthcyanidins,
indol-3-carbinol, sulforaphone, fibrous ligands, plant
phytosterols, ferulic acid, anthocyanocides, triterpenes, omega 3/6
fatty acids, polyacetylene, quinones, terpenes, cathechins,
gallates, and quercitin.
[0354] Dragee cores are provided with suitable coatings such as
concentrated sugar solutions, which may also contain gum arabic,
talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol,
and/or titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures. Dyestuffs or pigments may be added to
the tablets or dragee coatings for product identification or to
characterize the quantity of active compound, (i.e., dosage).
[0355] Pharmaceutical preparations contemplated for oral
administration include push-fit capsules made of gelatin, as well
as soft sealed capsules of gelatin and a coating such as glycerol
or sorbitol. In some embodiments, push-fit capsules can contain the
active ingredients mixed with fillers or binders such as lactose or
starches, lubricants such as talc or magnesium stearate, and,
optionally, stabilizers. In some soft capsule embodiments, the
active compounds are dissolved or suspended in a suitable liquid or
solvent, such as fatty oils, liquid paraffin, or liquid
polyethylene glycol, with or without stabilizers.
[0356] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds. Aqueous
injection suspensions optionally contain substances that increase
the viscosity of the suspension such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. In this aspect, suitable lipophilic solvents or
vehicles include fatty oils such as sesame oil, or synthetic fatty
acid esters, such as ethyl oleate or triglycerides, or liposomes.
Optionally, suspensions contain suitable stabilizers or agents that
increase the solubility of the compounds thus allowing for the
preparation of highly concentrated solutions.
[0357] Compositions comprising a compound of the invention
formulated in a pharmaceutical acceptable carrier may be prepared,
placed in an appropriate container, and labeled for treatment of an
indicated condition. For gossypol compounds, conditions indicated
on the label may include treatment of conditions related to faulty
regulation of apoptosis, hyperproliferative diseases, cancers,
acquired immune deficiency syndrome (AIDS), degenerative
conditions, and vascular diseases. The pharmaceutical compositions
may be provided as salts and can be formed with many acids,
including but not limited to hydrochloric, sulfuric, acetic,
lactic, tartaric, malic, succinic, and the like. Salts tend to be
more soluble in aqueous or other protonic solvents than are
corresponding free base forms. In other cases, a preferred
preparation comprises a lyophilized powder in 1 mM-50 mM histidine,
0.1%-2% sucrose, or 2%-7% mannitol at a pH range of from about 4.5
to 5.5, optionally combined with buffer prior to use.
[0358] In preferred embodiments, dosing and administration regimes
are tailored by the clinician, or others skilled in the
pharmacological arts, based upon well known pharmacological and
therapeutic considerations including, but not limited to, the
desired level of therapeutic effect; and the practical level of
therapeutic effect obtainable. Generally, it is advisable to follow
well-known pharmacological principles for administrating
chemotherapeutic agents (e.g., it is generally advisable to not
change dosages by more than 50% at time and no more than every 3-4
agent half-lives). For compositions that have relatively little or
no dose-related toxicity considerations, and where maximum efficacy
(e.g., destruction of cancer cells) is desired, doses in excess of
the average required dose are not uncommon. This approach to dosing
is commonly referred to as the "maximal dose" strategy.
[0359] Additional dosing considerations relate to calculating
proper target levels for the agent being administered, the agent's
accumulation and potential toxicity, stimulation of resistance,
lack of efficacy, and describing the range of the agent's
therapeutic index.
[0360] In certain embodiments, the present invention contemplates
using routine methods of titrating the agent's administration. One
common strategy for the administration is to set a reasonable
target level for the agent in the subject. In some preferred
embodiments, agent levels are measured in the subject's plasma.
Proper dose levels and frequencies are then designed to achieve the
desired steady-state target level for the agent. Actual, or
average, levels of the agent in the subject are monitored (e.g.,
hourly, daily, weekly, etc.) such that the dosing levels or
frequencies can be adjusted to maintain target levels. Of course,
the pharmacokinetics and pharmacodynamics (e.g., bioavailability,
clearance or bioaccumulation, biodistribution, drug interactions,
etc.) of the particular agent or agents being administered can
potentially impact what are considered reasonable target levels and
thus impact dosing levels or frequencies.
[0361] Target-level dosing methods typically rely upon establishing
a reasonable therapeutic objective defined in terms of a desirable
range (or therapeutic range) for the agent in the subject. In
general, the lower limit of the therapeutic range is roughly equal
to the concentration of the agent that provides about 50% of the
maximum possible therapeutic effect. The upper limit of the
therapeutic range is usually established by the agent's toxicity
and not by its efficacy. The present invention contemplates that
the upper limit of the therapeutic range for a particular agent
will be the concentration at which less than 5 or 10% of subjects
exhibit toxic side effects. In some embodiments, the upper limit of
the therapeutic range is about two times, or less, than the lower
limit: Those skilled in the art will understand that these dosing
consideration are highly variable and to some extent
individualistic (e.g., based on genetic predispositions,
immunological considerations, tolerances, resistances, and the
like). Thus, in some embodiments, effective target dosing levels
for an agent in a particular subject may be 1, . . . 5, . . . 10, .
. . 15, . . . 20, . . . 50, . . . 75, . . . 100, . . . 200, . . . X
%, greater than optimal in another subject. Conversely, some
subjects may suffer significant side effects and toxicity related
health issues at dosing levels or frequencies far less (1, . . . 5,
. . . 10, . . . 15, . . . 20, .. . 50, . . . 75, . . . 100, . . .
200, . . . X %) than those typically producing optimal therapeutic
levels in some or a majority of subjects. In the absence of more
specific information, target administration levels are often set in
the middle of the therapeutic range.
[0362] In certain embodiments, genetic screening methods (e.g., SNP
testing) are used to test a subject's predisposition to adverse
reactions in response to particular chemotherapeutic agents or
classes of chemotherapeutic agents.
[0363] In still further embodiments, the present invention provides
methods for repetitive dosing and/or the continuous
(semi-continuous) infusion of therapeutic agents (e.g., small
molecule Bcl-2 antagonists or agonists) sufficient to maintain,
within a given therapeutic range, a steady-state concentration of
agent(s) in a subject (e.g., in the subject's plasma). Those
skilled in the art will appreciate that the compositions of the
present invention can be administered such that a maintenance dose
is provided. Thus, in some embodiments, the chosen agent target
concentration or rate of drug delivery is adjusted to balance the
rate of drug loss. Those skilled in the art of administering
chemotherapeutic agents will appreciate the calculations and
measurements used to ensure the balance of drug input versus drug
loss to provide the desired target level of drug (or other
therapeutic agent) in the subject. Particularly useful in
performing these calculations are defined levels of agent clearance
and availability in a particular subject.
[0364] In additional embodiments, the present invention provides
intermittent dosing methods, since marked fluctuations in agent
concentration between doses are generally undesirable. In
situations where the absorption and distribution of the agent are
balanced and spontaneous, concentration fluctuations are dictated
by the agent's elimination half-life.
[0365] In embodiments where the administered compositions are
relatively nontoxic, maximal dosing methods can be used, because
even concentrations of the agent several times that necessary for
ensuring therapeutic efficacy are well tolerated. In these
embodiments, the dosing intervals are lengthened such that the
concentration of the agent in the subject's system remains within
the range of therapeutic effectiveness for relatively long periods
of time before being cleared from the subject and additional
administrations are required to bring the agent's level back into
the therapeutically effective range. Thus, in certain of these
embodiments, dosing intervals are longer than the agent's
elimination half-life.
[0366] In other embodiments, where the compositions have relatively
narrow therapeutic ranges, it may be important calculate the
maximum and minimum concentrations that will occur at particular
dosing interval(s). In preferred embodiments, the minimal
steady-state concentration of administered agents are determined
using equations, optionally corrected for the bioavailability of
the agents, which are well known to those skilled in the
pharmacological arts.
[0367] In still other embodiments, where the agents follow
multiexponential kinetics and the agents are administered orally,
the estimation of the maximal steady-state concentration involves
manipulation of several exponential constants concerning agent
distribution and absorption.
[0368] The present invention also provides methods for
administering loading doses of an agent, or agents, to a subject.
As used herein, a "loading dose" is one or a series of doses that
when given at the onset of a treatment quickly provide the target
concentration of the therapeutic agent. In some embodiments,
loading doses are administered to a subject having an immediate
need for the target level of an agent in relation to the time
required to attain a steady-state target level of the agent
provided using a constant rate of administration. Various negative
considerations should be weighed against the exigency of the
subject's condition and her need for a loading dose prior to its
administration. These considerations include, but are not limited
to: 1) loading doses are often administered in one large bolus
which may abruptly subject the patient to a toxic concentration of
the agent; 2) agents with long half-lives will remain at levels
above the target-level as compared to agents administered under
lower constant rate schemes. Loading doses are often large, rapid,
and given parenterally, thus dangerous side effects can potentially
occur at the site of administration before the agent can obtain
equilibrium in the subject's plasma.
[0369] In preferred embodiments, the clinician rationally designs
an individualized dosing regimen based on known pharmacological
principles and equations. In general, the clinician designs an
individualized dosing regimen based on knowledge of various
pharmacological and pharmacokinetic properties of the agent,
including, but not limited to, F (fractional bioavailability of the
dose), Cp (concentration in the plasma), CL (clearance/clearance
rate), Vss (volume of drug distribution at steady state) Css
(concentration at steady state), and t1/2 (drug half-life), as well
as information about the agent's rate of absorption and
distribution. Those skilled in the art are referred to any number
of well known pharmacological texts (e.g., Goodman and Gilman's,
Pharmaceutical Basis of Therapeutics, 10th ed., Hardman et al.,
eds., 2001) for further explanation of these variables and for
complete equations illustrating the calculation of individualized
dosing regimes. Those skilled in the art also will be able to
anticipate potential fluctuations in these variables in individual
subjects. For example, the standard deviation in the values
observed for F, CL, and Vss is typically about 20%, 50%, and 30%,
respectively. The practical effect of potentially widely varying
parameters in individual subjects is that 95% of the time the Css
achieved in a subject is between 35 and 270% that of the target
level. For drugs with low therapeutic indices, this is an
undesirably wide range. Those skilled in the art will appreciate,
however, that once the agent's Cp (concentration in the plasma) is
measured, it is possible to estimate the values of F, CL, and Vss
directly. This allows the clinician to effectively fine tune a
particular subject's dosing regimen.
[0370] In still other embodiments, the present invention
contemplates that continuing therapeutic drug monitoring techniques
be used to further adjust an individual's dosing methods and
regimens. For example, in one embodiment, Css data is used is to
further refine the estimates of CL/F and to subsequently adjust the
individual's maintenance dosing to achieve desired agent target
levels using known pharmacological principles and equations.
[0371] Therapeutic drug monitoring can be conducted at practically
any time during the dosing schedule. In preferred embodiments,
monitoring is carried out at multiple time points during dosing and
especially when administering intermittent doses. For example, drug
monitoring can be conducted concomitantly, within fractions of a
second, seconds, minutes, hours, days, weeks, months, etc., of
administration of the agent regardless of the dosing methodology
employed (e.g., intermittent dosing, loading doses, maintenance
dosing, random dosing, or any other dosing method)., However, those
skilled in the art will appreciate that when sampling rapidly
follows agent administration the changes in agent effects and
dynamics may not be readily observable because changes in plasma
concentration of the agent may be delayed (e.g., due to a slow rate
of distribution or other pharmacodynamic factors). Accordingly,
subject samples obtained shortly after agent administration may
have limited or decreased value.
[0372] The primary goal of collecting biological samples from the
subject during the predicted steady-state target level of
administration is to modify the individual's dosing regimen based
upon subsequently calculating revised estimates of the agent's CL/F
ratio. However, those skilled in the art will appreciate that early
postabsorptive drug concentrations do not typically reflect agent
clearance. Early postabsorptive drug concentrations are dictated
principally by the agent's rate of absorption, the central, rather
than the steady state, volume of agent distribution, and the rate
of distribution. Each of these pharmacokinetic characteristics have
limited value when calculating therapeutic long-term maintenance
dosing regimens. Accordingly, in preferred embodiments, when the
objective is therapeutic long-term maintenance dosing, biological
samples are obtained from the subject, cells, or tissues of
interest well after the previous dose has been administered, and
even more preferably shortly before the next planned dose is
administered.
[0373] In still other embodiments, where the therapeutic agent is
nearly completely cleared by the subject in the interval between
doses, then the present invention contemplates collecting
biological samples from the subject at various time points
following the previous administration, and most preferably shortly
after the dose was administered.
[0374] In yet other embodiments, when low clearance of the agent is
problematic, and toxicity issues are likely to result from its
accumulation, the present invention contemplates measuring agent
concentrations immediately before the administration of the
subsequent dose. In these embodiments, the determination of maximal
and minimal agent concentrations are preferred.
[0375] The methods of the present invention further contemplate
that when a constant maintenance dosage is administered, steady
state is reached only after expiration of four agent half-lives.
Samples collected too soon after dosing begins do not accurately
reflect agent clearance. However, for potentially highly toxic
agents, significant toxicity and damage may already have ensued
before expiration of the agent's fourth half-life. Thus, in some
instances when it is important to maintain control over agent
concentrations, a first sample is taken after two half-lives,
assuming a loading dose has not been administered. If agent
concentration already exceeds 90% of the eventual expected mean
steady-state concentration, the dosage rate is halved, and another
sample obtained following an additional two half-lives. The dosage
is halved again if this sample once more exceeds the target level.
If the first concentration does not exceed tolerable limits,
subsequent administrations are given at the initial dose rate. If
the concentration is lower than expected, the steady state can
likely be achieved in about two half-lives, and at this point the
dosage rate can be adjusted as described herein.
[0376] In embodiments comprising intermittent dosages, an
additional concern related to timing of collection of concentration
information, is if the sample was obtained immediately before the
next scheduled dose, concentration will be at a minimal value, not
the mean; however, as discussed herein, the estimated mean
concentration can be calculated using equations known in the
pharmacological arts.
[0377] When administering therapeutic agents having first-order
kinetics, the average, minimum, and maximum concentrations at
steady state are linearly related to the dose and dosing rate.
Thus, in these embodiments, the ratio between the measured and the
desired agent concentrations is used to adjust dosing.
[0378] In another aspect of the present invention, computer
programs are helpful in designing dosing regimens. Typically, these
programs take into account the measured drug concentrations and
various factors (e.g., measured or predicted) related to the
individual subjects.
[0379] The present invention is not limited to any particular
temporal constraints on collecting subject, tissue, cell culture,
or animal drug administration data or samples. Moreover, the
present invention is not limited to collecting any particular type
of samples (e.g., biological samples) from a subject, tissue, cell
culture, or test animal laboratory animal or otherwise. Indeed, in
some embodiments, the present invention contemplates acquiring
biological samples including, but not limited to, polynucleotides,
polypeptides, lipids, carbohydrates, glycolipids, ionic species,
metabolites, inorganic molecules, macromolecules and macromolecular
precursors as well as cell fractions, blood (e.g., cellular and
soluble or insoluble blood components including, but not limited
to, plasma, serum, metabolites, factors, enzymes, hormones, and
organic or inorganic molecules), exudates, secretions, sputum,
excreta, cell and tissue biopsies, CNS fluids (cerebrospinal
fluid), secretions of lachrymal, salivary, and other glands,
seminal fluids, etc., and combinations of these or any other
subcellular, cellular, tissular, organismal, systemic, or
organismic biological materials. Biological samples taken from a
subject can be analyzed for chemical or biochemical changes (e.g.,
changes in gene expression) or other effects resultant from
administration of the therapeutic agent. Further biological sample
and sampling consideration are described below.
[0380] In some of these embodiments, the biological and
pharmacological effects of the therapeutic compositions are
determined using routine laboratory procedures on the collected
samples including, but not limited to, microscopy (e.g., light,
fluorescence (confocal fluorescence, immunofluorescence),
phase-contrast, differential interference-contrast, dark field, or
electron (transmission, scanning, cryo-), NMR, autoradiography),
cell sorting techniques (e.g., fluorescence-activated),
chromatography techniques (e.g., gel-filtration, ion exchange,
hydrophobic, affinity, HPLC), electrophoretic techniques (e.g.,
SDS-PAGE, 2D-, 3D-, isoelectric focusing), ultracentrifugation,
immunocytochemical and immunohistochemical technologies (e.g.,
ELISA, Western blotting, Immuno blotting), nucleic acid, including
recombinant, technologies (e.g., PCR (inverse, reverse, nested),
Northern blotting, Southern blotting, Southwestern blotting, in
situ hybridization, FISH, nick-translation, DNAse footprinting,
DNAse hypersensitivity site mapping, Maxam-Gilbert sequencing,
Sanger sequencing, gel-shift (mobility shift) analysis, Si nuclease
analysis, RNAse protection assay, CAT assays, transgenic
techniques, knock-out techniques, and reporter gene systems), amino
acid analysis (e.g., Edman degradation), morphological,
pathological, or phenotypical observations, and other observations
with or without aid of instrumentation.
[0381] In some embodiments, subjects are questioned directly or
indirectly regarding their state of health and any changes
attributable to the administration of the therapeutic compositions
(e.g., drugs, small molecules, and other therapeutic agents and
techniques) and methods of the present invention.
[0382] Various interpatient and intrapatient pharmacokinetic
considerations affect the design of dosing and administration
regimens for individual patients. For any given drug, there may be
wide variations in its pharmacokinetic properties in a particular
subject, and up to one-half or more of the total variation in
eventual response. The importance of these variable factors depends
in part upon the agent and its usual route of elimination. For
example, agents that are primarily removed by the kidneys and
excreted unchanged into the urinary system, tend to show less
interpatient variability in subjects with similar renal function
than agents that are metabolically inactivated. Agents that are
extensively metabolized, and agents that have high metabolic
clearance and large first-pass elimination rates show large
differences in interpatient bioavailability. Agents with slower
rates of biotransformation typically have the largest variation in
elimination rates among individual subjects. Differences in subject
genotypes also plays an important part in determining different
metabolic rates. Pathological and physiological variations in
individual subjects' organ functions (e.g., renal or hepatic
diseases) are major factors that can affect an agent's rate of
disposition. Kidney or liver diseases often impair drug disposition
and thus increase interpatient drug variability. Other factors
(e.g., age) can also affect the responsiveness of targeted cells
and tissues (e.g., the brain) to a particular composition or method
of the present invention, and can alter the expected range of the
therapeutic target level for the agent.
[0383] When invasive patient samples (e.g., blood, serum, plasma,
tissues, etc.) are necessary to determine the concentration of the
therapeutic agent(s) in a subject, design of the collection
procedures should be undertaken after considering various criteria
including, but not limited to: 1) whether a relationship exists
between the concentration of the agent and any desired therapeutic
effects or avoidable toxic effects; 2) whether these is substantial
interpatient variability, but small intrapatient variation in agent
disposition; 3) whether it is otherwise difficult or impractical to
monitor the effects of the agent; and 4) whether the therapeutic
concentration of the agent is close to the toxic concentration. In
still other embodiments, concentration measurements are
supplemented with additional measurements of pharmacokinetic,
pharmacodynamic, or pharmacological effects.
[0384] In some instances, considerable interpatient response
variations exist after the concentration of agent has been adjusted
to the target level. For some agents, this pharmacodynamic
variability accounts for much of the total variation in subject
response. In some embodiments, the relationship among the
concentration of an agent and the magnitude of the observed
response may be complex, even when responses are measured in
simplified systems in vitro, although typically a sigmoidal
concentration-effect curve is seen. Often there is no single
characteristic relationship between agent concentration (e.g., in
the subject's plasma) and measured effect. In some embodiments, the
concentration-effect curve may be concave upward. In other
embodiments, the curve is concave downward. In still other
embodiments, the data plots are linear, sigmoid, or in an inverted
U-shape. Moreover, the resulting concentration-effect relationship
curves can be distorted if the response being measured is a
composite of several effects. In some preferred embodiments, the
composite concentration-effect curves are resolved into simpler
component curves using calculations and techniques available to
those skilled in the art.
[0385] The simplified concentration-effect relationships,
regardless of their exact shape, can be viewed as having four
characteristic variables: potency, slope, maximal efficacy, and
individual variation. Those skilled in the art will appreciate that
the potency of an agent is measured by the intersection of the
concentration-effect curve with the concentration axis. Although
potency is often expressed as the dose of an agent required to
produce the desired effect, it is more appropriately expressed as
relating to the concentration of the agent in the subject (e.g., in
plasma) that most closely approximates the desired situation in an
in vitro system to avoid complicating pharmacokinetic variables.
Although potency affects agent dosing, knowledge of an agent's
potency alone is relatively unimportant in clinical use so long as
a dose sufficient to obtain the target level can be conveniently
administered to the subject. It is generally accepted that more
potent agents are not necessarily therapeutically superior to less
potent agents. One exception to this principle, however, is in the
field of transdermal agents.
[0386] The maximum effect that an agent can induce in a subject is
called its maximal or clinical efficacy. An agent's maximal
efficacy is typically determined by the properties of the agent and
its receptor-effector system and is reflected in the plateau of the
concentration-effect curve. In clinical use, however, an agent's
dosage may be limited by undesirable effects (e.g., toxicity), and
the true maximal efficacy of the agent may not be practically
achievable without harming the subject.
[0387] The slope and shape of the concentration-effect curve
reflects the agent's mechanism of action, including the shape of
the curve that, at least in part, describes binding to the agent's
receptor. The rise of the concentration-effect curve indicates the
clinically useful dosage range of the agent. Those skilled in the
art will appreciate that the dosage ranges recited herein are
approximations based on sound pharmacological principles and that
actual responses will vary among different individuals given the
same concentration of an agent, and will even vary in particular
individuals over time. It is well known that concentration-effect
curves are either based on an average response, or are tailored to
reflect an actual response in a particular individual at a
particular time.
[0388] The concentration of an agent that produces a specified
effect in a particular subject is called the individual effective
concentration. Individual effective concentrations usually show a
lognormal distribution, resulting in a normal variation curve from
plotting the logarithms of the concentration against the frequency
of achieving the desired effect. A cumulative frequency
distribution of individuals achieving the desired effect as a
function of agent concentration is called the concentration-percent
curve or quantal concentration-effect curve. The shape of this
curve is typically sigmoidal. The slope of the
concentration-percent curve is an expression of the pharmacodynamic
variability in the population rather than an expression of the
concentration range from a threshold to a maximal effect in the
individual patient.
[0389] Those skilled in the art will appreciate that the median
effective dose (ED.sub.50) is the dose of an agent sufficient to
produce the desired effect in 50% of the population.
[0390] In preclinical drug studies, the dose (MTD) is determined in
experimental animals. The ratio of the MTD to the ED.sub.50 is an
indication of the agent's therapeutic index and is a measurement of
the selectivity of the agent in producing its desired effects. In
clinical studies, the dose, or preferably the concentration, of an
agent sufficient to produce toxic effects is compared to the
concentration required for the therapeutic effects in the
population to provide a clinical therapeutic index. However, due to
individual pharmacodynamic variations in the population, the
concentration or dose of an agent required to produce the
therapeutic effect in most subjects occasionally overlaps the
concentration that produces toxicity in some subjects despite the
agent having a large therapeutic index. Those skilled in the art
will appreciate that few therapeutic agents produce a single
effect, thus, depending on the effect being measured, the
therapeutic index for the agent may vary.
[0391] Preferred embodiments of the present invention provide
approaches to individualize dosing levels and regimens. In
preferred embodiments, optimal treatment regimens for particular
subjects are designed after considering a variety of biological and
pharmacological factors including, but not limited to, potential
sources of variation in subject response to the administered
agent(s), diagnosis specifics (e.g., severity and stage of disease,
presence of concurrent diseases, etc.), other prescription and non
prescription medications being taken, predefined efficacy goals,
acceptable toxicity limits, cost-benefit analyses of treatment
versus non treatment or treatment with other various available
agents, likelihood of subject compliance, possible medication
errors, rate and extent of agent absorption, the subject's body
size and compositions, the agent's distribution, the agent's
pharmacokinetic profile (e.g., physiological variables,
pathological variables, genetic factors and predispositions, drug
interactions, potential drug resistances, predicted rate of
clearance), potential drug-receptor interactions, functional state,
and placebo effects.
[0392] In preferred embodiments, the clinician selects an
appropriate marker for measuring the ultimate effectiveness of the
administered agent(s) in the subject. The present invention
contemplates that in some embodiments, appropriate markers of an
agent's effectiveness include a decrease (or increase) in some
measurable biological state, condition, or chemical level (e.g.,
toxin load, viral titer, antigen load, temperature, inflammation,
blood cell counts, antibodies, tumor morphology, and the like). A
large number of diagnostic procedures and tests are available for
gathering information on various markers including, but not limited
to, cell culture assays (e.g., invasion assays in soft-agar and the
like), radiographic examination (e.g., chest X-ray), computed
tomography, computerized tomography, or computerized axial
tomography (CAT) scans, positron emission tomography (PET) scans,
magnetic resonance imaging (MRI or NMRI), mammography,
ultrasonography (transvaginal, transcolorectal), scintimammography
(e.g., technetium 99m sestamibi, technetium-99m tetrofosmin),
aspiration (e.g., endometrial), palpation, PAP tests (e.g.,
smears), sigmoidoscopy (e.g., flexible fiberoptic), fecal occult
blood testing (e.g., Guaiac-based FOBT), digital rectal
examination, colonoscopy, virtual colonoscopy (also known as
colonography), barium enema, stool analysis (See e.g., K. W.
Kinzler and B. Vogelstein, Cell, 87(2):159-70 (1996); S. M. Dong et
al., J. Natl. Cancer Inst., 93(11):858-865 (2001); G. Traverso et
al., N. Engl. J. Med., 346(5):311-20 (2002), G. Traverso et al.,
Lancet, 359(9304):403 (2002); and D. A. Ahlquist et al.,
Gastroenterology, 119(5):1219-1227, (2000)), serum
prostate-specific antigen (PSA) screening, endoscopy, gallium
scans, marrow and tissue biopsies (e.g., core-needle, percutaneous
needle biopsy, thoracotomy, endometrial, etc.) and histological
examinations, direct and/or indirect clinical observations (e.g.,
patient surveys, inquiries, or questionnaires), cytological
sampling and collection of biological tissues, fluids, and markers
therein, (e.g., blood, urine (e.g., hematuria screening, urinary
cytologic examinations), sputum (e.g., sputum cytology), feces, CNS
fluids (e.g., LPs, spinal taps), blood products, including proteins
and peptides (e.g., Bcl-2 family proteins), cancer markers (e.g.,
CA 125 (ovarian cancer), CA 15-3 (breast cancer), CEA (ovarian,
lung, breast, pancreas, and gastrointestinal tract cancers), PSA
(prostate cancer), p53 gene product, MIC2 gene product),
metabolites (e.g., vanillylmandelic acid (VMA), and homovanillic
acid (HVA)), antigens (e.g., serum alpha-fetoprotein (AFP)), salts,
minerals, vitamins, soluble factors, insoluble factors, nucleic
acids, and the like).
[0393] For any compound used in the methods of the invention, the
therapeutically effective dose can be estimated initially from the
concentration of compounds that causes fifty percent cell growth
inhibition and/or cell killing in the cell culture assays.
Subsequently, dosages can be formulated in animal models (e.g.,
murine models) to achieve a desirable circulating concentration
(target-level) range that induces the desired effect (e.g.,
apoptosis) in target cells characterized by elevated expression
levels of Bcl-2 family proteins. A therapeutically effective dose
is the amount of gossypol compound (and in some embodiments, and
additional therapeutic agents (e.g., chemotherapeutic and/or
anit-neoplastic agents) sufficient to ameliorate (or prevent) the
symptoms of a disease or pathology (e.g., unregulated cell
proliferation, growth, invasion, autoimmunity).
[0394] In preferred embodiments, the toxicity and therapeutic
efficacy of agents is determined using standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., for
determining the MTD and the ED.sub.50. Agents that exhibit large
therapeutic indices are preferred. The data obtained from cell
culture assays or animal models can be used to formulate dosing
ranges in, for example, mammals (e.g., humans, Equus caballus,
Fells catus, and Canis familiaris, etc.). Preferable dosing
concentrations are near the calculated or observed ED.sub.50 value
for an agent. More preferable dosing concentrations are near an
agent's ED.sub.50 value and cause little or no toxicity. Any given
dosage may vary within, exceed, or be less than, the therapeutic
index for any particular agent, depending upon the formulation,
sensitivity of the patient, and the route of administration.
[0395] In some embodiments, from 1, 2, 3, 4, 5, . . . 10, . . . 20,
. . . 35, . . . 55, . . . 100, . . . 1,000, . . . 10,000, or more,
units of time (e.g., minutes, hours, days, weeks, etc.) pass
between the first administration of a therapeutic agent and
subsequent administration. In some of these embodiments, the
interval(s) between any two or more administration points are
constant (e.g., of equal duration). In still other embodiments, the
interval(s) between any two or more administration points are
varied (e.g., not of equal duration). Varied intervals can be
either random or repeating and formulaic. Those skilled in the art
will appreciate the steps necessary for designing and adjusting the
dosing schedules and/or the dosing order of any one or more
agents.
[0396] Accordingly, preferred methods of the present invention are
not limited to providing any particular order or sequence for
administering the gossypol compounds and non-gossypol additional
therapeutic agents to a subject or to in vitro/ex vivo cells,
tissues, or organs. For example, in some embodiments, a gossypol
compound is administered to a subject or to in vitro cells,
tissues, or organs, followed by one or more additional agents.
[0397] The present invention provides the following exemplary
formulas to illustrate the flexibility available to the skilled
clinician when designing dosing regimens comprising one or more
gossypol compound and optionally one or more non-gossypol compound
(e.g., conventional anticancer drug), therapy (e.g., radiotherapy),
or technique (e.g., surgical intervention). Thus, each variable
represents the subjection of the patient or in vitro cells,
tissues, or organs of interest to a therapeutic event (e.g., the
administration of a gossypol compound). It is understood that the
exemplary formulas represent a portion of the total possible
formulaic combinations and permutations of the particular variables
used in this exemplary. It is further understood, one skilled in
the art could complete the exemplary listing of formulas to recite
every possible permutation of the recited variables. It is also
understood that any implied time intervals between adjacent
variables can represent simultaneous therapeutic events, or the
elapse of milliseconds, seconds, minutes, hours, days, weeks,
months, or years. G1=a first administration of a gossypol compound;
G2=a second administration of a gossypol compound, G3=a third
administration of a gossypol compound; Gn=a fourth administration
of a gossypol compound; NGC1=a first administration of a
non-gossypol compound, therapy, or technique; NGC2=a second
administration of a non-gossypol compound, therapy, or technique;
NGC3=a third administration of a non-gossypol compound, therapy, or
technique; and NGCn=a fourth administration of a non-gossypol
compound, therapy, or technique, such that the following exemplary
administration regimens are possible: (G1); (G1, G2); (G1, G2, G3);
(G1, G2, G3, Gn); (G1, NGC1, G1, NGC1, NGC2); (G1, NGC1, NGC2,
NGC3); (G1, NGC1, NGC2, NGC3, NGCn); (G1, G2, NGC1); (G1, G2, NGC1,
NGC2); (G1, G2, NGC1, NGC2, NGC3); (G1, G2, NGC1, NGC2, NGC3,
NGCn); (G1, G2, G3, NGC1); (G1, G2, G3, NGC1, NGC2); (G1, G2, G3,
NGC1, NGC2, NGC3); (G1, G2, G3, NGC1, NGC2, NGC3, NGCn); (G1, G2,
G3, Gn, NGC1); (G1, G2, G3, Gn, NGC1, NGC2); (G1, G2, G3, Gn, NGC1,
NGC2, NGC3); (G1, G2, G3, Gn, NGC1, NGC2, NGC3, NGCn); (NGC1, G1);
(NGC1, G1, G2); (NGC1, G1, G2, G3); (NGC1, G1, G2, G3, Gn); (NGC1,
NGC2, G1); (NGC1, NGC2, G1, G2); (NGC1, NGC2, G1, G2, G3); (NGC1,
NGC2, G1, G2, G3, Gn); (NGC1, NGC2, NGC3, G1); (NGC1, NGC2, NGC3,
G1, G2); (NGC1, NGC2, NGC3, G1, G2, G3); (NGC1, NGC2, NGC3, G1, G2,
G3, Gn); (NGC1, NGC2, NGC3, NGCn, G1); (NGC1, NGC2, NGC3, NGCn, G1,
G2); (NGC1, NGC2, NGC3, NGCn, G1, G2, G3); (NGC1, NGC2, NGC3, NGCn,
G1, G2, G3, Gn); (G1, NGC1, G2); (G1, NGC1, G2, G3); (G1, NGC1, G2,
G3, Gn); (G1, G2, NGC1, G3); (G1, G2, NGC1, G3, Gn); (G1, G2, G3,
NGC1, Gn); (NGC1, G1, NGC2); (NGC1, G1, NGC2, NGC3); (NGC1, G1,
NGC2, NGC3, NGCn); (NGC1, NGC2, G1, NGC3); (NGC1, NGC2, NGC3, G1,
NGCn); (G1, NGC1, NGC2, G2); (G1, NGC1, NGC2, G2, G3); (G1, NGC1,
NGC2, G2, G3, Gn); (G1, NGC1, NGC2, NGC3, G2); (G1, NGC1, NGC2,
NGC3, G2, G3); (G1, NGC1, NGC2, NGC3, G2, G3, Gn); (G1, NGC1, NGC2,
NGC3, NGCn, G2); (G1, NGC1, NGC2, NGC3, NGCn, G2, G3); (G1, NGC1,
NGC2, NGC3, NGCn, G2, G3, Gn); (G1, NGC1, G2, NGC2); and (G1, NGC1,
G2, NGC2, G3); (G1, NGC1, G2, NGC2, G3, Gn).
[0398] In some embodiments, from 1, 2, 3, 4, 5, . . . 10, . . . 20,
. . . 35, . . . 55, . . . 100, . . . 1,000, . . . 10,000, or more,
administrations of an agent (or agents) are required to produce the
desired effect (e.g., amelioration of a disease such as a
neoplastic disease) in a subject or in in vitro cells, tissues, or
organs of interest. The methods of the present invention are not
limited to the administration of any particular gossypol compound,
and optionally any one or more additional therapeutic agents,
surgical interventions, or radiotherapies. In some embodiments, at
least one gossypol compound is administered to a subject
substantially simultaneously with at least one additional
therapeutic agent, surgical intervention, or radiotherapy.
[0399] The present invention is not limited to any particular
pharmaceutical formulations. Indeed, in some contemplated
pharmaceutical compositions and methods, a gossypol compound is
formulated (e.g., in suspension) with a non-gossypol therapeutic
agent. In other pharmaceutical compositions and methods, a
multitude of gossypol compounds (e.g., 2 or more) and optionally a
multitude of non-gossypol therapeutic agents (e.g., 2 or more) are
formulated in any combination thereof. Accordingly, the present
invention is not limited to any particular formulations for
combining two or more gossypol compounds and/or two or more
non-gossypol therapeutic agents. However, as described herein, and
as routinely known in the chemical, biological, and pharmacological
arts, certain gossypol compounds and non-gossypol therapeutic
agents are preferentially combined or segregated. Certain
pharmaceutical compositions optionally comprise stabilizers,
preservatives, adjuvants, pH modifiers, bioavailability modifiers,
additives, excipients, diluents, lubricants, anti-oxidants,
disintegrating agents, binders, thickening agents, emulsifiers,
surfactants, sweeteners, pigments, flavorings, perfuming agents and
the like, to improve various biological, chemical, or
pharmaceutical characteristics.
[0400] Normal dosage amounts may vary from about 0.001 to 1,000 mg,
up to a total dose of about 1 g, depending upon the route of
administration. Guidance as to particular dosages and methods of
delivery are provided in the literature (See e.g., U.S. Pat. Nos.
4,657,760; 5,206,344; and 5,225,212, U.S. Pat. No. 6,041,788, U.S.
Pat. No. 6,273,727, U.S. Pat. No. 6,558,957, U.S. 20030017459A1,
U.S. Pat. No. 5,782,799, U.S. Pat. No. 6,056,734, U.S. Pat. No.
6,528,086, U.S.20020065483A1, WO 0168169A1, and WO02072178A1 all of
which are herein incorporated by reference). Administration of some
agents to a patient's bone marrow may necessitate delivery in a
manner different from intravenous injections.
[0401] In some embodiments, the gossypol compounds are administered
at a dosage range of about 1 to 1,000 mg/day, preferably about 1 to
200 mg/day, more preferably from about 10 to 80 mg/day, and most
preferably from about 30 to 40 mg/day. In some preferred
embodiments, the gossypol compounds are administered (e.g., orally)
in a tolerable daily dose (e.g., 30 to 40 mg/day) shown to have
some biologic activity (e.g., alterations in Bcl proteins,
angiogenesis proteins, cell cycle alteration, apoptosis markers, or
alterations in Rb and Cyclin D1 levels). In a further embodiment,
the gossypol compounds are administered at a dosage range of about
40 to about 500 mg/week).
[0402] In other embodiments, the effective dose of the gossypol
compounds will typically be in the range of about 0.01 to about 50
mg/kg, preferably about 0.1 to about 10 mg/kg of mammalian body
weight, administered in single or multiple doses. Generally, the
compositions may be administered to subjects in need of such
treatment in a daily dose range of about 1 to about 2,000 mg per
subject.
[0403] Preferred embodiments of the present invention provide
pharmaceutical compositions and methods for administering an
effective amount of a gossypol compound (and optionally one or more
non-gossypol therapeutic agents, such as conventional anticancer
drugs) to a subject to inhibit cell (e.g., cancer cell)
proliferation. In some other preferred embodiments, the present
invention further provides pharmaceutical compositions and methods
of coadministering an effective amount of at least one conventional
anticancer agent in addition to gossypol to a patient, such that
cell (e.g., cancer cell) proliferation is inhibited.
[0404] In preferred embodiments, the subject has a disease
characterized by the overexpression of a Bcl-2 family protein
(e.g., Bcl-2, Bcl-X.sub.L, Mcl-1, A1/BFL-1, and BOO-DIVA, etc.). In
some embodiments, diseases characterized by overexpression of a
Bcl-2 family protein include, but are not limited to,
hyperproliferative diseases, cancers, acquired immune deficiency
syndrome (AIDS), degenerative conditions, and vascular
diseases.
[0405] In still further embodiments, neoplastic diseases (e.g.,
cancers) suitable for treatment (and optionally prevention) by the
present compositions and methods include, but are not limited to,
breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic
cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer,
brain cancer, primary brain carcinoma, head-neck cancer, glioma,
glioblastoma, liver cancer, bladder cancer, non-small cell lung
cancer, head or neck carcinoma, breast carcinoma, ovarian
carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms' tumor,
cervical carcinoma, testicular carcinoma, bladder carcinoma,
pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic
carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal
carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell
carcinoma, endometrial carcinoma, adrenal cortex carcinoma,
malignant pancreatic insulinoma, malignant carcinoid carcinoma,
choriocarcinoma, mycosis fungoides, malignant hypercalcemia,
cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic
lymphocytic leukemia, chronic granulocytic leukemia, acute
granulocytic leukemia, acute myelogenous leukemia, chronic
myelogenous leukemia, hairy cell leukemia, neuroblastoma,
rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential
thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma,
soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia,
and retinoblastoma. However, the present invention is not intended
to be limited to treating (and optionally preventing) any
particular type of cancer.
[0406] In some embodiments, diseases suspected of being
characterized by having elevated levels of Bcl-2 family protein(s)
suitable for treatment (and optionally prevention) by the present
invention are selected by obtaining a sample of interest (e.g.,
cells, tissues, fluids, etc.) suspected of having high levels of
Bcl-2 family proteins (e.g., Bcl-2, Bcl-X.sub.L, Mcl-1, A1/BFL-1,
and BOO-DNA, etc.), measuring the levels of Bcl-2 family proteins
in the sample using one or more well established
immunohistochemical techniques (e.g., ELISA and Western blots,
etc.), and comparing the levels of Bcl-2 family proteins in the
sample with levels of Bcl-2 family proteins in relevant reference
nonpathological samples. In other embodiments, diseases suspected
of being characterized by having elevated levels of one or more
Bcl-2 family proteins (e.g., Bcl-2, Bcl-X.sub.L, Mcl-1, A1/BFL-1,
and BOO-DNA, etc.) are selected by comparing levels of one or more
markers (e.g., polynucleotides, polypeptides, lipids, etc.) in a
sample (e.g., cells, tissues, fluids, etc.) that directly or
indirectly indicate elevated levels of Bcl-2 family proteins in the
sample as compared to levels of these markers in relevant
nonpathological samples. In still further embodiments, diseases
suspected of being characterized by having elevated levels of Bcl-2
family proteins (e.g., Bcl-2, Bcl-X.sub.1, Mcl-1, A1/BFL-1, and
BOO-DIVA, etc.) are selected from diseases that do not respond or
that stop responding to treatment with one or more conventional
anticancer therapies (e.g., chemotherapy, radiation therapy, and/or
surgical intervention).
[0407] The present invention is not intended to be limited to the
administration routes chosen for delivering agents to a subject.
Indeed, a number of suitable administration routes are
contemplated, the selection of which is within the skill of those
in the art.
[0408] In still other preferred embodiments taxanes (e.g.,
docetaxel) are administered to a patient in combination with
gossypol compounds. The classic docetaxel dosing schedule is 60-100
mg/m.sup.2 every 3 weeks. However, recent studies suggest that
taxanes can be given safely, with perhaps higher dose intensity, on
a weekly schedule. (See e.g., J. D. Hainsworth et al., J. Clin.
Oncology, 16:2164-2168 (1998); J. D. Hainsworth et al., J. Clin.
Oncology, 19:3500-3505 (2001); and C. Kouroussis et al., Cancer
Chemo. Pharm., 46:488-492 (2000)). The patient toxicities
associated with administering taxanes include neutropenia,
asthenia, alopecia, hypersensitivity reactions, skin toxicity, and
edema. Preferred embodiments of the present invention provide
weekly administrations of taxanes to reduce patient toxicities
while preserving agent efficacy. In other embodiments,
administration of taxanes (e.g., docetaxel) more frequently than
once a week during a patient's course of treatment with the
disclosed gossypol compounds is expected to produce synergistic
effects.
[0409] While the present invention is not limited to any particular
mechanism, nor to any understanding of the action of the agents
being administered, the following example provides a description of
an exemplary testing procedure used to determine potential drug
interactions between gossypol compounds and one or more anticancer
agents that are candidates for co-administration with gossypol.
[0410] Docetaxel is extensively metabolized by CYP3A4, a specific
cytochrome p450 enzyme. Pharmacokinetic data obtained for docetaxel
indicates wide variance in its clearance between patients. Poor
docetaxel clearance may result in an increase in the
area-under-the-curve (AUC) and thus greater patient toxicity.
Several investigations have reported that gossypol decreases
cytochrome P-450 and mixed-function oxidases, although these
results have been challenged, and no human studies have been
performed which specifically address this issue. Thus, it is
possible that gossypol could inhibit CYP3A4 activity and lead to
toxic docetaxel accumulation in some patients.
[0411] In one embodiment, the patient is administered a daily dose
of a gossypol compound for 1 week prior to receiving their first
dose of docetaxel. The pharmacokinetic profile of docetaxel in the
patient's system is evaluated after the patient receives their
first dose of docetaxel. (See, R. Bruno et al., J. Clin. Oncol.,
16:187-196 (1998)). Docetaxel dosing is started at a reduced dose
of about 15 mg/m.sup.2/week. The dose of docetaxel is gradually
escalated to a maximally tolerated dose of about 35
mg/m.sup.2/week. Simultaneously, information will be collected on
effects of gossypol administration on the phenotypic expression of
CYP3A4. The phenotypic expression of CYP3A4 is measured easily and
reproducibly using an erythromycin breath test (ERMBT). (See, e.g.,
P. Watkins, Pharmacogenetics, 4:171-184 (1994); and J. Hirth et
al., Clin. Cancer Res., 6:1255-1258 (2000)). The ERMBT test has
been shown to predict steady state trough blood levels of drugs
that are CYP3A4 substrates. Consequently, some embodiments of the
present invention are directed to the co-administration of gossypol
compounds and taxanes (e.g., docetaxel) using an ERMBT to determine
potential drug interactions. Those skilled in the art will
appreciate that similar testing methodologies can be utilized to
determine potential interactions between gossypol compounds and
additional candidate compounds for co-administration.
[0412] In some embodiments, standard immunohistochemical techniques
are used to analyze patient samples before, during, or after
treatment with the methods and compositions of the present
invention. In some of these embodiments, the immunohistochemical
techniques are used to quantify changes in the levels of Bcl-2
family proteins (e.g., Bcl-2, Bcl-X.sub.L, and Bax, etc.). For
example, in some embodiments, antibodies to Bcl-2 (DAKO,
Carpinteria, Calif.), Bcl-X.sub.L, and/or Bax (Zymed, South San
Francisco, Calif.) are used to determine levels of these Bcl-2
proteins in a patient sample. In preferred embodiments, results
from the immunohistochemical studies are interpreted using
well-established criteria known to those in the art, wherein any
cytoplasmic or nuclear staining are considered positive. The
expression levels of Bcl-2, Bcl-X.sub.L, and Bax can be determined
by counting at least 1,000 neoplastic cells in each case and
expressed as a percentage. Expression will be considered high when
the percentage of positive cells is >25% for Bcl-2, and
Bcl-X.sub.L, and >50% for Bax. (See e.g., G. Rassidakis et al.,
Amer. J. Path., 159:527-535 (2001); and S. Shi et al., J.
Histochem. Cytochem., 39:741-748 (1991)). In other embodiments,
intermittent sampling of whole blood is conducted. Samples are
subsequently prepared for fluorescence activated cell sorting
(FACS) analysis of Bcl-2 and Bcl-X.sub.L expression in peripheral
blood lymphocytes (PBLs) and for immunomagnetic selection of
circulating epithelial cells.
[0413] In some embodiments, the primary endpoint of dosing studies
occurs when the maximum tolerated dose of a gossypol compound (at a
particular daily dose, e.g., 30 mg/day), optionally co-administered
with a anticancer drug, is established. In some embodiments,
dose-limiting toxicity (DLT) is established when a given sample
(e.g., a cell, tissue, or fluid sample) exhibits >500
neutrophils per given unit, or upon observing any Grade 3 or 4
toxicities while the patient is being studied.
[0414] In still some other embodiments, to evaluate dose escalation
a minimum of 9 weeks of treatment is required for 2 or more
patients at each dose level. The maximum tolerated dose (MTD) is
defined as the dose at which 33% of patients experience DLT. In
preferred embodiments, doses are allocated to patients according to
the criteria described in the Continual Reassessment Method (J.
O'Quigley et al., Biometrics 46:33-48 (1990)) called Time-to-Event
CRM or (TITE-CRM). Briefly, the TITE-CRM method provides a model
for the time to occurrence of toxic response as a function of dose,
and allows information from all patients enrolled in a trial to be
used when allocating new patient dose levels. Because this method
is very flexible in terms of the number of patients treated at each
dose, subjects may be continuously recruited throughout a trial,
without recruitment pauses, as long as patients are treated at a
dose consistent with the safety profile.
[0415] In preferred embodiments, diseased cells and tissues are
subjected to assays for cell viability and signs of induction of
apoptosis (e.g., morphological changes, DNA integrity, mitochondria
pathways, alterations of expression of Bcl-2 family proteins, and
caspase activation as well as upstream and downstream effectors of
caspases and caspase inhibitors). Those skilled in the art will be
able to readily design and execute assays to test these and other
cellular and biochemical parameters in treated cells and
tissues.
X. Exemplary Combination Therapies
[0416] The present invention provides the following exemplary
embodiments comprising the therapeutic administration of a gossypol
compound in combination with additional therapeutic agents such as
conventional chemotherapy agents (anticancer drugs) and radiation
therapy.
[0417] A. Exemplary Combination Therapies
[0418] Experiments were conducted to further evaluate the in vivo
therapeutic efficacy of various gossypol compounds, such as
(-)-gossypol, alone and in combination with one or more
conventional antineoplastic therapeutic agents. Experiments were
conducted in various mouse xenograft tumor models, including breast
cancer cell line MDA-231, prostate cancer cell line PC-3, ovarian
cancer cell line A2780 and its Bcl-X.sub.L transfected clones,
colon cancer cell line HT-29, and non-small cell lung cancer A549
xenograft mouse models. In several models, the administration of
(-)-gossypol alone provided good antitumor activity and inhibition
of tumor growth. In other models, administration of (-)-gossypol
with docetaxel, paclitaxel, or cisplatin achieved superior
antitumor activity and cancer cell inhibition as compared to the
administration of gossypol alone. Some animals receiving a
combination of gossypol and a conventional antineoplastic
therapeutic agent showed complete tumor regression.
[0419] In certain generalized experiments, 4-6 weeks old male
athymic NCr-nu/nu nude mice were used in coadministration studies
involving administration of a gossypol compound, e.g.,
(-)-gossypol, in combination with a conventional anticancer
chemotherapeutic agent. In one embodiment, tumor xenografts were
established by injecting MDA-MD-231 cells (1.times.10.sup.6) into
both side fat pads, or by injecting PC-3, HT-29, or A549 cells
(5.times.10.sup.6) into both flanks of each mouse. Tumors were
measured with a caliper in two dimensions, length (a), and width
(b). The tumor volumes were calculated by (length X width.sup.2,
a.times.b.sup.2). Treatments were initiated at day 5-10 post
inoculation, when the majority of tumor diameters were about 5-7 mm
and tumor volume had reached about 50 mm.sup.3. For efficacy
testing, racemic gossypol and/or isolated (-)-gossypol enantiomer
were given orally at 7.5 to 30 mg/kg every day in 0.1 ml of 10%
ethanol for four weeks.
[0420] In certain other combination treatment experiments, racemic
gossypol and/or isolated (-)-gossypol enantiomer were given orally
to xenografted mice at daily doses of from 7.5 to 15 mg/kg. 24
hours later after administration of the gossypol compound test
animals were given a dose of conventional anticancer
chemotherapeutic agent. Conventional anticancer chemotherapeutic
agents were administered as follows: docetaxel at 7.5 mg/kg given
intravenously once a week; and/or paclitaxel at 10 mg/kg, three
times per week intraperitoneally. The combination treatments lasted
for 3 consecutive weeks. All mice in the control group were
injected with 0.1 ml PBS. Approximate tumor sizes and body weights
were measured two to three times a week. Average tumor volumes and
standard deviations were calculated for each group and plotted.
[0421] Antitumor activity curves for racemic gossypol and the
(-)-gossypol enantiomer were plotted with observation time on the
X-axis, and corresponding tumor volume (geometric mean) on the
Y-axis. The area under the curve (AUC) was calculated by Tai's
mathematical model for each curve, and shown as geometric means and
95% confidential intervals. The difference of AUC among treatment
groups was compared by ANOVA. T-test was used for other
analysis.
[0422] Previous studies have shown that overexpression of
Bcl-X.sub.L protects cancer cells from docetaxel induced apoptosis.
Thus, the present invention contemplates that compositions and
methods for coadministering gossypol compounds, e.g., (-)-gossypol
with conventional anticancer chemotherapeutics, such as docetaxel,
provides a more effective treatment than either agent used alone,
especially in disease characterized by high expression levels of
Bcl-2 family proteins (e.g., Bcl-2 and/or Bcl-X.sub.L). While the
present invention is not limited to any particular mechanisms, it
is contemplated that administration of gossypol compounds to
diseased cells and tissues overexpressing Bcl-2 family proteins
(e.g., Bcl-2 and/or Bcl-X.sub.L) sensitizes these diseased cells
and tissues to the therapeutic effects of conventional
chemotherapeutic agents, such as docetaxel and/or radiation
therapy. Accordingly, various other experiments were performed to
test the effectiveness of coadministering (-)-gossypol with
docetaxel as a therapeutic treatment for neoplastic diseases, such
as cancer. Representative data from these experiments in a mouse
xenograft model of human breast cancer MDA-231 are provided in FIG.
18. Briefly, in FIG. 18 docetaxel treatment was started at day 7
and was given intravenously at a weekly dose of 7.5 mg/kg for 3
weeks. The results show that treatment with docetaxel alone in a
sub-optimal dose (7.5 mg/kg weekly) was sufficient to inhibit tumor
growth. However, the coadministration of (-)-gossypol, at any one
of three doses levels (e.g., 7.5, 15 or 30 mg/kg), with docetaxel
achieved greater anticancer activity and inhibition of tumor cell
growth. Three out of ten mice treated with the gossypol docetaxel
combination had complete tumor regression. Overall, there was more
than 90% inhibition of tumor growth in the combination group as
compared to the control group. Statistical analyses were performed
using a mixed-effects repeated measures model that accurately takes
into account the correlation within an animal over time, and
between tumors within an animal. The data were modeled using the
natural logarithm of tumor volume, which is standard practice for
tumor growth models. A comparison of tumor growth in animals
receiving (-) -gossypol alone and docetaxel alone was performed. An
exemplary comparison of (-)-gossypol at 7.5 mg/kg and docetaxel is
provided in Table 7.
TABLE-US-00007 TABLE 7 (-)-Gossypol Docetaxel 7.5 mg/kg 7.5 mg/kg
Combination (-)-Gossypol -- <0.0001 <0.0001 7.5 mg/kg
Docetaxel <0.001 -- <0.0001 7.5 mg/kg Combination <0.0001
0.0028 --
[0423] In a subset of the mice treated with either docetaxel alone
or combination therapy, a second round of treatments with the same
regimen was initiated at day 45. The average tumor volume before
the second cycle treatments was about 2,000 mm.sup.3. Tumors in the
docetaxel alone treated group continued to grow, thus all the mice
were sacrificed due to the tumor burden. In contrast, in the
combination treatment groups, animals displayed tumor regression
and a 50% reduction of tumor volume (FIG. 19). The data shows that
(-)-gossypol is very effective at potentiating the agent docetaxel
when administered in combination treatments even at levels where
(-)-gossypol administered alone is not, or only partially,
effective.
[0424] The outcome of coadministration of (-)-gossypol and
conventional anticancer chemotherapy agents was further tested in
additional mouse xenograft cancer models. The results of one
experiment conducted in the mouse A549 non-small cell lung
carcinoma xenograft are shown in FIG. 20. The A549 cell line
expresses high levels of Bcl-X.sub.L protein. The mice were
administered a daily oral dose of 7.5 mg/kg (-)-gossypol in
combination with a weekly dose of paclitaxel (i.p., 15 mg/kg). Over
90% tumor inhibition was observed. The results of the combination
treatment were statistically significant compared to administration
of either drug alone (P<0.002 vs. paclitaxel alone; P<0.001
vs. (-)-gossypol alone). No adverse effects were seen with any
dose.
[0425] Summaries of still other in vivo studies of (-)-gossypol in
various cancer xenograft models are provided in Tables 8-15, as
shown below.
TABLE-US-00008 TABLE 8 Drug/Dose/ Tumor Host Route Regimen Results
MDA-231 Balb/c nude (-)-Gossypol at (-)-gossypol q day There was no
difference between the human mouse, all 7.5, 15 or 30 mg/kg, for 4
weeks untreated control and the vehicle breast female, 4-6 p.o.
beginning ~7 days control. cancer, weeks old Docetaxel at after
tumor cell There was dose-dependent bilateral 8 mice (16 7.5 mg/kg,
i.v. inoculation inhibition of tumor growth by (-)- fat-pad tumors)
per Controls: Docetaxel q week gossypol (G-) alone. At a dose of
xenografts group Untreated, and for 3 weeks 30 mg/kg of G-, there
was 40% (~50 mm.sup.3 vehicle beginning ~1 day tumor growth
inhibition, but lower at start of control. after gossypol doses of
G- (15 or 7.5 mg/kg) alone treatment) treatment did not achieve
significant anti- or 8 days after tumor effect in this experiment.
tumor cell Docetaxel on this dose regimen. inoculation achieved
about 70% tumor growth Drugs given alone inhibition alone, but
tumors grew or in combination back 2-3 weeks after treatment
stopped. Combinations of G- at any of the three doses with
docetaxel at 7.5 mg/kg all achieved significant tumor growth
inhibition. At 7.5 mg/kg of G- in combination with docetaxel, there
was more than 90% tumor growth inhibition with p < 0.01. There
was tumor regression in 3 mice treated with combination regimen.
There was no weight loss or death in mice treated with
gossypol.
TABLE-US-00009 TABLE 9 Drug/Dose/ Tumor Host Route Regimen Results
PC-3 NCr-nu Both racemic Both racemic Racemic gossypol at 15 mg/kg,
human nude mouse, gossypol and gossypol and (-)- daily p.o. for 26
days, achieved prostate all male, 5-6 (-)-gossypol at gossypol q
day for limited tumor growth inhibition cancer, weeks old 15 mg/kg,
p.o. 4 weeks beginning (15.6% on day 42). The (-)-gossypol
bilateral 5 mice (10 Docetaxel at ~7 days after tumor (G-) alone
achieved moderate tumor xenografts tumors) per 7.5 mg/kg, i.v.,
cell inoculation growth inhibition (52% on day 42, in flank group
in combination Docetaxel q week p = 0.08). region with (-)- for 3
weeks Docetaxel at this dose regimen (~100 mm.sup.3 gossypol only
beginning ~11 days achieved about 67% tumor growth at start of And
vehicle after gossypol inhibition alone (p = 0.005), whereas
treatment) control treatment the combinations of G- with or 16 days
after docetaxel at 7.5 mg/kg achieved tumor cell 84% tumor growth
inhibition as inoculation compared with vehicle control Drugs given
alone (p = 0.002). The combination or in combination treatment is
also more significant than the docetaxel alone (P = 0.059). More
importantly, there was complete tumor regression in 3 out of 5 mice
treated with combination regimen. There was no weight loss in mice
treated with gossypol.
TABLE-US-00010 TABLE 10 Drug/Dose/ Tumor Host Route Regimen Results
PC-3 NCr-nu Racemic Both racemic Racemic gossypol at 15 mg/kg,
human nude mouse, gossypol at 15 mg/kg, gossypol and (-)- daily
p.o. for 28 days, achieved prostate all male, 5-6 p.o. gossypol q
day for limited tumor growth inhibition cancer, weeks old
(-)-Gossypol at 4 weeks beginning (39% on day 56, p = 0.145)
compared bilateral 8 mice (16 7.5 and 15 mg/kg, ~17 days after to
vehicle controls. The (-)-gossypol xenografts tumors) per p.o.
tumor cell (G-) alone achieved moderate in flank group Docetaxel at
inoculation antitumor activity (60% on day 56, region 7.5 mg/kg,
i.v. Docetaxel q week p = 0.0028). The difference between (~120
mm.sup.3 And vehicle for 3 weeks the racemic and (-)-gossypol is at
start of control beginning ~5 days significant (p = 0.0055).
treatment) after gossypol Docetaxel at this dose regimen treatment
achieved about 70% tumor growth or 23 days after inhibition alone
compared to tumor cell controls (p = 0.0016), whereas the
inoculation combinations of G- with docetaxel Drugs given alone at
7.5 mg/kg achieved 89.9% tumor or in combination inhibition as
compared with vehicle control (p = 0.004). The combination
treatment is more effective as compared with the (-)-gossypol alone
or docetaxel alone (p = 0.009). More importantly, there was
complete tumor regression in 5 out of 8 mice in the combination
treatment regimen, whereas there was no tumor regression with
docetaxel alone. There was no weight loss in mice treated with
gossypol.
TABLE-US-00011 TABLE 11 Drug/Dose/ Tumor Host Route Regimen Results
PC-3 NCr-nu (-)-Gossypol at (-)-Gossypol q day Either (-)-gossypol
(G-) or cisplatin human nude mouse, 7.5 mg/kg, p.o. for 4 weeks
(CDDP) alone at this dose regimen prostate all male, 5-6 Cisplatin
at 5 mg/kg, beginning ~11 days achieved only 20-25% tumor growth
cancer, weeks old i.v. after tumor cell inhibition. (p = 0.85 for
G-, p = 0.79 bilateral 8 mice (16 Controls: inoculation for CDDP,
not significant), whereas xenografts tumors) per Untreated, and
Cisplatin twice q the combinations of G- with in flank group
vehicle week for 2 weeks cisplatin at 5 mg/kg achieved 65% region
control. beginning ~6 days tumor growth inhibition as (~74 mm.sup.3
after gossypol compared with vehicle control at start of treatment
(p = 0.0038) or either drug alone treatment) or 17 days after (p =
0.028 for G-, p = 0.013 for tumor cell CDDP). inoculation There was
no complete tumor Drugs given alone regression in the either drug
alone or or in combination the combination treatment regimen. There
was 10-15% weight loss for CDDP alone, and 20-23% weight loss for
the combination regimen during the treatment. Mice started to gain
back the weight 1-2 weeks after the CDDP treatment stopped.
TABLE-US-00012 TABLE 12 Drug/Dose/ Tumor Host Route Regimen Results
HT-29 NCr-nu (-)-Gossypol at (-)-Gossypol q day The (-)-gossypol
(G-) alone human nude mouse, 7.5 mg/kg, p.o. for 4 weeks achieved
moderate tumor growth colon all male, 10 Cisplatin at 10 mg/kg,
beginning ~6 days inhibition (47% on day 29). The cancer, weeks old
i.v. after tumor cell difference between the (-)-gossypol bilateral
5 mice (10 And untreated inoculation and control is significant (p
= 0.02). xenografts tumors) per control Cisplatin twice q There was
no cisplatin or in flank group week for 2 weeks combination group
data as the dose region beginning ~1 day of cisplatin used was too
high. Most (~160 mm.sup.3 after gossypol mice were dead in the
cisplatin at start of treatment treated group during the 2.sup.nd
week of treatment) or 7 days after the treatment. tumor cell
inoculation Drugs given alone or in combination
TABLE-US-00013 TABLE 13 Drug/Dose/ Tumor Host Route Regimen Results
A549 NCr-nu (-)-Gossypol at (-)-Gossypol q day The (-)-gossypol
(G-) alone human non- nude mouse, 7.5 mg/kg, p.o. for 4 weeks
achieved moderate tumor growth small cell all female, Paclitaxel at
beginning ~16 days inhibition (57% on day 31). The lung 5-6 weeks
15 mg/kg, i.p. after tumor cell difference between the (-)-gossypol
carcinoma old And untreated inoculation and control group is
significant bilateral 5 mice (10 control Paclitaxel twice q (p =
0.0459). xenografts tumors) per week for 3 weeks Paclitaxel at this
dose regimen in flank group beginning ~3 days achieved about 67%
tumor growth region after gossypol inhibition alone (p = 0.0166)
whereas (~75 mm.sup.3 treatment the combinations of G- with at
start of or 19 days after paclitaxel at 7.5 mg/kg achieved
treatment) tumor cell 76% tumor growth inhibition as inoculation
compared with vehicle control Drugs given alone (p = 0.0024). The
combination or in combination treatment is more effective as
compared with the (-)-gossypol alone (p = 0.0034) or paclitaxel
alone (p = 0.0038). There were no complete tumor regressions seen
in this experiment. There was no weight loss in mice treated with
gossypol and/or Paclitaxel.
TABLE-US-00014 TABLE 14 Drug/Dose/ Tumor Host Route Regimen Results
PC-3 NCr-nu (-)-Gossypol at (-)-Gossypol 5 The (-)-gossypol (G-) or
radiation human nude mouse, 10 mg/kg, p.o. times q week for 4 alone
achieved limited tumor prostate all male, 5-6 Radiation at 2 weeks
beginning growth inhibition in this dose cancer, weeks old Gy per
day, ~13 days after regimen (about 20%, P = 0.3 or 0.7, bilateral 5
mice (10 total 30 Gy, tumor cell day 39, not significant).
xenografts tumors) per locally inoculation Combinations of G- with
radiation in flank group towards tumor Radiation 2 Gy 5 achieved
93% tumor growth region site, this is times q week for 3 inhibition
as compared with vehicle (~150 mm.sup.3 radiation plus weeks
beginning control (p = 0.0024, day 39). The at start of vehicle. ~5
days after combination treatment is more treatment) Controls:
gossypol treatment effective as compared with the (-)- Untreated,
and or 18 days after gossypol alone (p = 0.0013, day 39) vehicle
alone tumor cell or radiation alone (p = 0.0011, day control.
inoculation 39). G- and radiation There were no complete tumor
given alone or in regressions seen in this experiment. combination
There was no significant weight loss in mice treated with gossypol
and/or radiation.
TABLE-US-00015 TABLE 15 Drug/Dose/ Tumor Host Route Regimen Results
PC-3 NCr-nu (-)-Gossypol at (-)-Gossypol 5 The (-)-gossypol alone
achieved human nude mouse, 10 mg/kg, p.o. times q week for 4
limited tumor growth inhibition in prostate all male, 5-6 Radiation
at 2 weeks beginning this dose regimen (about 30%, cancer, weeks
old Gy per day, ~13 days after p = 0.549, not significant).
bilateral 8 mice (10 total 30 Gy, tumor cell The radiation alone
achieved xenografts tumors) per locally inoculation moderate tumor
growth inhibition in in flank group towards tumor Radiation 2 Gy 5
this dose regimen as compared with region site, this is times q
week for 3 the control group (55%, p = 0.0459, (~70 mm.sup.3
radiation plus weeks beginning day 37). at start of vehicle. ~5
days after Combinations of G- with radiation treatment And vehicle
gossypol treatment achieved 91.5% tumor growth alone control. or 18
days after inhibition as compared with vehicle tumor cell control
(p = 0.0024, day 37). The inoculation combination treatment is more
G- and radiation effective as compared with the (-)- given alone or
in gossypol alone (p = 0.0112) or combination radiation alone (p =
0.0068). There was complete tumor regression in 10 out of 16 tumors
in the combination treatment regimen, whereas there was no tumor
regression with single agent alone. There was no significant weight
loss in mice treated with gossypol and/or radiation.
[0426] B. Gossypol Compounds in the Treatment of Head and Neck
Squamous Cell Carcinoma
[0427] There are about 40,000 new cases of head and neck squamous
cell carcinoma (HNSCC) diagnosed in the United States each year.
The 5-year survival rates for patients afflicted with
[0428] HNSCC are typically not good, in part because the treatment
of locally advanced head and neck cancers with conventional chemo-
and radiation therapies is hampered by the emergence of resistant
cancer cells.
[0429] Abnormal programmed cell death plays a critical role in
cancer progression and outcome following conventional chemo- and
radiation therapies. While the present invention is not limited to
any particular mechanisms, it is contemplated that HNSCCs develop
resistance to conventional chemo- and radiation therapies by
developing the ability to suppress chemotherapy-induced apoptosis.
(A. M. Petros et al., Protein Sci., 9:2528-2534 (2000); A. F.
Schott et al., Oncogene, 11:1389-1394 (1995); and J. C. Reed et
al., Ann. Oncol., 5:61-65 (1994)). Studies have shown that 74% of
laryngeal tumors express high levels of Bcl-X.sub.L and 15%
overexpress Bcl-2 (D. K. Trask et al., Laryngoscope, 112:638-644
(2002)), and low expression of Bcl-X.sub.L has been associated with
excellent response in advanced laryngeal cancers to chemo- and
radiation therapies. Bcl-2 and Bcl-X.sub.L are homologous members
of the Bcl-2 family of proteins. Bcl-2 and Bcl-X.sub.L function as
potent suppressors of mitochondrial-mediated apoptosis.
[0430] In in vitro studies, gossypol induced apoptosis in breast,
colon, and prostate cancer cells that had high levels of Bcl-2 or
Bcl-X.sub.L expression. As the majority of HNSCCs express high
levels of Bcl-X.sub.L, the present invention provides compositions
and methods for administering gossypol compounds, e.g.,
(-)-gossypol, to treat HNSCCs and other head and neck cancers.
[0431] Data obtained during the development of the present
invention shows a correlation between response to (-)-gossypol and
Bcl-X.sub.L/Bcl-X.sub.S expression ratios among the HNSCC cell
lines examined. Thus, it is contemplated that anti/pro-apoptotic
protein expression ratios represent a predictive measure of
cellular apoptotic potential. Additional data suggests that when
Bcl-X.sub.L is the dominant anti-apoptotic factor, that Bcl-X.sub.S
is the dominant anti-apoptotic factor in HNSCCs. In Example 17, in
the panel of 10 HNSCC cell lines, only one, UM-SCC-74B, had no
detectable expression of Bcl-X.sub.L but a medium level of Bcl-2
protein expression. All of the tumor HNSCC cell lines tested were
much more sensitive to (-)-gossypol than were normal fibroblast
cell lines. While not being limited to any particular mechanisms,
the present invention contemplates that sensitivity in HNSCC cell
lines is related to the absence of a deregulated cell survival
pathway in normal cells. Bel-X.sub.L is expressed in fibroblasts in
low levels and (-)-gossypol does not induce either apoptosis or
growth inhibition until high concentrations are reached.
[0432] Experiments conducted during the development of the present
invention found that p53 status plays an important role in
(-)-gossypol-induced apoptosis in some cancers (e.g., HNSCCs). In
this regard, cell lines with wild type p53 exhibited much more
robust induction of apoptosis in response to (-)-gossypol treatment
relative to tumor cells with mutant p53. The present invention is
not limited however to any particular mechanisms, and indeed a
mechanistic understanding of the present invention is not required
to make and use the present compositions and methods. Thus, it is
contemplated that cell-killing by (-)-gossypol, as determined in
the MTT assays conducted in Example 17, is not solely through
apoptosis.
[0433] In some embodiments, cisplatin resistance in HNSCCs
correlates to the presence of wild type p53 and high Bcl-X.sub.L
expression in in vitro studies. Animal studies demonstrate that
tumors with high expression of p53, correlating to p53 mutation,
are more likely to be controlled by chemo- and radiation therapy
treatment protocols. In some embodiments, low expression levels of
Bcl-X.sub.L correlates to induction chemotherapy using cisplatin
and 5-fluorouracil. Thus, the present invention shows that a subset
of tumors and cell lines with wild type p53 and high expression of
Bcl-X.sub.L are resistant to cisplatin-based therapeutic regimens.
However, in some preferred embodiments, the compositions and
methods of the present invention show that gossypol compounds,
e.g., (-)-gossypol, provides significant antitumor activity against
cell lines with wild type p53 and high levels of Bcl-X.sub.L that
are unlikely to respond to conventional anticancer chemotherapeutic
agents including cisplatin (e.g. UM-SCC-1 and -6; both have high
levels of Bcl-X.sub.L expression and wild-type p53). Accordingly,
in some embodiments, the present invention provides compositions
and methods for the combined administration of conventional
anticancer chemotherapeutic agents, such as cisplatin, with
gossypol compounds, (e.g., (-)-gossypol), that effectively treat
typically chemo- and/or radiation therapy resistant HNSCCs. In some
additional embodiments, the present invention contemplates
diagnostic methods for detecting expression of a Bcl-2 family
protein (e.g., Bcl-2 and/or Bcl-X.sub.L) and p53 as an aid in
selecting an appropriate therapeutic intervention (e.g., avoidance
of cisplatin, or use of cisplatin with the compositions and methods
of the present invention).
[0434] (-)-Gossypol selectively inhibits UM-SCC cell growth. Ten
UM-SCC cell lines exposed to (-)-gossypol in a 6-day MTT assay
(Example 17) showed dose-dependent inhibition of cell growth over a
range from 0.5 to 10 .mu.M, while fibroblast cell lines showed
little change relative to untreated controls at doses <10 .mu.M
(FIG. 21). FIG. 21 shows growth inhibition of HNSCC cells by
(-)-gossypol. UM-SCC and human fibroblast cell lines were
continuously exposed to varying concentrations of (-)-gossypol in
6-day MTT cell survival assays. Control wells contained media
(DMEM) with vehicle alone. For each data point n=5. In this assay,
(-)-gossypol had a mean IC.sub.50 of 5.57.+-.2.57 .mu.M in HNSCC
cell lines which is significantly lower than the mean IC.sub.50 of
20.31.+-.9.20 .mu.M in fibroblasts (p=0.0142). (-)-Gossypol
selectively inhibits head and neck squamous cell carcinoma.
[0435] Additional experiments described in Example 17 determined
the protein expression levels of Bcl-2, --X.sub.L, and --X.sub.S in
UM-SCC cell lines using Western blot analyses (See, FIG. 22).
Bcl-X.sub.L is expressed in the majority of UM-SCC cell lines
(9/10) in this panel, with only UM-SCC 74B lacking detectable
expression. Bcl-2 is expressed in UM-SCC-74B, -17B and -6. All cell
lines expressed Bcl-XS at some level.
[0436] In still other embodiments, levels of Bcl-2 family protein
expression were evaluated for correlation with (-)-gossypol's in
vitro activity. The relative levels of expression for Bcl-X.sub.L
and Bcl-X.sub.S were evaluated by densitometry but no statistically
significant correlation with sensitivity to (-)-gossypol was
identified (r=-0.14, p=0.70 and r=0.20, p=0.58). As both
Bcl-X.sub.L and --X.sub.S expression are common and vary among cell
lines, the possibility that the Bcl-X.sub.L/Bcl-X.sub.S ratio was
associated with (-)-gossypol's in vitro effect was investigated.
Interestingly, as shown in FIG. 23, there appeared to be a
correlation between the Bcl-X.sub.L/Bcl-X.sub.S expression ratios
and cell line sensitivity to (-)-gossypol (r=-0.83, p=0.0029) among
the cell lines examined. FIG. 23 shows Bcl-X.sub.L/Bcl-X.sub.S
protein ratios and (-)-gossypol 6-day MTT IC.sub.50 values.
Densitometry measurements for Bcl-X.sub.L and Bcl-X.sub.S were
recorded for three independent Western blots performed on cell
lines. These ratios were calculated as the Bcl-X.sub.L measurement
divided by the Bcl-X.sub.S measurement from the same experiment.
Ratios were then averaged and plotted in a linear fashion on the Y1
axis; bars, +SD. IC.sub.50 values for (-)-gossypol were plotted on
the Y2 axis. A clear inverse correlation is shown between
Bcl-X.sub.L/X.sub.S ratio and sensitivity to (-)-gossypol (r=-0.83,
p=0.0029).
[0437] In Example 17 four cell lines, UM-SCC-1, -6, -12 and -14A,
were shown to exhibit significant cell death following 48 hour
exposure to 10 .mu.M (-)-gossypol, with surviving (trypan blue
excluding) fractions of 20%, 9%, 14% and 16% respectively relative
to untreated controls (p=<0.0001).
[0438] TUNEL assays for apoptosis were performed on fibroblasts and
a subset of four UM-SCC cell lines that spanned the spectrum of
sensitivity to (-)-gossypol (FIGS. 24A-24C). Briefly, FIGS. 24A-24C
show apoptosis after (-)-gossypol as measured by a fluorescent flow
cytometric TUNEL assay. Open peaks represent BrdU labeling of
untreated cells. Shaded peaks indicate BrdU labeling of cell
populations following 48-hour treatment with 10 .mu.M (-)-gossypol;
FIG. 24A, UM-SCC-cell lines with wild type p53; FIG. 24B,
UM-SCC-cell lines with mutant p53; and FIG. 24C normal fibroblast
cells. No clear correlation between proportion of apoptotic cells
and cell line sensitivity to (-)-gossypol was found. However,
UM-SCC-1, which is very sensitive to (-)-gossypol, displayed the
highest apoptotic fraction following (-)-gossypol treatment
(AI=90.1%). In contrast, fibroblast cell lines showed no induction
of apoptosis. Of the four tumor cell lines, UM-SCC-1 (IC.sub.50=2
.mu.M) and UM-SCC-6 (IC.sub.50=8 .mu.M) cell lines have wild type
p53, while UM-SCC-12 (IC.sub.50=4 .mu.M) and UM-SCC-14A
(IC.sub.50=11 .mu.M) contain p53 mutations. The two wild-type p53
tumor cell lines displayed a mean apoptotic index (AI) of
85.2%.+-.6.9 (UM-SCC-1, AI=90.1%, UM-SCC-6, AI=80.3%). In contrast,
the two cell lines with mutant p53 have a mean apoptotic index of
20.7%.+-.9.3 (UM-SCC-12, AI=27.2%, UM-SCC-14A, AI=14.1%). This
difference is statistically significant with a p-value of
0.0157.
[0439] C. Administration of (-)-gossypol Compounds in Combination
with Radiation Therapy
[0440] The present invention provides methods for administering
gossypol compounds with radiation therapy. The methods of the
present invention comprising the administration of gossypol
compounds (and optionally other chemotherapeutic agents) in
conjunction with radiation therapy are not intended to be limited
to any particular dosing or administration routes. For example, in
some embodiments, the chemotherapeutic agents, including gossypol
compounds and any other chemotherapeutic agent(s), are administered
prior to the subject receiving at least one session or course of
radiation therapy. In other embodiments, the subject receives at
least one session of radiation therapy prior to the administration
of chemotherapeutics (e.g., gossypol compounds and optionally other
anticancer agents). In yet other embodiments, the chemotherapeutic
administrations overlap, at least to some extent, with sessions of
radiation therapy. Those skilled in the fields of medicine (e.g.,
oncology, radiology, dosimetry, medical physics, pathology,
histology, and the like) and pharmacology will appreciate that, in
general, the treatment of diseases, and especially cancers,
including the therapeutic administration of compositions and
methods of the present invention, is a dynamic process.
[0441] In preferred embodiments, when radiation therapy is used
with the pharmaceutical compositions and methods of the present
invention, the subject's medical team considers a number of factors
including, but not limited to, the type, amount, delivery, field
size, and duration of the radiation, the subject's health and
medical history, the type and stage of cancer being treated, and
many other factors.
[0442] The present invention is not limited by the types, amounts,
or delivery and administration systems used to deliver the
therapeutic dose of radiation to a subject. For example, the
subject may receive photon radiotherapy, particle beam radiation
therapy, other types of radiotherapies, and combinations thereof.
In some embodiments, the radiation is delivered to the subject
using a linear accelerator. In still other embodiments, the
radiation is delivered using a gamma knife.
[0443] The source of radiation can be external or internal to the
patient. External radiation therapy is most common and involves
directing a beam of high-energy radiation to a tumor site through
the skin using, for instance, a linear accelerator. While the beam
of radiation is localized to the tumor site, it is nearly
impossible to avoid exposure of normal, healthy tissue. However,
external radiation is usually well tolerated by patients. Internal
radiation therapy involves implanting a radiation-emitting source,
such as beads, wires, pellets, capsules, particles, and the like,
inside the body at or near the tumor site including the use of
delivery systems that specifically target cancer cells (e.g., using
particles attached to cancer cell binding ligands). Such implants
can be removed following treatment, or left in the body inactive.
Types of internal radiation therapy include, but are not limited
to, brachytherapy, interstitial irradiation, intracavity
irradiation, radioimmunotherapy, and the like.
[0444] The subject may optionally receive radiosensitizers (e.g.,
metronidazole, misonidazole, intra-arterial Budr, intravenous
iododeoxyuridine (IudR), nitroimidazole,
5-substituted-4-nitroimidazoles, 2H-isoindolediones,
[[(2-bromoethyl)-amino]methyl]-nitro-1H-imidazole-1-ethanol,
nitroaniline derivatives, DNA-affinic hypoxia selective cytotoxins,
halogenated DNA ligand, 1,2,4 benzotriazine oxides,
2-nitroimidazole derivatives, fluorine-containing nitroazole
derivatives, benzamide, nicotinamide, acridine-intercalator,
5-thiotretrazole derivative, 3-nitro-1,2,4-triazole,
4,5-dinitroimidazole derivative, hydroxylated texaphrins,
cisplatin, mitomycin, tiripazamine, nitrosourea, mercaptopurine,
methotrexate, fluorouracil, bleomycin, vincristine, carboplatin,
epirubicin, doxorubicin, cyclophosphamide, vindesine, etoposide,
paclitaxel, heat (hyperthermia), and the like), radioprotectors
(e.g., cysteamine, aminoalkyl dihydrogen phosphorothioates,
amifostine (WR 2721), IL-1, IL-6, and the like). Radiosensitizers
enhance the killing of tumor cells. Radioprotectors protect healthy
tissue from the harmful effects of radiation.
[0445] A typical course of treatment for most types of cancer
involves the use of radiation therapy. Radiation therapy uses a
beam of high-energy particles or waves, such as X-rays and gamma
rays, to eradicate cancer cells by inducing mutations in cellular
DNA. In that cancer cells divide more rapidly than normal cells,
tumor tissue is more susceptible to radiation than normal tissue.
Radiation also has been shown to enhance exogenous DNA expression
in exposed cells. In a preferred embodiment, the inventive method
further comprises administering a dose or multiple doses of
radiation to a patient over the therapeutic period.
[0446] In one embodiment, intratumoral delivery of a nucleic acid
sequence encoding cytotoxic factors (e.g., TNF-.alpha.) and
confocal radiation to the tumor site results in localized delivery
of two potent anti-cancer treatment modalities. When the nucleic
acid sequence encoding the cytotoxic factor is operably linked to a
radiation-inducible promoter, radiation potentiates the factor's
production and maintains therapeutic levels of factor at the tumor
site continuously throughout the period of radiation therapy. The
present invention contemplates that the disclosed methods provide
additive or synergistic effects of radiation and cytotoxic factor,
and gossypol compounds to eradicate tumor cells.
[0447] Any type of radiation can be administered to a patient, so
long as the dose of radiation is tolerated by the patient without
unacceptable negative side-effects. Suitable types of radiotherapy
include, for example, ionizing (electromagnetic) radiotherapy
(e.g., X-rays or gamma rays) or particle beam radiation therapy
(e.g., high linear energy radiation). Ionizing radiation is defined
as radiation comprising particles or photons that have sufficient
energy to produce ionization, i.e., gain or loss of electrons (as
described in, for example, U.S. Pat. No. 5,770,581 incorporated
herein by reference in its entirety). The effects of radiation can
be at least partially controlled by the clinician. The dose of
radiation is preferably fractionated for maximal target cell
exposure and reduced toxicity.
[0448] The total dose of radiation administered to a patient
preferably is about 0.01 Gray (Gy) to about 100 Gy. More
preferably, about 10 Gy to about 65 Gy (e.g., about 15 Gy, 20 Gy,
25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, or 60 Gy) are
administered over the course of treatment. While in some
embodiments, a complete dose of radiation can be administered over
the course of one day, the total dose is ideally fractionated and
administered over several days. Desirably, radiotherapy is
administered over the course of at least about 3 days, e.g., at
least 5, 7, 10, 14, 17, 21, 25, 28, 32, 35, 38, 42, 46, 52, or 56
days (about 1-8 weeks). Accordingly, a daily dose of radiation will
comprise approximately 1-5 Gy (e.g., about 1 Gy, 1.5 Gy, 1.8 Gy, 2
Gy, 2.5 Gy, 2.8 Gy, 3 Gy, 3.2 Gy, 3.5 Gy, 3.8 Gy, 4 Gy, 4.2 Gy, or
4.5 Gy), preferably 1-2 Gy (e.g., 1.5-2 Gy). The daily dose of
radiation should be sufficient to induce destruction of the
targeted cells. If stretched over a period, radiation preferably is
not administered every day, thereby allowing the subject to rest
and the effects of the therapy to be realized. For example,
radiation desirably is administered on 5 consecutive days, and not
administered on 2 days, for each week of treatment, thereby
allowing 2 days of rest per week. However, radiation can be
administered 1 day/week, 2 days/week, 3 days/week, 4 days/week, 5
days/week, 6 days/week, or all 7 days/week, depending on the
subject's responsiveness and any potential side effects. Radiation
therapy can be initiated at any time in the therapeutic period.
Preferably, radiation is initiated in week 1 or week 2, and is
administered for the remaining duration of the therapeutic period.
For example, radiation is administered in weeks 1-6 or in weeks 2-6
of a therapeutic period comprising 6 weeks for treating, for
instance, a solid tumor. Alternatively, radiation is administered
in weeks 1-5 or weeks 2-5 of a therapeutic period comprising 5
weeks. These exemplary radiotherapy administration schedules are
not intended however to limit the present invention.
[0449] In modern oncology, radiation therapy is used to treat a
wide variety of cancers with varying degrees of effectiveness.
X-ray irradiation strongly induces apoptosis. For example,
radiation therapy is currently used to treat all stages of
localized prostate cancer, however, certain clinical and
radiobiological evidence indicates that prostate cancer cells are
relatively resistant to radiation therapy. In particular, PC-3, a
human prostate cancer cell line, is hormone-refractory and
resistant to current chemo- and radiation therapies. The present
invention determined that PC-3 cells express very high levels of
both Bcl-2 and Bcl-X.sub.L protein. Overexpression of Bcl-2 and
Bcl-X.sub.L proteins provides, at least in part, resistance to
chemo- or radiation therapy induced apoptosis that is observed in
many types of cancer cells (e.g., PC-3 prostate cancer cell line).
In some preferred embodiments, chemo- or radiation therapy
resistant cancers (e.g., prostate cancer) expressing high levels of
Bcl-2 family proteins (e.g., Bcl-2 and/or Bcl-X.sub.L, and the
like) are targets for treatment using the methods and compositions
of the present invention. While any understanding of particular
mechanisms is not important to make and use the compositions and
methods of the present invention, and the present invention is not
so limited, it is contemplated that antagonizing the anti-apoptotic
functions of Bcl-2 and/or Bcl-X.sub.L using the gossypol compounds
(and optionally administration of other therapeutic agents) of the
present invention overcomes the resistance to conventional
radiation therapies seen in many types of cancers.
Examples
[0450] The following examples are provided to demonstrate and
further illustrate certain preferred embodiments of the present
invention. The examples are not to be construed as limiting the
scope of the present invention.
Example 1
Homology Modeling
[0451] The sequence of human Bcl-2 was obtained from Gene Bank
(entry gi4557355) (SEQ ID NO:1). (See, FIG. 1). The NMR structure
of Bcl-X.sub.L (pdb code: 1BXL from the protein databank), which
has 45% amino acid sequence identity, 56% sequence similarity and
3% gaps with Bcl-2, was used as the template. The structure of
Bcl-2 was built using the homology-modeling program MODELLER
(version 4.0). (A. Sali et al., Structure, Function, and Genetics,
23:318-326 (1995); and A. Sali, Curr. Opin. Biotech., 6:437-451
(1995)). Further refinement was done using the molecular dynamics
program CHARMM (version 27b2). (B. R. Brooks, J. Comput. Chem.,
4:187-217 (1983)). Hydrogen atoms were assigned to the modeled
structure using the program QUANTA (Molecular Simulations Inc., San
Diego, Calif.). The Bak BH3 peptide was placed into the Bcl-2 BH3
domain binding site in the same orientation as in the NMR structure
of Bcl-X.sub.L in complex with the Bak BH3 peptide (1BXL in protein
databank). (S. Michael et al., Science, 275:983-986 (1997)). The
complex structure was solvated by inserting it into a 60 .ANG.
diameter TIP3P water sphere and deleting solvent molecules that
have heavy atoms at less then 2.5 .ANG. from any protein heavy
atom.
[0452] MD simulations were done using the all atom parameter set
from the MSI CHARMM force field(ref 23) in QUANTA 98, a constant
dielectric, .epsilon.=1 and constant temperature, T=300 K. The leap
frog method with 1 fs time step was applied for numerical
integration. Long-range electrostatic forces were treated with the
force switch method with a switching range of 8-12 .uparw.1. (See,
B. R. Brooks et al., J. Comput. Chem., 4:187-217 (1983)). Van der
Waals forces were calculated with the shift method and a cutoff of
12 .ANG.. The nonbond list was kept to 14 .ANG. and updated
heuristically. Solvent waters were kept from evaporating by using a
spherical miscellaneous mean field potential as implemented in
CHARMM. (B. R. Brooks, supra). The solvated protein was energy
minimized using 100 cycles using the Steepest Descent method and
additional 1000 cycles using the Adopted-Basis Newton Raphson
method. This was followed by 3.0 ns MD simulations. The simulation
was performed on an Origin2000 computer at the Advanced Biomedical
Computing Center at the National Institutes of Health. The
representation of the refined structure of Bcl-2 in complex with
Bak BH3 peptide is shown in FIGS. 2A and 2B.
Example 2
Expression and Purification of the Bcl-X.sub.L Protein
[0453] Recombinant Bcl-X.sub.L protein with a N-terminal His-tag
was overexpressed from the pET15b expression vector (Novagen,
Darmstadt, Germany). In this construct, the putative C-terminal
membrane-anchoring region (residues 214-237) and a loop between
helix 1 and helix 2 (residues 49-88) were removed to facilitate
protein purification. This loop was previously shown to be
dispensable for the anti-apoptotic activity of the protein. (See,
S. W. Muchmore et al., Nature, 381:335-341 (1996)). The current
construct of Bcl-X.sub.L produces about 20 mg of the purified
Bcl-X.sub.L protein from 1 L of cell culture.
[0454] The protein samples for NMR studies were uniformly labeled
with .sup.15N for screening and uniformly double labeled with
.sup.15N and .sup.13C for structure characterization according to
the methods described in M. Jansson et al., J. Biomol. NMR,
7:131-141 (1996), and M. L. Cai et al., J. Biomol. NMR, 11:97-102
(1998).
Example 3
Resolution of (-)-gossypol and (+)-gossypol from Racemic
Gossypol
[0455] Racemic gossypol acetic acid purchased from the National
Cancer Institute (Bethesda, Md.) or commercial sources (e.g.,
Sigma-Aldrich Corp., St. Louis, Mo.) was dissolved in diethyl
ether, washed twice with water to remove the acid and the ether
layer was concentrated by rotary evaporation. To a solution of 1.04
g of gossypol (2 mmol) in 50 ml of CH.sub.2Cl.sub.2 was added 1 g
of L-phenylalanine methyl ester hydrochloride (4.6 mmol), 0.4 g of
NaHCO.sub.3 (4.7 mmol), 3 g of 4 .ANG. molecular sieves, and 1 ml
of 2-propanol, the resulting mixture was stirred at room
temperature overnight under N.sub.2 in the dark and filtered. The
L-phenylalanine methyl ester reacts with the aldehyde groups of
gossypol to form a Schiff's base with two diastereoisomers which
were then resolved on a normal silica flash chromatography column.
The filtrate was concentrated, and the residue was purified by
chromatography on silica gel eluting with hexanes:EtOAc=3:1 to give
two fractions. Acid hydrolysis of the two fractions in 5 N H
Cl:THF=1:5 (room temperature, overnight) regenerated the individual
gossypol enantiomers, respectively. The first fraction with a
higher R.sub.f value contained (-)-gossypol, and the second
fraction with a lower R.sub.f value contained (+)-gossypol. The
crude gossypol fractions were extracted into ether from the residue
after removing THF from the reaction mixture. The gossypol
fractions were then purified by chromatography on silica gel and
eluted with hexanes:EtOAc=3:1 to give optically pure gossypol, with
a yield of 30-40% in two steps. The optical rotatory dispersion
values for these products were .alpha..sub.D=-352.degree. (c=0.65,
CHCl.sub.3) for (-)-gossypol and .alpha..sub.D=+341.degree.
(c=0.53, CHCl.sub.3), in agreement with literature values. (J. Si
and L. Huang, Kexue Tongbao, 28:1574 (1983)) (See, FIG. 25).
Example 4
Fluorescence Polarization Based Binding Assays and Determination of
the Binding of Various Gossypol Compounds to Bcl-2 and Bcl-X.sub.L
Proteins
[0456] The present invention used established sensitive and
quantitative in vitro fluorescence polarization-based (FP) binding
assays to determine the in vitro binding affinity of small molecule
inhibitors (e.g., gossypol compounds) to both Bcl-2 and
Bcl-X.sub.L. (See e.g., J. L. Wang et al., Cancer Res.,
60:1498-1502 (2000); J. L. Wang et al., Proc. Natl. Acad. Sci.
U.S.A., 97:7124-7129 (2000); A. Degterev et al., Nat. Cell Biolog.,
3:173-182 (2001); and I. J. Enyedy et al., J. Med. Chem.,
44:4313-4324 (2001)). The FP-based assays were used to monitor the
displacement of fluorescently labeled BH3 domain peptides from
recombinant Bcl-2 or Bcl-X.sub.L proteins. Once a fluorescently
labeled BH3 peptide (e.g., Bad, Bak, Bid BH3 peptides, and the
like) binds to Bcl-2 or Bcl-X.sub.L protein, fluorescence
polarization is enhanced. When a small molecule inhibitor, such as
a gossypol compound, binds to Bcl-2 or Bcl-X.sub.L it displaces the
fluorescently labeled BH3 domain peptide, thus decreasing the
observed fluorescence polarization. Determination of the binding
affinities of the Bcl-2 and Bcl-X.sub.L proteins is important to
determining their selectivity. Although the structures of Bcl-2 and
Bcl-X.sub.L are very similar, there are some differences between
the two proteins. Accordingly, small molecule inhibitors of Bcl-2
and/or Bcl-X.sub.L (e.g., gossypol compounds) may display
selectivity between the two proteins. In additional embodiments,
the binding of gossypol compounds to Bcl-2 and Bcl-X.sub.L is
further determined and confirmed using the NMR methods outlined
herein.
[0457] Initial screening of Bcl-2 inhibitors (e.g., gossypol,
enantiomers, derivatives, and pharmaceutically acceptable salts
thereof) was carried out at 200 .mu.M. If significant inhibition
was observed for an inhibitor (larger than 50%), its IC.sub.50
value was determined. Five ml of the test compounds, in reaction
buffer, were added to wells containing a tracer and Bcl-2 or
Bcl-X.sub.L proteins provided at the same concentration as the test
compound. Final FP readings were taken after a 10-min incubation at
room temperature. For making IC.sub.50 determinations of initial
test compounds, 9 to 10 test compound concentrations (i.e., between
1 nM and 200 .mu.M) were used. Non-labeled Bak peptide was used as
a positive control. Inactive compounds were used as negative
controls.
[0458] In one embodiment, Bcl-2 fluorescence polarization assays
were carried out as follows. Fluorescein-labeled 16-mer peptide
tracer Flu-Bak-BH3 (GQVGRQLAIIGDDINR (SEQ ID NO:9) derived from Bak
BH3 domain) was synthesized and labeled at the amino terminus.
Forty-six-kDa soluble recombinant GST-fused Bcl-2 protein was
purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).
Reaction were carried out in a total volume of 20 .mu.l per well
(containing 10 .mu.l of 1.times. phosphate-buffered saline, 5 .mu.l
of the GST-Bcl-2 protein, and 5 .mu.l of peptide tracer). The
reaction wells were incubated at room temperature for 20 min. FP
readings were taken at 485 nm and 535 nm using an ULTRA READER
(Tecan U.S. Inc, Research Triangle Park, N.C.). A series of
validation experiments were performed by analyzing the maximal and
minimal signals obtained from the background, buffer, Bcl-2
protein, tracer, and the mixture of Bcl-2 protein and tracer. The
Kd of binding between Bcl-2 protein and the 16-mer
fluorescein-labeled peptide was determined by titrating Bcl-2
protein at concentrations ranging from 5.4 nM to 540 nM and
fluorescent tracer concentrations ranging from 0.145 nM to 1,450
nM. Optimal binding was obtained at a final concentration of 290 nM
fluorescent tracer and 270 nM Bcl-2 protein. To verify the observed
specificity, binding of the fluorescently labeled peptide and
nonlabeled 16-mer peptide were compared in a binding competition
assay. The data indicate that nonlabeled 16-mer peptide was able to
abrogate binding of the labeled tracer, with an IC.sub.50 of
approximately 0.3 .mu.M. The binding of racemic gossypol to Bcl-2
protein under these assay conditions is shown in FIG. 3.
[0459] In another embodiment, Bcl-X.sub.L fluorescence polarization
assays were carried out as follows. Fluorescein-labeled 16-mer
peptide tracer Flu-Bak-BH3 (GQVGRQLMIGDDINR (SEQ ID NO:9) derived
from Bak BH3 domain) was synthesized and labeled at the amino
terminus. Soluble recombinant His-fused Bel-X.sub.L protein, as
described in Example 2, was used. The competitive binding curve for
racemic gossypol is shown in FIG. 3. FIG. 6A shows gossypolone's
competitive inhibition of Bak BH3 peptide binding to Bcl-X.sub.L
protein under the described assay conditions. FIG. 6B shows that
(-)-gossypol ethyl Schiff's base displays time-dependent inhibition
of the binding between Bak BH3 peptide and Bcl-X.sub.L protein. The
present invention is not limited to any mechanism. Indeed, an
understanding of any particular mechanism is unnecessary to
practice (make and use) the present invention. Nonetheless, it is
contemplated that the above finding results from the hydrolysis of
the Schiff's base to form free (-)-gossypol.
[0460] In another embodiment, Bcl-2 fluorescence polarization
assays were carried out as follows. A 25-residue Bad BH3 peptide
(NLWAAQRYGRELRRMSDEFVDSFKK (SEQ. ID. NO.: 10)) labeled at the
N-terminus with 6-carboxyfluorescein succinimidyl ester (FAM) as a
fluorescence tag (Flu-Bad-25) was synthesized. The Bcl-2 protein
used in these assays was a soluble recombinant GST-fusion (Santa
Cruz Biotechnology). An aliquote of the test compound and
preincubated Bcl-2 protein (0.020 .mu.M) and Flu-Bad-25 peptide
(0.005 .mu.M) in the assay buffer (100 mM potassium phosphate, pH
7.5; 100 .mu.g/ml bovine gamma globulin; 0.02% sodium azide
(Invitrogen, Corp., Carlsbad, Calif.) were added to black Dynex
96-well round-bottom plates (Fisher Scientific, Inc., Hampton,
N.H.). In each experiment, a bound peptide control containing Bcl-2
and Flu-Bad-25 peptide (equivalent to 0% inhibition), and free
peptide control containing only free Flu-Bad-25mer (equivalent to
100% inhibition), were included on the assay plate. The
polarization values were measured after 4 hrs of incubation when
the binding reached equilibrium using an ULTRA READER (Tecan U.S.
Inc., Research Triangle Park, N.C.). The K.sub.d value of the
binding of Bad 25-mer BH3 peptide to Bcl-2 was determined to be 6.9
nM (FIG. 26). IC.sub.50 values, the inhibitor concentration at
which 50% of bound peptide is displaced, were determined from a
plot using nonlinear least-squares analysis. Curve fitting was
performed using GRAPHPAD PRISM software (GraphPad Software, Inc.,
San Diego, Calif.). The non-labeled Bad peptide was used as the
positive control. The K.sub.i values were calculated using the
following equation:
K.sub.i=I].sub.50/([L].sub.50/K.sub.p+[PL].sub.0/[L].sub.0+1)=[I].sub.50-
/([L].sub.50/K.sub.d+[P].sub.0/K.sub.d+1) (Equation 1)
[0461] The competitive binding curves of racemic gossypol,
(-)-gossypol and (+)-gossypol in directly blocking binding between
Bad 25-residue BH3 peptide and Bcl-2 are shown in FIG. 4.
[0462] In another embodiment, Bel-X.sub.L fluorescence polarization
assays were carried out as follows. Bid 21-residue BH3 peptide
labeled with 6-carboxyfluorescein succinimidyl ester (FAM) was
synthesized. Recombinant Bel-X.sub.L proteins, as described in
Example 2, were used. Various gossypol compounds (e.g., racemic
gossypol, (-)-gossypol, and (+)-gossypol) were analyzed using the
following assay conditions: 15 nM Bel-X.sub.L and 2.5 nM Flu-Bid
peptide in assay buffer containing 50 mM Bis-Tris, pH 7.4 with
0.01% bovine gamma globulin. The K.sub.d value of Bid 21-mer BH3
peptide biding to Bcl-X.sub.L was determined to be 2.9 nM (FIG.
27). The competitive binding curves of racemic gossypol,
(-)-gossypol, and (+)-gossypol in directly blocking the binding
between Bad 21-residue BH3 peptide and Bcl-X.sub.L are shown in
FIG. 5.
[0463] The competitive binding curve of racemic apogossypol in
directly blocking binding between Bad 25-residue BH3 peptide and
Bcl-2 is shown in FIG. 45.
[0464] The competitive binding curve of racemic apogossypol in
directly blocking the binding between Bad 21-residue BH3 peptide
and Bcl-X.sub.L is shown in FIG. 46.
Example 5
Docking Gossypol into Bcl-2
[0465] This example describes docking studies of gossypol performed
using the DOCK program (version 4.0.1) (S. Makino et al., J.
Comput. Chem., 18:1812-1825 (1997)) and the LigandFit program
available in the Cerius2 molecular modeling package. (Molecular
Simulations Inc., San Diego, Calif.). The structure of Bcl-2 used
for refined docking is the same as that used for database
searching. The structure of gossypol used for docking was built and
minimized in the QUANTA program (Molecular Simulations Inc., San
Diego, Calif.). Atomic charges of gossypol compounds and the Bcl-2
protein were assigned using the Geisteiger method as implemented in
the Sybyl program (Tripos, Inc., St. Louis, Mo.). Refined docking
was performed with the DOCK program (version 4.0.1) using 500
configurations per ligand building cycle, 5,000 maximum
orientations of the anchor, 1,000 maximum minimization cycles and
10,000 maximum iterations per cycle. The default convergence
criteria were used for energy refinement of the docked structure.
The docking with LigandFit program (ReceptorScience, Singapore) was
done using a grid that covered the whole BH3 binding site with 0.2
.ANG. grid spacing. Parameters for gossypol and Bcl-2 were assigned
using the 1997 release of the CFF force field. The position of the
ligand was optimized using the maximum allowed 999,999 Monte Carlo
steps. The default parameters were used for convergence criteria
during refinement with the LIGAND FIT program (Accelrys, Inc., San
Diego, Calif.).
Example 6
Confirmation of Gossypol Binding to Bcl-X.sub.L by NMR
[0466] The binding of gossypol compounds (e.g., racemic gossypol,
(-)-gossypol, and (+)-gossypol) to Bcl-X.sub.L was determined using
.sup.15N Heteronuclear Single Quantum Coherence Spectroscopy (HSQC)
NMR methods.
[0467] The protein samples for NMR studies were uniformly labeled
with .sup.15N for screening and uniformly double labeled with
.sup.15N and .sup.13C for structure characterization according to
the methods described in M. Jansson et al., J. Biomol. NMR,
7:131-141 (1996), and M. L. Cai et al., J. Biomol. NMR, 11:97-102
(1998).
[0468] Since the NMR experiments were performed at pH 7.2 in a
pulse field gradient (PFG), HSQC with water flip back was used to
maximize signal intensity (S. Grzesiek and A. Bax, J. Am. Chem.
Soc., 115:12593-12594 (1993); and G.S. Sheppard et al., Abstracts
of Papers of the Amer. Chem. Soc., 213:81 (1997)) and to minimize
destruction from the water signal. HSQC spectra of Bcl-X.sub.L were
recorded prior to (free Bcl-X.sub.L) and after the addition of the
concentrated inhibitor solution. The two spectra were compared to
identify the chemical shifts induced by the additions of the
inhibitor. Data processing was conducted using nmrPipe, pipp and
nmrDraw software (See, D. S. Garrett et al., J. Magn. Reson. Ser.,
B 95:214-220 (1991); and F. Delaglio et al., J. Biomol. NMR,
6:277-293 (1995)). Shifted peaks were cross-referenced to the
assignment table to reveal the residues affected by the presence of
gossypol compounds. The residues affected by the binding of
(-)-gossypol are shown in FIGS. 11A and 11B, which show that
(-)-gossypol binds to the BH3 binding site in Bcl-X.sub.L.
[0469] To provide further insights into the binding of gossypol and
its enantiomers, additonal embodiments of the present invention
used NMR methods to determine the 3D structure of gossypol in
complex with Bcl-X.sub.L in solution. To that end, a set of triple
resonance NMR experiments were performed, and the backbone and
sidechain resonance assignments were assessed. The initial
complexed structure, was calculated with the X-PLOR program
(Harvard University, Cambridge, Mass.) using a
torsion-angle-dynamics protocol. The obtained 3D structure of
Bcl-X.sub.L in complex with gossypol was similar to the free form
of Bcl-X.sub.L with the hydrophobic BH3 binding pocket formed by
the a-helixes of the BH1, BH2, and BH3. To further refine the
complexed structure of Bcl-X.sub.L and (-)-gossypol solved by NMR
methods, the present invention further performed 4 ns molecular
dynamics (MD) simulations. The average complex structure from the
computational simulation from the last 50 ps is provided in FIG.
11C.
[0470] Similar HSQC experiments of Bcl-X.sub.L were performed to
obtain the binding affinity of a gossypol compound by titrating a
range of inhibitor concentrations from 0.5 .mu.M to 500 .mu.M with
a constant concentration (100 .mu.M) of .sup.15N labeled
Bcl-X.sub.L protein. Changes in the chemical shifts of protein
residues were monitored by .sup.15N-HSQC spectra. Using chemical
shift data for residue 146, a dose-dependent titration curve by
gossypol was developed. FIGS. 28A and 28B show the titration curves
of (-)-gossypol and (+)-gossypol with Bcl-X.sub.L protein.
[0471] The present invention is not limited to any particular
mechanism. Indeed, an understanding of any particular mechanim is
uncesssary to practice (make and use) the compositions and methods
of the present invention. Nonetheless, it is contemplated that the
MD refined complexed structures of the present invention confirm
the important interactions between (-)-gossypol and Bcl-X.sub.L
observed in the HSQC spectra obtained from previous NMR
experiments. Furthermore, MD refined structures identified by the
present inventon indicate that (-)-gossypol interacts with
hydrophobic residues Y105 and L134 as well as with charged residues
G142 and R143. The present invention is not limited to any
particular mechanism or mechanisms. Indeed, an understanding of any
particular mechanims is uncesssary to practice (make and use) the
compositions and methods of the present invention. Nonetheless,
certain embodiments of the present invention show that gossypol
targets the hydrophobic cleft on the surface of Bcl-X.sub.L, which
is a docking site for the BH3 domain of pro-apoptotic proteins
(e.g. Bid, Bad, Bax and Bak). Binding of (-)-gossypol to this
region alters the accessibility or binding properties of
Bcl-X.sub.L and Bcl-2 proteins to pro-apoptotic proteins leads to
the inhibition of the function of these proteins. Thus, in some
embodiments, the present invention provides compounds that bind to
the hydrophobic cleft on the surface of Bcl-2 and/or Bcl-X.sub.L
by, for example, interacting with residues Y105, L134, G142, and/or
R143. The function of such compounds (whether natural or
synthesized) can be assessed by testing the ability of the compound
to disrupt or displace gossypolcompound/Bcl-2/Bcl-X.sub.L
interactions.
Example 7
Expression of Bcl-2 Family Proteins in Human Cancer Cells
[0472] Several cancer cell lines (Table 16 and FIG. 7) that express
various levels of Bcl-2 and/or Bcl-X.sub.L proteins were selected
in order to test the activity of gossypol to inhibit proliferation
of human cancer cells.
TABLE-US-00016 TABLE 16 Cancer Cell Line Bcl-2 expression
Bcl-X.sub.L expression Group I T47 breast cancer .+-. +++ MDA-453
breast - +++ cancer Group II MDA-435 breast +++ .+-. cancer Group
III MDA-231 breast +++ +++ cancer Group IV WI-38 normal - -
SK-MEL-28 .+-. - melanoma
Experiments using the cancer cell lines shown in Table 16 allowed
for the testing of several important features of gossypol as an
anticancer agent. The activity and selectivity of gossypol in
binding assays with a number of cancer cell types that express
various levels of Bcl-2 family proteins indicates the range of
cancer types that are suitable candidates for treatment with
gossypol. Testing gossypol in cancer cells with high Bcl-2 and high
Bcl-X.sub.L expression levels indicates whether inhibition of
either protein alone was sufficient for induction of apoptosis or
whether simultaneous inhibition of both proteins achieves greater
anticancer potency. Assays using a number of cancer cell types
expressing various levels of Bcl-2 family proteins indicate whether
gossypol displays selectivity in cancer cells with both low Bcl-2
and low Bcl-X.sub.L expression.
Example 8
Investigations into the Mechanisms of Apoptosis Induced by
Gossypol
[0473] A series of biochemical assays were carried out to determine
the mechanisms of action of the gossypol compounds as small
molecule inhibitors of Bcl-2 family proteins (e.g., Bcl-2 and/or
Bcl-X.sub.L). Although an understanding of the mechanism is not
necessary to practice the present invention and the present
invention is not so limited, it is contemplated that an
understanding of the mechanisms by which gossypol compounds act on
cells and tissues that overexpress Bcl-2 family proteins is
advantageous to the present invention.
[0474] Following the treatment of cells with inhibitors,
qualitative assessments of cell morphology were made to determine
features related to apoptosis such as cellular swelling, nuclear
swelling, membrane blebbing, vacuolization, and apoptotic body
formation.
[0475] In some embodiments, DNA level detection of apoptosis was
made by detecting DNA fragmentation using TUNEL assays. When cells
are undergoing apoptosis, apoptotic endonucleases not only affect
cellular DNA by producing the classical DNA ladder but also
generate free 3'-OH groups at the ends of these DNA fragments.
These groups were end-labeled by the TdT-FragFLTM DNA Fragmentation
Kit (Oncogene, Boston, Mass.) thus allowing detection of apoptotic
cells using molecular biology-based, end-labeling, histochemical,
or cytochemical techniques. The rationale of this assay is that
terminal deoxynucleotidyl transferase (TdT) binds to exposed 3'-OH
ends of DNA fragments generated in response to apoptotic signals
and catalyses the addition of biotin-labeled and unlabeled
deoxynucleotides. Biotinylated nucleotides were detected using a
streptavidin-horseradish peroxidase (HRP) conjugate.
Diaminobenzidine reacts with the labeled sample to generate an
insoluble colored substrate at the site of DNA fragmentation (See
e.g., L. Lagneaux et al., Br. J. Haematol., 112:344-352 (2001); and
K. Kitamura, Leukemia, 14:1743-1750 (2000)).
[0476] In other embodiments, nuclei level detection methods (e.g.,
fluorescent dye Hoechst 33258 and propidium iodide staining) were
used to quantify signs of apoptosis in treated cells. Morphological
changes in the nuclear chromatin of cells undergoing apoptosis were
detected by staining with 2.5 .mu.g/ml of bisbenzimide Hoechst
33258 fluorochrome (Calbiochem, La Jolla, Calif.) followed by
examination on a fluorescence microscope. A representative example
of chromatin changes in cancer cells with high expression levels of
Bcl-2 and Bcl-X.sub.L versus normal human fibroblast cells is
provided in FIGS. 8A and 8B. In some experiments, cells were
double-stained with propidium iodide (PI, 2.5 .mu.g/ml) and Hoechst
33258 (2.5 .mu.g/ml) to distinguish apoptotic cells from necrotic
cells. Intact blue nuclei, condensed/fragmented blue nuclei,
condensed/fragmented pink nuclei, and intact pink nuclei were
considered viable, early apoptotic, late apoptotic, and necrotic
cells, respectively (See e.g., B. R. Gastman et al. Cancer Res.,
60:6811-6817 (2000); and N. Hail Jr., and R. Lotan, Cancer
Epidemiol. Biomarkers Prey., 9:1293-1301 (2000)).
[0477] In still further embodiments, the present invention used
cell level detection of apoptotic events by flow cytometry. Cells
that had undergone apoptotic events were detected by flow cytometry
using a FACSCAN instrument (Becton Dickinson, Franklin Lakes, N.J.)
with 488-nm laser line and analyzed using CELL QUEST software
(Becton Dickinson, Franklin Lakes, N.J.). Phosphatidylserine
exposed on the outside of the cells (one of the major
characteristics of apoptosis) was determined by TACSTM Annexin
V-FITC Kit (Trevigen, Gaithersburg, Md.). Annexin V-FITC
fluorescence is detected in FL-1, and propidium iodide was detected
in FL-2. (Hail Jr., and R. Lotan, R. Cancer Epidemiol. Biomarkers
Prey., 9:1293-1301 (2000)).
[0478] Apoptosis was further detected and quantified using
Annexin-V-fluorescence assays. Cells were treated with different
concentrations of (-)-gossypol or its enantiomers alone, docetaxel
alone, or (-)-gossypol and docetaxel for 48 hrs. Cells were
collected and washed with PBS and stained with
Annexin-V-Fluorescein and propidium iodide (PI) following standard
protocols. Stained cells were analyzed with a flow cytometer
(Becton Dickinson) using 488 nm excitation and a 515 nm bandpass
filter for fluorenscein detection, and a >600 nm filter for PI
detection. Representations of induction of apoptosis by gossypol
(at various doses) in MDAMB-231 or T47D cells are shown in FIGS. 9,
10, 29, and 30.
[0479] The expression and phosphorylation status of Bcl-2 proteins
in cells treated with gossypol compounds at various doses and times
was determined. In particular, the expression levels of Bcl-2,
Bcl-X.sub.L, Bcl-X.sub.S, Bak, Bad, Bax, and Bid was determined by
specific antibodies using Western blot analysis. Phosphorylation
status for proteins such as Bcl-2, Bcl-X.sub.L, and Bad was
determined by Western blotting using specific antibodies that
recognize the phosphorylated proteins.
[0480] The effects of gossypol on cellular mitochondria were
examined by several methods (e.g., cytochrome c release and pore
formation assays). Cells were treated with inhibitors for 24-48 hrs
and cell fractionation was performed as described in R. M. Kluck et
al., Science, 275:1132-1136 (1997) with some modifications as
follows. Briefly, cells were harvested and washed once with
ice-cold PBS and resuspended in 1 ml ice-cold buffer C (10 mM
Hepes-KOH at pH 7.4, 0.42 M NaCl, 2.5% (v/v) glycerol, 1.5 mM
MgCl.sub.2, 0.5 mM sodium EDTA, 0.5 mM EGTA, 1 mM dithiothreitol)
and a protease inhibitor mix (PIM). The cell suspension was
homogenized on ice by passage 15 times through a 22 gauge needle.
The homogenates were centrifuged twice at 750 g for 10 min at
4.degree. C. to remove nuclei. The post-nuclear supernatant
fractions were centrifuged at 10,000 g for 15 min at 4.degree. C.,
and the resulting mitochondria-enriched pellets were resuspended in
100 ml buffer C+PIM (cold). The post-mitochondrial supernatant was
centrifuged at 10,000 g for 1 hr at 4.degree. C. to remove membrane
contaminants and the resulting supernatant (soluble portion) was
used for cytosolic cytochrome-c release detection (the pellet is
the mitochondrial membrane (heavy membrane proteins) portion).
Soluble fraction proteins and an equivalent amount of heavy
membrane proteins were subjected to SDS-PAGE and analyzed by
Western blotting with antibodies against cytochrome c (Becton
Dickinson, Franklin Lakes, N.J.), voltage-dependent anion channel
(VDAC) (Calbiochem, La Jolla, Calif.), Smac, and AIF (apoptosis
inducing factor) (Santa Cruz Biotechnology, Santa Cruz, Calif.)
analyses. These methods determined apoptosis signaling following
treatment of the test cells (e.g., cancer cells) with gossypol
compounds through three mitochondrial pathways: cyto-c release,
Smac release, and AIF release.
[0481] Breast cancer cell lines MDA-231 and T47D were treated with
either 5 or 20 .mu.M of gossypol for 24 hrs. FIG. 13 shows that
cytochrome c was released from the mitochondria into the cytosol
after treatment with 5-20 .mu.M of gossypol in MDA-231 cells or
T47D cells which provides further evidence of gossypol compopund
mediated apoptosis in these cells. Additional assays measured the
activation of caspase-3, or the cleavage of PARP protein, a
down-stream protein target of caspases. Treatment with (-)-gossypol
also resulted in more cleavage of PARP protein under identical
doses (FIGS. 14 and 31).
[0482] Treatment of cells with 10 or 20 .mu.M of gossypol for 8 hrs
also provided a decrease in the mitochondrial transmembrane
potential of the test MDA-231 cells as assessed by the cationic
lipophilic cell-permeable fluorescent dye
3,3'-dihexy(-)-oxacarbocyanine iodide (DiOC.sub.6) assays. The
present invention is not limited to any particular mechanisms.
Indeed, an understanding of any particular mechanism is unnecessary
to practice (make and use) the present invention. Nonetheless, the
data indicate that gossypol compounds inhibit the anti-apoptotic
functions of Bcl-2 proteins by suppressing the mitochondrial
functions regulated by these proteins.
[0483] In additonal embodiments, the activation of caspases was
determined by collecting and washing treated cells with PBS and
suspending the cells in 25 mM HEPES (pH 7.5), 5 mM MgCl.sub.2, 5 mM
EDTA, 5 mM dithiothione, 2 mM phenylmethylsulfonyl fluoride, 10
g/mL pepstatin A, and 10 g/mL leupeptin after treatment. The
treated cells were then lysed. The cell lysates were clarified by
centrifugation at 12000.times.g for 20 min at 4.degree. C.
Caspase-1, -2, -3, -6, -8, and -9 activity in the supernatant was
determined using the fluorogenic CaspACE Assay System (Promega
Corp., Madison, Wis.). Briefly, 50 mg aliquots of total protein,
determined by the bicinchoninic acid assay (Promega Corp.), were
incubated with 50 mM substrate Ac-YVAD-AMC, Ac-VDVAD-AMC,
Ac-DEVD-AMC, Ac-VEID-AMC, Ac-IETD-AMC, or Ac-LEHD-AMC at 30.degree.
C. for 1 hr. The release of methylcoumaryl-7-amine (AMC) was
measured by excitation at 360 nm and emission at 460 nm using a
fluorescence spectrophotometer (Hitachi F-4500) according to G.
Denecker et al., Cell Mol. Life Sci., 58:356-370 (2001); and V. M.
Kolenko et al., Apoptosis, 5:17-20 (2000)).
[0484] The effects of caspase inhibitors (e.g., Z-VAD-FMK,
non-specific, Z-DEVD-FMK, caspases-3, -6, -7, -8, and -10, and
Z-LEHD-FMK, caspase-9) on the activity of the gossypol compounds
was determined using standard caspase inhibitor assays. All of the
caspase inhibitors used are commercially available (Enzyme System
Products, Livermore, Calif.). FIGS. 32A and 32B show caspase
inhibitors in gossypol-mediated caspase activation and growth
inhibition.
Example 9
Cell Survival Assay
[0485] Cells were seeded in 24 or 96-well-plates and gossypol
compounds were prepared at 2-10 fold dilutions in suitable medium.
Inhibition of cell viability was determined by treating the cells
with the gossypol compounds for 24 hrs. Cell viability was then
determined by the trypan-blue assays. Inhibition of cell growth was
determined by treating cells with gossypol compounds for 3-6 days.
Cell inhibition was determined using XTT. In some embodiments, MTT
assays are used to determine cell inhibition.
[0486] In one embodiment, to evaluate cell proliferation, soluble
XTT (Sodium,
3'-(1-((phenylamino)-carbonyl)-3,4-tetrazolium)-bis(4-methoxy-6--
nitro) benzenesulfonic acid hydrate (Polysciences, Inc.,
Warrington, Pa.) assays were performed on cells growing in 96-well
plates. After 5 days of incubation, when the control untreated
cells reached confluence, 50 .mu.l of XTT (1 ng/ml) was added to
each well and incubation continued for 4 hrs at 37.degree. C.
Absorbance at 450 nm was measured using a Dynatech Model MR700
device (Dynatech Laboratories Ltd. Billinghurst, United Kingdom).
The percentage of surviving cells was defined as mean absorbance of
treated wells/mean absorbance of untreated wells.times.100.
[0487] MTT assays provide a fast, accurate, and reliable method for
obtaining cell viability measurements. MTT assays are simple and
colorimetric. Numerous laboratories have utilized MTT assays for
toxicity studies (See e.g., Kuhlmann et al., Arch. Toxicol., 72:536
(1998)). Briefly, mitochondria produce ATP to provide sufficient
energy for the cell. In order to do this, the mitochondria
metabolize pyruvate to produce acetyl CoA. Within the mitochondria,
acetyl CoA reacts with various enzymes in the tricarboxylic acid
cycle resulting in subsequent production of ATP. Succinate
dehydrogenase is one of the enzymes measured by MTT assays. The MTT
compound (3-(4,5-dimethylthiazol-2-yl)-2 diphenyl tetrazolium
bromide) is a yellow substrate that is cleaved by succinate
dehydrogenase to form a purple formazan product. The colorimetric
response of MTT (yellow to purple) thus identifies changes in
mitochondria function. Nonviable cells are unable to produce the
MTT formazan product; therefore, the amount of purple MTT formazan
product produced directly correlates to the quantity of viable
cells. Absorbance at 540 nm is used to measure the amount of
formazan product.
[0488] The present invention is not limited to any particular
mechanisms. Indeed, an understanding of any particular mechanism is
unnecessary to practice (make and use) the present invention.
Nonetheless, it is contemplated that FIG. 12 shows that, in certain
embodiments, (-)-gossypol is 2 times more potent than
(.+-.)-gossypol and is 5-10 times more potent than (+)-gossypol in
inhibition of cell growth in breast cancer cell lines with high
levels of Bcl-X.sub.L. The data suggest that (-)-gossypol is more
potent than (+)-gossypol, and is more selective between cancer
cells with high levels of Bcl-X.sub.L expression and cancer or
normal cells with low levels of Bcl-X.sub.L such as DU-145 or
WI-38.
Example 10
Colony-Formation in Soft-Agarose
[0489] The soft-agar colony formation assays were used to directly
measure the transforming ability of cancer cells. These assays are
known to correlate well with in vivo tumorigenicity.
[0490] Experiments were conducted using combination treatments to
assess the apoptotic effects or inhibition of cell proliferation
obtained by administration of Bcl-2 inhibitors in combination with
known chemotherapeutic agents. In preferred embodiments, these
results wereused to select synergistic agent combinations.
[0491] Cells were trypsinized and resuspended in 1 ml 0.33% top
agarose (10,000 cells/ml) on 1% bottom agarose. The next day, 1 ml
aliquots of regular medium containing (-)-gossypol were gently
layered to the top of the agarose and incubation continued for 2
weeks. The number of colonies greater than 80 .mu.m in diameter
were counted using a Bausch and Lomb Image Analysis System (Omega
3800). FIG. 33 shows a representative example of inhibition of soft
agar colony formation in these experiments:
Example 11
In Vivo Antitumor Activity Studies
[0492] Preliminary in vivo studies showed that gossypol is a potent
Bcl-X.sub.L inhibitor, and that it exhibits significant anti-tumor
activity alone and in combination with additional conventional
anticancer agents (e.g., docetaxel). Preferred embodiments of the
present invention provide in vivo anti-tumor efficacy and
selectivity studies using human cancer xenograft models.
[0493] The results of in vitro experiments can be compared to in
vivo toxicity tests to extrapolate live animal conditions.
Typically, toxicity from a single dose of a substance is assessed.
In some embodiments, animals were monitored over 14 days for any
signs of toxicity (increased temperature, breathing difficulty,
death, etc.). Typically, the standard of toxicity is the
measurement of the maximal tolerated dose (MTD). The MTD is the
highest dose that results in no lethality/tissue abnormality or
causes them to gain 10% less weight than control animals.
[0494] The determination of the MTD occurred by exposing test
animals to a geometric series of doses under controlled conditions.
Other tests included subacute toxicity testing, which measures the
animal's response to repeated doses of gossypol compounds (or one
or more conventional anticancer agents) for no longer than 14 days.
Subchronic toxicity testing involved testing of a repeated dose for
90 days. Chronic toxicity testing is similar to subchronic testing
but lasts for more than 90 days. In vivo testing was conducted to
determine toxicity with respect to certain tissues. For example, in
some embodiments of the present invention, tumor toxicity (e.g.,
effect of the compositions of the present invention on the survival
of tumor tissue) was determined (e.g., by detecting changes in the
size and/or growth of tumor tissues).
[0495] In order to design optimal dose schedules for gossypol
therapies, studies first utilized human breast cancer cell lines
MDA-231(clone 2LMP), and then additional tumor xenograft models
such as MDA-435 (LCC6), and T47D. MDA-231 expresses high levels of
both Bcl-2 and Bcl-X.sub.L; MDA-435 expresses high levels of Bcl-2
but low levels of Bcl-X.sub.L; T47D expresses low levels of Bcl-2
but high levels of Bcl-X.sub.L. Among all of the 7 human breast
cancer cell lines examined, no cell line had both low Bcl-2 and low
Bcl-X.sub.L. The human prostate DU-145 mice xenograft model,
however, expresses low levels of both Bcl-2 and Bcl-X.sub.L and was
thus used as a negative control in some embodiments to test the
specificity of gossypol compounds and gossypol derivatives.
[0496] In one embodiment using the MDA-231 model, a series of
comprehensive dose and schedule investigations was performed to
determine: 1) the minimal active dose, defined as inhibition of
tumor growth by 50% as compared to control with a statistical
confidence level of 95%; 2) the optimal schedule of administration
in inhibition of tumor growth while not causing toxicity defined as
weight loss of more than 25%; 3) the effect of gossypol compounds
on large tumors (more than 2,000 mm.sup.3); 4) how long could
gossypol can be administered to mice in the control group without
causing morbidity or mortality (e.g., weight loss).
[0497] After identifying optimal doses and dosing schedules,
testing of combination therapies with at least one additional
conventional chemotherapeutic agent was conducted, including, but
not limited to, 1) doxorubicin (4 mg/kg); 2) 5-FU (10 mg/kg); 3)
VP-16/etoposide (40 mg/kg); and 4) cyclophosphamide (100 mg/kg);
and 5) cisplatin (10 mg/kg). As a positive control, TAXOTERE was
used at a dose of 7.5 mg/kg. Control group mice received either no
treatment or vehicle alone. To achieve statistic significance, a
minimum of 10 mice per group was used in the combination
regimes.
[0498] For all tests, mice were randomized and then injected in the
fat pad with 1-5.times.10.sup.6 cells prepared in serum-free
medium. The animals were measured and weighed twice each week
during the treatment period, followed by twice a week measurements
for an additional 4-6 weeks. A gross visual necropsy of each animal
was performed at death or terminal sacrifice. A representative
example of in vivo animal testing data of gossypol either alone or
in combination with TAXOTERE is provided in FIG. 15.
[0499] The rate of apoptosis in tissues was determined using the
TUNEL method (terminal deoxyribonucleotidyl transferase
(TdT)-mediated dUTP-digoxigenin nick end labeling). The TUNEL
method is extremely sensitive (See. Y. Gavrieli et al., J. Cell
Biol., 119:493-501 (1992); and M. Dowsett et al., Cytometry,
32:291-300 (1998)). Paraffin-embedded tissues were sectioned, and
slides were incubated in labeling buffer for 5 min. then placed in
a humid chamber with TdT, dNTP mix, and Mg.sup.++ in labeling
buffer. Strepavidin-Horseradish Peroxidase was applied onto each
sample for 10 min., the samples were washed and counter stained
with methyl green or hematoxylin. The rate of apoptosis was
calculated by counting and dividing the number of apoptotic cells
by the total number of cells seen per light microscopy field at
40.times. magnification, and is expressed by percent.
Example 12
Drug Interactions Between Gossypol and Docetaxel
[0500] The present invention is not limited to any particular
mechanisms. Indeed, an understanding of any particular mechanism is
unnecessary to practice (make and use) the present invention.
Nonetheless, it is contemplated that, in certain embodiments,
gossypol compounds are likely to be used in combination with
standard chemotherapeutic agents. Although gossypol alone inhibited
cell proliferation, combination treatments resulted in enhanced
effects (e.g., greater induction of apoptosis in target cells).
Experiments show that (-)-gossypol acts in synergy with docetaxel
(TAXOTERE, or TXT), or paclitaxel (TAXOL) to inhibit the growth of
breast cancer cells such as MDA-MB-231 (FIG. 34), or MCF-7 (FIG.
16).
[0501] Isobologram analyses (See, T. C. Chou and P. Talalay, Adv.
Enzyme Regulation, 22:27 55 (1984)), are widely used to determine
synergism of two or more drugs when used in combination. In some
embodiments, isobologram analyses show that the combination of
TAXOTERE with (-)-gossypol resulted in significant synergy, with a
Combination Index (CI) of 0.7 and 0.5, and a Dose Reduction Index
(DRI) of 1.94 and 2.34 for administration of TAXOTERE with either 4
.mu.M or 5 .mu.M of (-)-gossypol, respectively. Briefly, FIGS. 17A
and 17B show the in vitro effects of (-)-gossypol in combination
with various doses of TAXOL in MDA-MB-231 based growth assays,
wherein: CI value <1 indicates synergistic effects; CI=1
indicates additive effects; CI value >1 indicates antagonistic
effects; DRI >1.0 indicates synergistic effects. Treatment of
MDA-MB-231 cells with (-)-gossypol significantly potentiated the
cells' response to TAXOL mediated cytotoxicity and resulted in
further reductions in cell survival from controls. Thus, in some
embodiments, gossypol compounds provide a synergistic effect when
used in combination with cytotoxic agents such as taxanes (e.g.,
TAXOTERE). The present invention is not limited to any particular
mechanisms. Indeed, an understanding of any particular mechanism is
unnecessary to practice (make and use) the present invention.
Nonetheless, the data indicate, in some embodiments, that gossypol
compound mediated inhibition of cell proliferation is enhanced when
combined with chemotherapeutic agents and that the combined effect
are specific for gossypol. Other chemotherapeutic drugs were also
tested in combination with gossypol.
[0502] The effect of gossypol alone on the erythromycin breath test
(ERMBT), a phenotypic test for CYP3A4 metabolism, was evaluated by
comparing baseline ERMBT levels at baseline and after 1-week
pretreatment with gossypol (See, e.g., P. Watkins,
Pharmacogenetics, 4:171-184 (1994); and J. Hirth et al., Clin.
Cancer Res., 6:1255-1258 (2000)). ERMBT assays produce a
measurement of the percentage of .sup.14C exhaled per hour, that is
usually approximated using the Normal distribution (See, D. Wagner,
Clin. Pharm. Therap., 64:129-130 (1998)). The mean levels of
.sup.14C exhaled/hr were compared using a standard (alpha=0.05),
two-sided, paired t test. Using estimates from previous work (J.
Hirth et al., Clin. Cancer Res., 6:1255-1258 (2000) for the
baseline mean .sup.14C exhaled/h (n=21, mean=2.41), its standard
deviation (SD=1.08), correlation of ERMBT measurements over time
(r=0.81), and assuming constant variance at each measurement, the
current example with data from 30 subjects has 96% power to detect
a 20% decrease (2.41 to 1.93), and 81% power to detect a 15%
decrease (2.41 to 2.05) in mean ERMBT levels. The mean, standard
deviation, and range of ERMBT values was tabulated and reported,
along with the significance of the paired t test.
Example 13
Pharmacokinetic Description of Docetaxel in Patient Samples
[0503] Blood is drawn for a pharmacokinetic description of
docetaxel when administered in combination with gossypol using an
optimal sampling strategy (See, P. Baille et al., Clin. Cancer
Res., 3:1535-1538 (1997)). Blood is drawn directly before the end
of infusion (EOI) of docetaxel, at 0.25, 0.75, 3.00, 6.50, and at
24 hrs following EOI. All samples are assayed at the same time. A
Bayesian criterion is used to calculate the docetaxel plasma
concentration area under the curve (AUC) based upon measured drug
levels and population pharmacokinetic parameters previously
estimated (See, R. Bruno et al., J. Clin. Oncol., 16:187-196
(1998)) for docetaxel alone, using NONMEM software (S. L. Beal and
L. B. Shener, Nonlinear Mixed Effects Model Users Guides (San
Francisco, Calif., NONMEM Project Group, University of California
at San Francisco) (1999)). Clearance (CL) is directly estimated by
fitting the nonlinear mixed effects model (R. Bruno et al., J.
Clin. Oncol., 16:187-196 (1998)). The AUC is calculated as a
function of dose and clearance, defined as AUC=dose/CL. ERMBT
values from baseline and 1-week following gossypol pretreatment are
plotted separately against Clearance. Ordinary least squares
regression is used to defined the relationship between docetaxel CL
and the ERMBT or the natural logarithm of ERMBT. The estimated
slopes are displayed along with previously published values for
single treatment docetaxel CL (J. Hirth et al., Clin. Cancer Res.,
6:1255-1258 (2000) in order to describe any differences in
docetaxel CL when administered in combination with gossypol versus
single agent administration.
Example 14
Expression of Bcl-2 Family Protein in Patient Samples
[0504] The expression of Bcl-2 and Bcl-X.sub.L in tissue samples
collected in paraffin blocks was assayed using standard
immunohistochemical (NC) assay methods. IHC results were
dichotomized and tabulated to show the percentage of patients
expressing these markers. Results were tabulated against anti-tumor
response. Chi-square statistic and Fisher's exact tests were
performed to assess the relationship between expression and
anti-tumor response, depending on the size of the cell counts in
the resulting table.
[0505] In some embodiments, tissues were fixed in 4% buffered
formalin, processed, and embedded in paraffm according to the
normal schedule used in the laboratory. From each block, 5
.mu.m-thick sections were cut on coated slides and dried overnight
at 37.degree. C. The sections were deparaffinized in xylene and
rehydrated through graded concentrations of ethanol to distilled
water. Sections to be stained with antibodies against Bcl-2, Bax,
Bcl-X.sub.L, and Bag-1 were pretreated by boiling them for 20 min
in citrate buffer (pH 6.0) or pretreated by digestion in 0.5%
trypsin (pH 7.2) at 37.degree. C. for 30 min. Immunohistochemical
stainings were performed using commercial Elite ABC kits
(VECTASTAIN, Vector Laboratories, Burlingame, Calif.). Blocking
serum was applied for 15 min followed by overnight incubation with
the diluted primary antibody: Bcl-2, 1:200 (clone 124,
DakoCytomation, Inc., Carpinteria, Calif.); Bax, 1:100 (clone 2D2;
Zymed Laboratories, Inc., South San Francisco, Calif.);
Bcl-X.sub.L, 1:50 (clone 2H12; Zymed); Bag-1, 1:200 (monoclonal
mouse (12), (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.);
the sections were then incubated with the biotinylated secondary
antibody and the peroxidase-labeled ABC solution (VECTASTAIN) for
30 min each. All of the dilutions were made in PBS (pH 7.2), and
all of the incubations were performed in humid chambers at room
temperature. Between each step in the staining procedures (except
before incubation with the primary antibody), the slides were
rinsed three times in PBS. Bound peroxidase was visualized in all
of the slides with a 3-amino-9-ethyl-carbazole solution (0.2 mg/ml
in 0.05 M acetate buffer containing 0.03% perhydrol (pH 5.0); AEC;
Sigma Chemical Co., Saint Louis, Mo.) at room temperature for 15
min. The sections were lightly counterstained in Mayer's
hematoxylin and mounted in Aquamount Mountant (BDH Ltd., Poole,
United Kingdom). For each antibody, a known positive case of cancer
was included in every staining batch as a positive control. Cells
were considered positive when a distinct cellular micropunctate
pattern of staining was seen, except for Bag-1, for which nuclear
staining was also accepted. The percentage of immunoreactive cells
was evaluated as the amount of positive tumor cells per all of the
tumor cells on the section. All of the stained sections were scored
by two investigators, who were blinded to the clinical data. Median
values were then used as the cut-points for low and high
expression. Spearman correlation coefficients were calculated for
the investigated tumor biological factors.
[0506] In some embodiments, 5 .mu.m-thick sections of the
formalin-fixed, paraffin-embedded tumors were immunostained using
monoclonal Bcl-2 (1:160) (DakoCytomation), polyclonal Bax (1:1500)
(PharMingen, San Diego, Calif.), and polyclonal Bcl-X (1:1500)
(PharMingen). An avidin-biotin enzyme complex kit (Signet
Laboratories Inc., Dedham, Mass.) with steam antigen retrieval was
used in combination with the automated TechMate 1000 immunostaining
system (Biotek Solutions Inc., Santa Barbara, Calif.). Hematoxylin
and eosin were used as counterstains. Sections of tonsil were used
as positive controls for Bcl-2, while normal breast ducts and
lobules were used as positive controls for Bax and Bcl-X.sub.L. In
some embodiments, negative controls had primary antibody replaced
by buffer. The immunostaining was recorded as 0 to 3+ according to
stain intensity, distribution in cytoplasm and/or nucleus, and
percentage of cancerous cells that stained positive. Tumors with
less than 5% of carcinoma cells staining with 1+ positivity were
considered negative.
[0507] In some embodiments, the percentage of nuclear and
cytoplasmic immunostain for the antigens (Bcl-2, Bax, Bcl-X.sub.L)
were quantitated using the CAS 200 image cytometer (Becton
Dickinson Cellular Imaging Systems, San Jose, Calif.). The
immunoperoxidase/diaminobenzidine procedure stained positive nuclei
and cytoplasm brown and negative areas blue and pink, respectively.
At 620 nm, brown, blue, and pink absorb, providing a measure of
total nuclear and cytoplasmic area. At 500 nm, thresholds were set
so that only the brown stained areas absorbed, allowing the
immunopositive nuclear and cytoplasmic areas to be measured
independently. Comparison with the 620 nm mask gave the percent
positive area immunostained (PPA). Fifteen fields were analyzed in
each slide so as to minimize the standard deviation. Areas were
isolated from adjacent stroma by using the scene segmentation
function, which allows the operator to precisely define portions of
the image to be analyzed. This prevents positive staining of
non-cancerous tissue elements such as lymphocytes, from being
included in the PPA. Computer generated histograms showed PPA on
the vertical axis and nuclear/cytoplasmic optical density (OD) on
the horizontal axis. The OD of the chromogen was proportional to
the amount of immunostain. The computer calculated the number of
fields, total nuclear/cytoplasmic area analyzed, and PPA as
averages of intensity of immunostaining. A ratio was then obtained
of the PPA Bcl-2 divided by the PPA of Bax and Bcl-X.sub.L. Basic
descriptive statistics, including means, standard deviations, and
ranges were used to characterize the study sample. Pearson's
correlation coefficients were used to explore the association
between continuous measures. P-values less than 0.05 were
considered statistically significant.
[0508] In some embodiments, the level of target antigen expression
was quantified as follows: samples with more than 50% positive
cells were classified as strongly positive (++), while samples with
5-50% positive cells were classified as moderately positive (+),
and samples with fewer than 5% positive cells were classified as
negative (-) (See, Alderson et al., Cancer Res., 999-1001 (1995)).
Areas free of necrosis and capillary endothelial proliferation were
chosen for analysis. The infiltrative edge of the tumor where
normal neurons and glia are surrounded by neoplastic cells was also
excluded. An eyepiece grid was used for counting at a magnification
of 400.times.. Three high-power fields were counted for each sample
and antigen. The correlation between the expression of different
proteins was analyzed with the Chi-square test of independence with
Yates' correction for continuity (P, 0.05).
Example 15
Peripheral Blood Lymphocytes Studies
[0509] Blood samples collected from study participants before study
treatment, on day 8 (following 1 week gossypol pretreatment), and
at week 9 (after completion of one cycle of gossypol plus
docetaxel) are used for exploratory studies involving peripheral
blood lymphocytes (PBL) and circulating epithelial cells (CEC).
Using fluorescent activated cell sorting techniques (FACS), PBLs
are characterized for their expression of Bcl-2 and Bcl-X.sub.L
before and after gossypol administration. The mean number of PBLs
with expression and its standard deviation will be reported at each
time point. Using an immunomagnetic separation method previously
described (See, T. Walker et al., Proc. Amer. Soc. Clin. Oncology,
19:54b (2001)) the feasibility of finding CECs is determined for
each patient. The mean number of CECs per 10 ml of blood collected
and its standard deviation across patients at each time point will
be reported.
Example 16
Administration of (-)-gossypol in Combination with Conventional
Chemotherapeutic Agents in a Mouse Xenograft Model
[0510] This example describes experiments conducted to evaluate the
in vivo antitumor efficacy of (-)-gossypol in human xenograft
models either alone or in combination with chemotherapy. Docetaxel
(TAXOTERE or TXT) was administered as described herein.
[0511] In this experiment, male 5 to 6 week old NCI athymic
NCr-nu/nu nude mice were inoculated subcutaneously in the mammary
fat pad on each side with an injection of about 1.times.10.sup.6
MDA-MB-231 (2LMP) in 0.3 ml serum free medium (SFM). The best
xenograft recipients were used. Treatments began when tumors
averaged about 50 mm.sup.3 (5-7 mm in diameter, usually at day 7).
Treatments ran for 4 weeks with a 2 month follow up period.
Resolution of gossypol enantiomers was carried out as described
previously. (-)-Gossypol compounds were dissolved in ethanol and
diluted with final 10% ethanol in PBS before injection. All oral
administrations of (-)-gossypol and 10% alcohol were given by
gavage.
[0512] Test animals were divided into cohort groups of 8 animals
into the following treatment groups: Control (Group 1); Vehicle
control (Group 2), daily oral administration of 10% alcohol only;
(-)-gossypol (Group 3), 7.5 mg/kg administered orally per day for 4
weeks; (-)-gossypol (Group 4), 15 mg/kg administered orally per day
for 4 weeks; (-)-gossypol (Group 5), 30 mg/kg administered orally
per day for 4 weeks; docetaxel (or TAXOTERE, TXT) (Group 6), 7.5
mg/kg administered intravenously once per week for 4 weeks;
(-)-gossypol plus TXT (Group 7), 7.5 mg/kg (-)-gossypol
administered orally per day for 4 weeks, plus 7.5 mg/kg TXT
administered intravenously once per week for 4 weeks; (-)-gossypol
plus TXT (Group 8), 15 mg/kg (-)-gossypol administered orally per
day for 4 weeks, plus 7.5 mg/kg TXT administered intravenously once
per week for 4 weeks; and (-)-gossypol plus TXT (Group 9), 30 mg/kg
(-)-gossypol administered orally per day for 4 weeks, plus 7.5
mg/kg TXT administered intravenously once per week for 4 weeks.
[0513] During the treatment course, tumor sizes and animal weights
were measured 3 times per/week for each animal. Following
treatment, tumor sizes were measured 2 times per/week, and animal
weights once per/week. Tumor and weight observation were made
without knowledge of the animal's treatment group. A representative
example of in vivo xenograft based experiments used to investigate
the anti-tumor activity of (-)-gossypol in combination with
docetaxel is provided in FIG. 18. Docetaxel treatment started at
day 7 and was given i.v. at a weekly dose of 7.5 mg/kg for 3 weeks.
The results show that treatment by docetaxel alone in sub-optimal
dose (7.5 mg/kg weekly) inhibited tumor growth. But, the
combination of gossypol at three doses level (7.5, 15, and 30
mg/kg, respectively) with docetaxel achieved a much greater
activity in inhibition of tumor growth. In the group of 10 mice
treated with a combination therapy, 3 out of 10 mice (6 tumors) had
complete tumor regression. Overall, there was more than 90% of
inhibition in tumor growth in the combination group as compared to
the group control. Statistical analyses were performed using a
mixed-effects repeated measures model which accurately takes into
account the correlation within an animal over time, and between
tumors within an animal. The data was modeled using the natural
logarithm of tumor volume. Comparisons of the growth rates of
animals administered (-)-gossypol alone and docetaxel alone were
performed. Table 17 provides an example of one such comparison with
(-)-gossypol at 7.5 mg/kg and docetaxel.
TABLE-US-00017 TABLE 17 Tumor Growth Inhibition Tumor Growth
Treatment (T/C %) Delay (T-C Days) Radiation 77.7 8.5 (-)-gossypol
98.6 0 Radiation + (-)-gossypol 12.6 54.5
Table 17 shows a comparison of the tumor growth rate between
(-)-gossypol 7.5 mg/kg, docetaxel 7.5 mg/kg, and the combination;
P-values for the linear contrast are reported. Findings were
statistically significant findings when p<0.05.
[0514] In a subset of the mice treated with either Docetaxel alone
or a combination therapy, a second round of treatments with the
same regimen was initiated at day 45. The average tumor volume
before the second cycle treatment was about 2000 mm.sup.3. Tumors
in the Docetaxel alone treated group continued to grow and all the
mice were sacrificed due to the tumor burden. The combination
treatment groups displayed tumor regression and total 50% reduction
of tumor volume (FIG. 19). The data clearly shows that (-)-gossypol
was very effective in potentiating docetaxel in combination
treatments even at a doses where it is less effective when
administered as a single agent.
[0515] In yet another embodiment, the in vivo anti-tumor activity
of (-)-gossypol was investigated in non-small cell lung carcinoma
line A549 cells. A549 cells express high levels of Bcl-X.sub.L.
FIG. 20 shows the results from these experiments. Briefly, in FIG.
20, gossypol was administered in combination with paclitaxel
(TAXOL), wherein paclitaxel was administered at a weekly dose of 15
mg/kg with daily p.o. administration of (-)-gossypol at 7.5
mg/kg.
Example 17
Treatment of Squamous Head and Neck Cancer with Gossypol
Compounds
[0516] The following experiments were conducted to determine the
suitability and efficacy of gossypol compounds, e.g., (-)-gossypol,
to therapeutically treat squamous head and neck cancers. The
results are summarized in FIGS. 21, 22, 23, and 24A-24C.
[0517] Ten squamous cell carcinoma cell lines established at the
University of Michigan (UM-SCC) and three human fibroblast lines
were grown in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum. UM-SCC cell lines
originated from the larynx (n=2; UM-SCC-12, -23), oral
cavity/oropharynx (n=5; UM-SCC-1, -6, -14A, -74B, -81B),
hypopharynx (n=1; UM-SCC-22A) and metastases from laryngeal cancers
(n=2; UM-SCC-17B, -25). Human fibroblast cell lines originated from
surgical specimens (fibroblast cell lines 2 and 3) and neonatal
foreskin samples (fibroblast cell line 1).
Cell-Growth Inhibition Assays (MTT Assays)
[0518] Logarithmically growing cell lines were cultured, washed,
counted, and plated at 5,000-15,000 cells per well in duplicate
wells of 96-well plates and incubated in DMEM overnight. The
following day, serial dilutions were made from stock solutions of
racemic, (+)-, or (-)-gossypol to achieve the desired
concentrations. All experimental conditions were performed with 5
replicates. The sample plates were incubated for 6 days in 300
.mu.L of DMEM containing gossypol or solvent controls. MTT assays
were then performed according to the manufacturer's protocol (Roche
Diagnostics, Mannheim, Germany). The MTT assay measures cell
survival based on mitochondrial conversion of MTT from a soluble
tetrazolium salt into an insoluble colored formazan precipitate,
which is dissolved in dimethyl sulfoxide and quantitated by
spectrophotometry. (M. C. Alley et al., Cancer Res., 48:589-601
(1988)). Percent absorbance relative to control was plotted as a
linear function of drug concentration.
[0519] The 50% inhibitory concentrations (IC.sub.50s) were
identified as the concentration of drug required to achieve 50%
growth inhibition relative to untreated control populations. The
cell growth inhibition curves by (-)-gossypol in a panel of
squamous cell carcinoma cell lines (UM-SCC) and three fibroblast
cell lines are provided in FIG. 21.
Western Blot Analysis
[0520] Proteins were harvested during log phase growth by lysing
cells in the flask using a solution of phosphate buffered saline
(PBS) (BioWhittaker, Walkersville, Md.) containing 1% NP-40 (Sigma;
St. Louis, Mo.), 1 mM PMSF (Sigma) and 1 tablet of a cocktail of
protease inhibitors (Boehringer Mannheim, Germany) per 100 ml of
PBS. The protein extracts were quantified using a colorimetric
assay (Bradford Reagent) (BioRad, Hercules, Calif.). Thirty
micrograms of protein were resolved on 12% Tris-glycine sodium
dodecyl sulfate polyacrylamide gels (115V) under denaturing
conditions and transferred to Hybond-P PVDF membranes (Amersham
Pharmacia Biotech, Buckinghamshire, England) at 30V overnight.
Membranes were blocked in Tris-buffered saline containing 5% nonfat
dry milk at room temperature for 1 hour and then incubated with
primary antibody. Primary antibodies included murine anti-human
Bcl-2 oncoprotein (124) monoclonal antibody (Dako, Glostrup,
Denmark), murine anti-Bcl-xL (YTH-2H12) monoclonal antibody
(Trevigen Inc., Gaithersburg, Md.) and rabbit anti-Bcl-XS (Ab-1)
polyclonal antibody (Oncogene Research Products; Boston, Mass.).
Equivalency of protein loading was evaluated using either murine
anti-actin monoclonal antibody (Chemicon International, Temecula,
Calif.) or murine anti-GAPDH monoclonal antibody (Chemicon).
Membranes were then incubated with a secondary horseradish
peroxidase-conjugated anti-mouse (or rabbit) antibody (Amersham
Pharmacia Biotech) and analyzed using Enhanced Chemiluminescence
Plus reagent (Amersham Pharmacia Biotech). Densitometry readings
for three independent blots were taken using AlphaEase software
version 5.5 (Alpha Innotech Corp., San Leandro, Calif.) for
statistical analysis.
[0521] Expression levels of Bcl-2, Bcl-X.sub.L and Bcl-X.sub.S
proteins in a panel of squamous cell carcinoma cell lines (UM-SCC)
and one fibroblast cell line are provided in FIG. 22. FIG. 23 plots
the relationship between the ratio of Bcl-X.sub.L and Bcl-X.sub.S
and the IC.sub.50 values (concentration required to inhibit 50% of
cell growth related to untreated control cells) of (-)-gossypol in
a panel of squamous cell carcinoma cell lines (UM-SCC) and one
fibroblast cell line.
Apoptosis Assays
[0522] Apoptosis of UM-SCC cell lines following (-)-gossypol
treatment was quantitatively detected by enzymatic labeling of DNA
strand breaks using terminal deoxynucleotidyltransferase (TdT) and
Alexa Fluor-BrdUTP, known as the TdT-UTP nick end-labeling (TUNEL)
assay by flow cytometry. Cells were exposed to (-)-gossypol for 48
hours, harvested, fixed and TUNEL stained according to the
manufacturer's protocol (Molecular Probes Inc., Eugene, Oreg.). Ten
thousand cells were analyzed per sample using excitation of Alexa
Fluor at 488 nm. Apoptotic index (AI) was defined as percent of
apoptotic cells in the treatment population minus that in the
vehicle control population.
[0523] The results on 4 representative squamous cell carcinoma cell
lines (UM-SCC-1, UM-SCC-6, UM-SCC-12, UM-SCC-14A) and two
fibroblast cell lines (fibroblast 1 and 2) are provided in FIGS.
24A, 24B, and 24C, respectively.
Example 18
Clonogenic Assay of Administration of (-)-gossypol and Radiation
Therapy
[0524] PC-3 cells obtained from the American Type Culture
Collection and the National Cancer Institute were cultured in
improved minimal essential medium (IMEM) (Biofluids, Rockville,
Md.) with 10% fetal bovine serum (FBS) and 2 mM L-glutamine.
Cultures were maintained in a humidified incubator at 37 .degree.
C. and 5% CO.sub.2. Resolution of gossypol enantiomers was carried
out as described in Example 3. Gossypol compounds were first
dissolved in ethanol and diluted with sterile water within 5 min to
a final ethanol concentration of 10% prior to each
administration.
[0525] The PC-3 cells were used in a standard clonogenic assay to
investigate the effects of gossypol compounds, e.g., (-)-gossypol,
on the cell's response to subsequent radiation therapy. Briefly, at
day 1, 200 to 10,000 PC-3 cells per well were plated into 6 well
plates using standard techniques. The cells were then treated with
1-5 .mu.M (-)-gossypol and then exposed to 2 to 8 Gy, 300 keV,
X-ray irradiation within 1 h. One ml of complete medium was added
per well on Day 5. After 10 to 12 days in culture, the plates were
stained with crystal violet and colonies with over 50 cells were
counted using a ColCount (Oxford Optronix Ltd., Oxford, U.K.)
colony counter. For each combination treatment, parallel analyses
with each agent alone were also performed.
[0526] The data generated during the course of development of the
present invention show that (-)-gossypol sensitizes PC-3 cells to
radiation therapy in the clonogenic assay tested. The cell survival
curves were plotted with linear-quadratic model as shown in (FIGS.
35A and 35B). Briefly, FIG. 35A shows the in vitro effects of
(-)-gossypol in combination with various doses of radiation on PC-3
clonogenic assays, wherein: CI=Combination index; CI value <1
indicating synergistic effects; CI=1 indicating additive effects;
CI value >1 indicating antagonistic effects; DRI=Dose Reduction
Index; DRI>1.0 indicating synergistic effect. Treatment of PC-3
cells with (-)-gossypol significantly reduced PC-3 cells'
resistance to accompanying radiation therapy, resulting in 10- and
20-fold reductions in colony formation from controls at doses of 6
Gy and 8 Gy, respectively. The isobologram analyses (See, T. C.
Chou and P. Talalay, Adv. Enzyme Regulation, 22:27-55 (1984)),
widely used analyses used to determine synergism, show that the
combination of radiation with (-)-gossypol resulted in significant
synergy, with the Combination Index (CI) of 0.27 and 0.34, and the
Dose Reduction Index (DRI) of 6.1 and 4.3 for administration of 8
Gy radiation with either 5 .mu.M or 4 gossypol, respectively. The
results demonstrate that (-)-gossypol sensitizes PC-3 cells to
X-ray irradiation in a dose-dependent manner.
Example 19
Administration of Gossypol Compounds and Radiation Therapy in an In
Vivo Mouse PC-3 Xenograft Model
[0527] In this experiment, 34 male 5 to 6 week old NCI athymic
NCr-nu/nu nude mice were inoculated in each flank with an injection
of about 5.times.10.sup.6 PC-3 cells. The best 25 xenograft
recipients were used. Tumors averaged about 50 mm.sup.3.
(-)-Gossypol was first dissolved in ethanol and diluted with
sterile water within 5 min to a final ethanol concentration of 10%
prior to each administration. Irradiated test animals were
restrained, placed under the X-ray head, and covered with lead
shields to ensure that the tumor area was exposed to radiation. All
oral administrations of (-)-gossypol and 10% alcohol were given by
gavage (Table 18).
[0528] The 25 best xenograft mice were divided into 5 experimental
groups (5 mice per group) as follows: Vehicle control (Group 1),
daily oral administration of 10% alcohol; radiation only (Group 2),
2 Gy administered 5 times/week for 2 weeks, and daily oral
administration of 10% alcohol; radiation plus gossypol (Group 3), 2
Gy administered 5 times/week for 3 weeks, and 10 mg/kg of
(-)-gossypol administered orally every day for 4 weeks; gossypol
only (Group 4), 10 mg/kg (-)-gossypol administered orally every day
for 4 weeks; control (Group 5), no treatment. During the treatment
course, tumor sizes and animal weights were measured 3 times
per/week for each animal. Following treatment, tumor sizes were
measured 2 times per/week, and animal weights once per/week. Tumor
and weight observations were made without knowledge of the animal's
treatment group.
[0529] Treatment with (-)-gossypol alone had minimal antitumor
effect (Table 18). However, the data generated during the course of
the development of the present invention shows that (-)-gossypol
sensitizes PC-3 cells to radiation therapy in the xenograft animal
model. (-)-Gossypol treatment was started at day 13 where the
average tumor volume was at 150 mm.sup.3 and was given orally at
five times weekly in doses of 10 mg/kg for 4 weeks. X-ray
irradiation was given at day 5 after treatment with (-)-gossypol.
Radiation therapy alone achieved limited antitumor effect (23%).
Radiation therapy in combination with administration of gossypol
(at the same dose level (10 mg/kg)) provided potent tumor growth
inhibition (88% growth inhibition).
TABLE-US-00018 TABLE 18 Tumor Growth Inhibition Tumor Growth
Treatment (T/C %) Delay (T-C Days) Radiation 77.7 8.5 (-)-gossypol
98.6 0 Radiation + (-)-gossypol 12.6 54.5
[0530] Overall, there was more than 90% inhibition in tumor cell
growth in animals receiving radiation therapy in combination with
gossypol as compared to the group controls (FIG. 36). Briefly, in
FIG. 36 day 0 is the starting day of (-)-gossypol treatment;
starting tumor size was 150 mm.sup.3; (-)-gossypol was administered
at 10 mg/kg, orally 5 times per week for 4 weeks; radiation: X-ray,
2 Gy, 5 times per week for 3 weeks (at the second week the
radiation was adjusted to 2.5 Gy) for a total dose of 30 Gy.
Significantly, there was no significant toxicity in either group as
shown by bodyweight measurements (FIG. 37). At the end of treatment
(day 25), one mouse from each single agent and combination therapy
group was sacrificed and tumor tissues were stained for both
apoptosis and angiogenesis analysis. The results showed that
(-)-gossypol was very effective in potentiating radiation in
combination treatment regimens to induce apoptosis and to inhibit
angiogenesis even at dose levels where it was not very effective as
a single agent.
Example 20
Administration of Gossypol Compounds and Radiation Therapy in an In
Vivo Mouse PC-3 Xenograft Model
[0531] In this experiment, 60 male 5 to 6 week old NCI athymic
NCr-nu/nu nude mice were inoculated in each flank with an injection
of about 5.times.10.sup.6 PC-3 cells. The best 40 xenograft
recipients were used. Tumors averaged about 70 mm.sup.3.
(-)-Gossypol compounds were first dissolved in ethanol and diluted
with sterile water within 5 min to a final ethanol concentration of
10% prior to each administration. Irradiated test animals were
restrained, placed under the X-ray head, and covered with lead
shields to ensure that the tumor area was exposed to radiation. All
oral administrations of (-)-gossypol and 10% alcohol were given by
gavage.
[0532] The 40 best xenograft mice were divided into experimental
groups as follows: radiation only (Groups 1 and 2), 2 Gy
administered 5 times/week for 3 weeks, and daily oral
administration of 10% alcohol (as vehicle control); radiation plus
gossypol (Groups 3 and 4), 2 Gy administered 5 times a week for 3
weeks, and 10 mg/kg of (-)-gossypol administered orally 5
times/week for 4 weeks (8 mice each Group); gossypol only (Groups 5
and 6), 10 mg/kg (-)-gossypol administered orally every day for 4
weeks (8 mice each Group); vehicle control (Groups 7 and 8), daily
oral administration of 10% alcohol (8 mice each Group as vehicle
control); large tumor animals (combination of Groups 9-12), tumors
allowed to reach 500 mm.sup.3.
[0533] FIG. 38 shows yet another embodiment wherein (-)-gossypol in
combination with radiation achieves tumor regression in an
androgen-independent prostate PC-3 xenograft model, wherein: day 0
is the starting day of gossypol treatment; starting tumor size: 70
mm.sup.3; (-)-gossypol 10 mg/kg, p.o., q.d..times.5, for 4 weeks;
radiation, X-ray, 2 Gy, q.d..times.5 for 3 weeks, total dose=30 Gy;
large tumors in the vehicle control group were sacrificed when they
reached over 2-3,000 mm.sup.3.
[0534] At least 10 out of 16 tumors in the combination group
exhibited complete regression with only tiny scars left (from peak
tumor sizes of 200 mm.sup.3). Other remaining tumors in this group
appear pale yellowish and soft as compared with that in control
group with hard solid and reddish appearance, indicating tumor
blood supply was inhibited.
Example 21
(-)-Gossypol Inhibits Prostate Cancer Cell Growth
[0535] (-)-Gossypol is 5-10 times more potent than (+)-gossypol and
2 times more potent than racemic gossypol (50% (-)-gossypol) in
inhibiting cancer cell growth in human prostate cancer cells PC-3
and LnCap (FIG. 39) both of which express high levels of
Bcl-X.sub.L. FIG. 39 shows prostate cancer cell growth inhibition
by gossypol; PC-3 and LnCap cells in 96 well plates were treated in
triplicate with gossypol and its enantiomers; MTT-based 5-day cell
proliferation assays were performed and IC.sub.50 values
determined. The present invention is not limited to any mechanism.
Indeed, a mechanistic understanding of the invention is unnecessary
to practice (make and use) the present compositions and methods.
Nonetheless, data generated during the course of the development of
the present invention using 10 head-neck cancer cell lines shows
that the ability of (-)-gossypol to inhibit cell growth is
inversely related to the level of Bcl-xL protein, i.e., the higher
the level of Bcl-X.sub.L protein, the more sensitive the cells
(e.g., cancer cells) are to (-)-gossypol. The activity profile of
(-)-gossypol is different from that of chemotherapeutic agents,
which often show resistance in cancer cell lines with high levels
of Bcl-X.sub.L and Bcl-2 proteins. Further in vitro studies
demonstrated that (-)-gossypol induces apoptosis in variety of
cancer cell lines with high expression levels of Bcl-xL and
achieves synergistic effects with docetaxel (TAXOTERE, TXT). In the
human prostate cancer cell line PC-3, which has high levels of
Bcl-X.sub.L and Bcl-2 proteins, (-)-gossypol enhanced the
activities of docetaxel and cisplatin (CDDP) in inhibiting cell
growth and inducing apoptosis (FIG. 40). Briefly, in FIG. 40, cells
were treated with (-)-gossypol alone or in combination with TXT or
CDDP for 48 hrs, then stained with Annexin V-FITC and PI for flow
cytometry; values are percent of apoptotic cells. FIG. 41 shows the
Bcl-2 family proteins expression in three prostate cancer cell
lines; HSP70 heat shock protein 70 kDa fro gel loading. FIG. 42
shows cytotoxicity of (-)-gossypol on prostate cancer cell lines;
MTT based 5-day cell proliferation assays were performed and
IC.sub.50 values determined. (-)-Gossypol while showing
cytotoxicity in prostate cancer PC-3 and LnCap cells, has very
limited effect on DU145 and normal human fibroblast WI-38 cells
(FIG. 42).
Example 22
Anti-Tumor Activity of (-)-gossypol Alone and in Combination with
Docetaxel in Androgen Independent Prostate Cancer PC-3 Xenograft
Model
[0536] Further embodiments of the present invention provide studies
of (-)-gossypol using a PC-3 xenograft model in nude mice to
evaluate (-)-gossypol's anti-tumor activity in vivo alone and in
combination with docetaxel. In some experiments, the antitumor
efficacy of (-)-gossypol was compared with that of racemic
gossypol. Data generated during the development of the present
invention shows that (-)-gossypol is significantly more potent in
tumor growth inhibition than racemic gossypol (50% (-)-gossypol)
(FIGS. 43A and 43B). Briefly, in FIG. 43A, 15 mg/kg (.+-.)- or
(-)-gossypol p.o. was administered to a PC-3 nude mouse xenograft
model for 26 days; (-)-gossypol is more potent than (.+-.)-gossypol
(P<0.001). FIG. 43B shows tumor inhibition by (-)-gossypol was
significantly enhanced when used in combination with docetaxel
(TXT); (-)-gossypol (7.5 mg/kg, p.o. daily for 4 weeks) or
docetaxel (7.5 mg/kg once a week for 3 weeks); ** student's t-test.
While docetaxel alone did not achieve complete tumor regression,
(-)-gossypol alone achieved complete tumor regresision in 2 out of
12 tumors after the 4-week-treatment (Table 19).
TABLE-US-00019 TABLE 19 Treatment Complete tumor regression/Total
Percent regression Vehicle Control 0/16 0% (-)-gossypol 2/12 17%
(-)-gossypol + TXT 9/14 64%* TXT 0/16 0% *(-)-gossypol + TXT versus
(-)-gossypol, p = 0.0143; (-)-gossypol + TXT versus TXT, p =
0.0001; two sided Chi-square test
When used in combination with docetaxel, over 90% of tumor growth
inhibition was observed, significantly more effective than either
drug alone (FIG. 43B). Nine out of 14 tumors (64%) treated with the
combination of (-)-gossypol+TXT showed complete tumor regression
with only scar tissue left. Six out of these 9 regressed tumors did
not grow back 8 weeks after the combination therapy ended. Data
generated during the course of development of the present invention
indicates (-)-gossypol has a potent anti-tumor activity in the
androgen-independent human prostate cancer PC-3 xenograft model,
and achieves much greater anticancer efficacy in vivo when used in
combination with docetaxel (64% complete tumor regression).
Example 23
Blocking of Heterodimerization by (-)-gossypol in HT-29 Colon
Cancer Cells
[0537] In one embodiment HT-29 colon cancer cells, which express
only the Bcl-xL protein, were treated with (-)-gossypol at various
doses for 4 hrs, 8 hrs, and 12 hrs. To avoid the detergent effects
on the dimerization of Bcl-xL proteins in immunoprecipitation
experiments, following the treatment of cells with gossypol,
immunoprecipitation of cell lysates was carried out in the Chaps
buffer with the anti-Bcl-X.sub.L antibody. The Bcl-X.sub.L bound
proteins were then immunoblotted with anti-Bim antibody. Treatment
with (-)-gossypol resulted in a dose-dependent decrease in the
binding of Bcl-X.sub.L and pro-apoptotic protein Bim, starting at 8
hrs following the gossypol treatment (FIG. 44). There is no change
in the total Bcl-X.sub.L or Bim protein in the same lysates
following the (-)-gossypol treatment. These results are consistent
with in vitro binding data (FP-based displacement assay and NMR
binding assay) and cellular activity of gossypol at similar doses
and support the notion that (-)-gossypol can enter the cells and
has the ability to inhibit the interaction of Bcl- X.sub.L with
pro-apoptotic proteins such as Bim in intact cells.
Example 24
Synthesis of Other Gossypol Compounds
[0538] Many simple gossypol compounds have been described in
literature (See e.g., L. D. David et al., Current Medicinal
Chemistry, 7:479-498 (2000)). The synthesis of apogossypol,
gossypolone, and several other gossypol compounds has been carried
out by the inventors and is provided below.
Synthesis of Apogossypol
[0539] Synthetic methods of preparing apogossypol have been
reported (See e.g., P. C. Meltzer et al., J. Org. Chem.,
50(17):3121-3124 (1985)). Briefly, apogossypol was prepared by
heating racemic gossypol (1.6 g) in aqueous NaOH (40%, 10 mL) at
85.degree. C. for 2 hrs under the protection of nitrogen. The
reaction mixture was then cooled down, poured into a mixture of ice
and sulfuric acid (from 15 g of concentrated H.sub.2SO.sub.4), the
resultant was extracted with ether twice, and the combined organic
phase was washed with water, dried over anhydrous Na.sub.2SO.sub.4,
and concentrated in vacuum to yield apogossypol.
Synthesis of Gossypolone
[0540] Synthetic methods of preparing gossypolone have been
reported (See e.g., R. H. Hass et al., J. Org. Chem., 30:4111-4113
(1965)). Briefly, a solution of 2.0 g (3.5 mmol) of gossypol acetic
acid purchased from a commercial supplier (e.g., Sigma-Aldrich) in
100 ml of acetone and 200 ml of acetic acid was stirred at room
temperature during the addition of 150 ml of a 10% aqueous solution
of ferric chloride hexahydrate (56 mmol) and stirring was
maintained for 12 hrs. The solution was cooled and 250 ml of water
was added to precipitate a dark iron-containing compound which was
removed and treated with a mixture of ether and aqueous 20%
sulfuric acid. The liberated phenol was taken into the ether layer.
The ether layer was separated and dried, and the ether was
evaporated. The residue was recrystallized from aqueous acetic acid
to yield 1.2 g of orange product.
[0541] All publications, patent applications, and patents mentioned
in the above specification are herein incorporated by reference.
Various modifications and variations of the described method and
system of the invention will be apparent to those skilled in the
art without departing from the scope and spirit of the invention.
Although the invention has been described in connection with
specific preferred embodiments, it should be understood that the
invention as claimed should not be unduly limited to such specific
embodiments. Indeed, various modifications of the described modes
for carrying out the invention that are obvious to those skilled in
the relevant fields are intended to be within the scope of the
following claims.
Sequence CWU 1
1
101155PRTArtificial SequenceSynthetic 1Asn Arg Glu Ile Val Met Lys
Tyr Ile His Tyr Lys Leu Ser Gln Arg1 5 10 15Gly Tyr Glu Trp Asp Ala
Gly Asp Val Gly Ala Ala Pro Pro Gly Ala 20 25 30Ala Pro Ala Pro Gly
Ile Phe Ser Ser Gln Pro Val Val His Leu Thr 35 40 45Leu Arg Gln Ala
Gly Asp Asp Phe Ser Arg Arg Tyr Arg Arg Asp Phe 50 55 60Ala Glu Met
Ser Arg Gln Leu His Leu Thr Pro Phe Thr Ala Arg Gly65 70 75 80Arg
Phe Ala Thr Val Val Glu Glu Leu Phe Arg Asp Gly Val Asn Trp 85 90
95Gly Arg Ile Val Ala Phe Phe Glu Phe Gly Gly Val Met Cys Val Glu
100 105 110Ser Val Asn Arg Glu Met Ser Pro Leu Val Asp Asn Ile Ala
Leu Trp 115 120 125Met Thr Glu Tyr Leu Asn Arg His Leu His Thr Trp
Ile Gln Asp Asn 130 135 140Gly Gly Trp Asp Ala Phe Val Glu Leu Tyr
Gly145 150 1552152PRTArtificial SequenceSynthetic 2Asn Arg Glu Leu
Val Val Asp Phe Leu Ser Tyr Lys Leu Ser Gln Lys1 5 10 15Gly Tyr Ser
Trp Ser Gln Phe Ser Asp Val Glu Glu Asn Arg Thr Glu 20 25 30Ala Pro
Glu Gly Thr Glu Ser Glu Ala Val Lys Gln Ala Leu Arg Glu 35 40 45Ala
Gly Asp Glu Phe Glu Leu Arg Tyr Arg Arg Ala Phe Ser Asp Leu 50 55
60Thr Ser Gln Leu His Ile Thr Pro Gly Thr Ala Tyr Gln Ser Phe Glu65
70 75 80Gln Val Val Asn Glu Leu Phe Arg Asp Gly Val Asn Trp Gly Arg
Ile 85 90 95Val Ala Phe Phe Ser Phe Gly Gly Ala Leu Cys Val Glu Ser
Val Asp 100 105 110Lys Glu Met Gln Val Leu Val Ser Arg Ile Ala Ala
Trp Met Ala Thr 115 120 125Tyr Leu Asn Asp His Leu Glu Pro Trp Ile
Gln Glu Asn Gly Gly Trp 130 135 140Asp Thr Phe Val Glu Leu Tyr
Gly145 15034PRTArtificial SequenceSynthetic 3Gly Phe Leu
Gly149PRTArtificial SequenceSynthetic 4Cys Asp Cys Arg Gly Asp Cys
Phe Cys1 5513PRTArtificial SequenceSynthetic 5Cys Asn Gly Arg Cys
Val Ser Gly Cys Ala Gly Arg Cys1 5 1063PRTArtificial
SequenceSynthetic 6Gly Ser Leu1718PRTArtificial SequenceSynthetic
7Met Leu Ser Leu Arg Gln Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg1 5
10 15Thr Leu88PRTArtificial SequenceSynthetic 8Pro Pro Lys Lys Lys
Arg Lys Val1 5916PRTArtificial SequenceSynthetic 9Gly Gln Val Gly
Arg Gln Leu Ala Ile Ile Gly Asp Asp Ile Asn Arg1 5 10
151025PRTArtificial SequenceSynthetic 10Asn Leu Trp Ala Ala Gln Arg
Tyr Gly Arg Glu Leu Arg Arg Met Ser1 5 10 15Asp Glu Phe Val Asp Ser
Phe Lys Lys 20 25
* * * * *