U.S. patent application number 16/384428 was filed with the patent office on 2019-12-19 for methods of treating and preventing melanoma with s-equol.
This patent application is currently assigned to The Board of Regents of the University of Texas System. The applicant listed for this patent is The Board of Regents of the University of Texas System. Invention is credited to Tyler CURIEL, Rong LI, Bin YUAN.
Application Number | 20190380998 16/384428 |
Document ID | / |
Family ID | 66397460 |
Filed Date | 2019-12-19 |
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United States Patent
Application |
20190380998 |
Kind Code |
A1 |
LI; Rong ; et al. |
December 19, 2019 |
METHODS OF TREATING AND PREVENTING MELANOMA WITH S-EQUOL
Abstract
The present invention provides methods and compositions for
treating or preventing melanoma with S-equol. The S-equol may be
administered alone or in combination with one or more cytotoxic or
immunotherapeutic compound or molecule.
Inventors: |
LI; Rong; (San Antonio,
TX) ; YUAN; Bin; (San Antonio, TX) ; CURIEL;
Tyler; (San Antonio, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Regents of the University of Texas System |
Austin |
TX |
US |
|
|
Assignee: |
The Board of Regents of the
University of Texas System
Austin
TX
|
Family ID: |
66397460 |
Appl. No.: |
16/384428 |
Filed: |
April 15, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62685392 |
Jun 15, 2018 |
|
|
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62728981 |
Sep 10, 2018 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/353 20130101;
A61P 35/00 20180101; C07K 16/2818 20130101; C07K 16/2827
20130101 |
International
Class: |
A61K 31/353 20060101
A61K031/353; C07K 16/28 20060101 C07K016/28; A61P 35/00 20060101
A61P035/00 |
Goverment Interests
STATEMENT OF GOVERNMENTAL SUPPORT
[0002] This invention was made with U.S. Government support under
grant number CA206529 awarded by the National Institutes of Health
and the Texas Cancer Research Grant Contract under grant number
DP150055 by the Cancer Prevention and Research Institute of Texas.
The U.S. Government and the Texas State Government have certain
rights in this invention.
Claims
1. A method for treating or preventing melanoma, comprising
administering a pharmaceutically effective amount of a formulation
comprising S-equol to a subject in need thereof.
2. The method of claim 1, wherein the formulation comprises 10-200
mg S-equol.
3. The method of claim 1, wherein the formulation comprises 50-150
mg S-equol.
4. The method of claim 1, wherein the formulation comprises about
50 mg S-equol.
5. The method of claim 1, wherein the formulation comprises about
150 mg S-equol.
6. The method of claim 1, wherein the formulation is administered
orally, intravenously, intraperitoneally, or subcutaneously.
7. The method of claim 1, wherein said subject is a human.
8. The method of claim 1, wherein the S-equol is administered in
combination with one or more other cancer treatments.
9. The method of claim 8, wherein the S-equol is administered in
combination with an immunotherapeutic agent.
10. The method of claim 9, wherein the immunotherapeutic agent is
an antibody.
11. The method of claim 10, wherein the antibody is directed to
programmed cell death protein 1 (PD-1).
12. The method of claim 11, wherein the antibody is directed to
programmed death ligand 1 (PDL-1).
13. The method of claim 11, wherein the antibody is
pembrolizumab.
14. The method of claim 12, wherein the antibody is
atezolizumab.
15. The method of claim 12, wherein the antibody is avelumab.
16. The method of claim 1, wherein the formulation is essentially
free of genistein, daidzein, and/or IBSO03569.
17. The method of claim 1, wherein genistein, daidzein, and/or
IBSO03569 are not co-administered with S-equol.
18. The method of claim 1, wherein the formulation is essentially
free of R-equol.
19. The method of claim 1, wherein the S-equol is produced
chemically.
20. The method of claim 1, wherein the formulation is administered
once per day.
21. The method of claim 1, wherein the formulation is administered
twice per day.
22. The method of claim 1, wherein the formulation is administered
three times per day.
23. The method of claim 1, wherein the formulation is administered
four times per day.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Application Ser. No. 62/685,392, filed Jun. 15, 2018, and U.S.
Provisional Application Ser. No. 62/728,981, filed Sep. 10, 2018,
which are hereby incorporated by reference in their entireties.
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] This invention relates to methods and compositions for
treating or preventing melanoma with a pharmaceutically effective
amount of S-equol or a pharmaceutical composition comprising
S-equol. In a preferred embodiment, the method relates to a
combination therapy using S-equol and another anti-cancer
treatment, such as immunotherapy.
Description of the Related Art
[0004] The methods and compositions of the present invention are
suitable for various types of cancers, particularly those
responsive to targeting of the estrogen receptor beta (ER.beta.).
In addition to being suitable for treating breast cancer (see, U.S.
Provisional Application 62/685,392, filed Jun. 15, 2018), the
methods and compositions of the present invention are suitable for
treating melanoma.
[0005] Malignant melanoma is a potentially serious skin cancer that
results from the uncontrolled growth of pigment cells, called
melanocytes. Melanoma may be the result of UV-induced changes to
the skin, and its occurrence may be increased as a result of
various genetic mutations. Mutations arising in the c-Kit gene, the
BRAF gene, the CDKN2A gene, which regulates cell division, the MDm2
gene, which is a negative regulator of the p53 tumor suppressor
gene, the retinoblastoma RB1 gene, and the MC1R
(melanocortin-1-receptor) gene are among those that may increase
the risk of developing melanoma. Treatment of melanoma may include
the use of surgery, chemotherapy and/or immunotherapy. (Gupta et
al., 2016; Clark et al., 2016; Steinberg et al., 2017).
[0006] Recent years have witnessed major clinical breakthroughs in
cancer immunotherapies (Mellman et al. (2011); Kaufman et al.
(2013); Brower (2015); Topalian et al. (2015)), which include
blocking the immune-suppressive immune checkpoint molecules CTLA-4
(cytotoxic T-lymphocyte-associated protein 4 or CD152 [cluster of
differentiation 152]), PD-1 (programmed cell death protein 1), and
PD-L1 (programmed death ligand 1; Hodi et al. (2010); Wolchok et
al. (2013); Topalian et al. (2014)). PD-1 is a checkpoint protein
on T-cells that serves as an "off switch" to keep T cells from
attacking other cells in the body. PD-1 interacts with PD-L1 on
tumor cells. When PD-L1 interacts with PD-1, it prevents the
T-cells from attacking the tumor cells. PD-1 inhibitors are
currently being used to treat melanoma and other cancers.
[0007] A review of cancer immunotherapy using checkpoint blockade
has been published as in Ribas and Wolchok, (2018) (see reference
list herein), and is incorporated by reference herein in its
entirety, particularly for the purposes of discussing pathways to
be targeted with immunotherapy and antibodies for use in that
checkpoint blockade. In addition, methods of rationally selecting
cancer vaccine targets based on a patient's "mutanome," namely, a
set of somatic mutations that generate cancer-specific neoepitopes
which can be recognized by autologous T cells as foreign, are
discussed in Sahin and Tureci, (2018) (see reference list herein).
This publication is also incorporated by reference herein in its
entirety for discussing personalized targeting of tumor antigens
such that cells of the immune system (e.g., CD4.sup.+ T helper
cells and CD8.sup.+ T cytotoxic cells) can be activated to attack
tumor cells. See, FIGS. 1 and 2. Both of these types of cancer
immunotherapy can be combined with the use of S-equol in accordance
with the present invention.
[0008] CD4.sup.+ T cells play a key role in the functioning of a
healthy immune system. They assist B cells to make antibodies,
activate the microbe killing capacity of macrophages and recruit
other immune cells to infected or inflamed areas of the body. These
activities are orchestrated through their production of various
cytokines and chemokines. It has been known for some time that
uncommitted CD4.sup.+ T-cells can differentiate into Th1 or Th2
cells, based on the prevailing pro-inflammatory/anti-inflammatory
environment, and that these activated Th1 and Th2 cells had
distinct cytokine production patterns and functions. Generally, Th1
cells were associated with the eradication of intracellular
pathogens whereas Th2 cells were heavily involved in responses
against extracellular pathogens and parasites. Uncontrolled Th1
responses were implicated in autoimmunity and aberrant Th2
responses were associated with allergy and asthma development.
However, this model did not explain the observation that a
deficiency in Th1 signaling and/or cytokines still allowed the
development of autoimmune diseases such as rheumatoid arthritis and
multiple sclerosis. More recently (2006) a third subset of CD4 T
cells, Th17 cells, which have a pro-inflammatory bias was
identified. Subsequent research using animal models and human
studies has demonstrated a key role for Th17 cells in the immune
system's defense against extracellular bacteria and fungi as well
as the development of autoimmune diseases, mediated by the
secretion of IL-17 by these cells. The secretion of IL-23 from
antigen-presenting cells such as dendritic cells, which have been
activated by the uptake and processing of pathogens, in turn
activates Th17 cells. (Taken from Bitesized Immunology:
https://www.immunology.org/public-information/bitesized-immunology/cells/-
th17-cells.) See, FIG. 2.
[0009] CD8.sup.+ (cytotoxic) T cells, like CD4.sup.+ Helper T
cells, are generated in the thymus and express the T cell receptor.
However, rather than the CD4 molecule, cytotoxic T cells express a
dimeric coreceptor, CD8, usually composed of one CD8a and one CD8P
chain. CD8.sup.+ T cells recognize peptides presented by MHC Class
I molecules, found on all nucleated cells. The CD8 heterodimer
binds to a conserved portion (the .alpha.3 region) of MHC Class I
during T cell/antigen presenting cell interactions (see FIG. 1).
CD8.sup.+ T cells (often called cytotoxic T lymphocytes, or CTLs)
are very important for immune defense against intracellular
pathogens, including viruses and bacteria, and for tumor
surveillance. When a CD8.sup.+ T cell recognizes its antigen and
becomes activated, it has three major mechanisms to kill infected
or malignant cells. The first is secretion of cytokines, primarily
TNF-.alpha. and IFN-.gamma., which have anti-tumor and
anti-microbial effects. The second major function is the production
and release of cytotoxic granules. These granules, also found in
natural killer (NK) cells, contain two families of proteins,
perforin, and granzymes. Perforin forms a pore in the membrane of
the target cell, similar to the membrane attack complex of
complement. This pore allows the granzymes also contained in the
cytotoxic granules to enter the infected or malignant cell.
Granzymes are serine proteases which cleave the proteins inside the
cell, shutting down the production of viral proteins and ultimately
resulting in apoptosis of the target cell. The cytotoxic granules
are released only in the direction of the target cell, aligned
along the immune synapse, to avoid non-specific bystander damage to
healthy surrounding tissue (see FIG. 1). CD8.sup.+ T cells are able
to release their granules, kill an infected cell, then move to a
new target and kill again, often referred to as serial killing. The
third major function of CD8.sup.+ T cell destruction of infected
cells is via Fas/FasL interactions. Activated CD8.sup.+ T cells
express FasL on the cell surface, which binds to its receptor, Fas,
on the surface of the target cell. This binding causes the Fas
molecules on the surface of the target cell to trimerize, which
pulls together signaling molecules. These signaling molecules
result in the activation of the caspase cascade, which also results
in apoptosis of the target cell. Because CD8.sup.+ T cells can
express both molecules, Fas/FasL interactions are a mechanism by
which CD8.sup.+ T cells can kill each other, called fratricide, to
eliminate immune effector cells during the contraction phase at the
end of an immune response. In addition to their critical role in
immune defense against viruses, intracellular bacteria, and tumors,
CD8.sup.+ T cells can also contribute to an excessive immune
response that leads to immunopathology, or immune-mediated damage.
(Taken from Bitesized Immunology:
https://www.immunology.org/public-information/bitesized-immunology/cells/-
cd8-t-cells.)
[0010] With respect to NK cells, the PK136 monoclonal antibody
reacts with mouse NK1.1, an antigen expressed by natural killer
cells and a subset of T cells in the NK1.1 mouse strains including
C57BL and NZB. Several commonly used laboratory mouse strains such
as BALB/c, SJL, AKR, CBA, C3H and A do not express the NK1.1
antigen. For detection of NK cells in these strains the monoclonal
antibody DXS 14-5971 is used. Simultaneous staining of C57BL/6
spleen cells with PK136 and DXS reveals coexpression of both
markers by a majority of cells as well as presence of small
populations of DXS+PK136- and DXS-PK136+ cells.
[0011] CD45 (lymphocyte common antigen) and its associated
molecules have also been shown to be important for T cell
activation. CD45 is a receptor-linked protein tyrosine phosphatase
that is expressed on all leucocytes, and which plays a crucial role
in the function of these cells. On T cells the extracellular domain
of CD45 is expressed in several different isoforms, and the
particular isoform(s) expressed depends on the particular
subpopulation of cell, their state of maturation, and whether or
not they have previously been exposed to antigen. It has been
established that the expression of CD45 is essential for the
activation of T cells via the TCR, and that different CD45 isoforms
display a different ability to support T cell activation. Although
the tyrosine phosphatase activity of the intracellular region of
CD45 has been shown to be crucial for supporting signal
transduction from the TCR, the nature of the ligands for the
different isoforms of CD45 have been elusive. Moreover, the precise
mechanism by which potential ligands may regulate CD45 function is
unclear. Interestingly, in T cells CD45 has been shown to associate
with numerous molecules, both membrane associated and
intracellular; these include components of the TCR-CD3 complex and
CD4/CD8. In addition, CD45 is reported to associate with several
intracellular protein tyrosine kinases including R561ck and p59fyn
of the src family, and ZAP-70 of the Syk family, and with numerous
proteins of 29-34 kDa. These CD45-associated molecules may play an
important role in regulating CD45 tyrosine phosphatase activity and
function. However, although the role of some of the CD45-associated
molecules (e.g. CD45-AP and LPAP) has become better understood in
recent years, the role of others still remains obscure. See, Altin
and Sloan, (1997) in the reference list herein.
[0012] However, these highly promising immunotherapies are only
effective for a subset of cancer patients and are not usually
curative. In particular, clinical trials of anti-PD-1 antibodies in
triple negative breast cancer (TNBC) patients demonstrate only
modest efficacy, with objective responses in the range of 10-20%,
and an additional 20% of patients experiencing some stabilization
of disease that would otherwise be rapidly progressive (Nanda et
al. (2016); Emens et al. (2015); Dirix et al. (2015); Rugo et al.
(2015); Dawood and Rugo (2016)). There is thus a pressing clinical
need to identify better therapies to improve individual responses
to immunotherapy for the treatment of TNBC, melanoma, and other
cancers.
[0013] PD-1 and its ligand PD-L1 are immune checkpoint molecules
that dampen T cell immunity (Lin et al. (2010); Curiel et al.
(2003); Brahmer et al. (2012); Pardoll and Drake (2012); Pardoll
(2012)). PD-L1 is overexpressed in tumor cells and some immune
cells, including Tregs (Lin et al. (2010); Curiel et al. (2003);
Pardoll (2012); Topalian et al. 2012)). Immunotherapy with
anti-PD-1 (.alpha.PD-1) and anti-PD-LI antibodies (.alpha.PD-L1)
has proven successful for treating various cancers (Brahmer (2012);
Pardoll and Drake (2012); Pardoll (2012)). Early clinical trials
for TNBC indicate that PD-1 and PD-L1 are valid targets for
intervention and toxicities of treatment are mild. In a Phase 1
study of 27 women with heavily pretreated, chemotherapy resistant,
metastatic TNBC that expressed PD-L1, the response rate was 18.4%
to pembrolizumab, an .alpha.PD-1 antibody (Nanda et al. 2016). A
similar study of atezolizumab, an .alpha.PD-LI antibody, produced a
response rate of 19% in 21 evaluable, PD-L1 expressing TNBC tumors
(Emens et al. (2015); Dawood and Rugo (2016)). Another group
reported an 8.6% response rate to avelumab, another .alpha.PD-LI
antibody, in 168 TNBC tumors unselected for PD-L1 expression (Dirix
et al. 2015), indicating that level of PD-L 1 tumor expression may
be important in mediating tumor response. Some disease
stabilization was seen in this aggressive subtype of breast cancer,
in approximately 20% of patients in these 3 trials. Serious
toxicities were few and related to immune modulation. These
clinical data indicate that the PD-1/PD-L1 axis can be a
therapeutic target, likely in combination approaches for better
efficacy.
[0014] There are two distinct types of estrogen receptors that
mediate diverse physiological effects of estrogens on breast cancer
cells. The first receptor is the ER.alpha. receptor which supports
estrogen-dependent breast tumor growth. The second is the ER.beta.
receptor which appears to work in an opposite fashion to
significantly attenuate the growth of breast tumor cells in
preclinical models (Clarke et al. (2003); Deroo and Korach (2006);
Deroo and Buensuceso (2010); Honma et al (2008); Katzenellenbogen
and Katzenellenbogen (2000); McDonnell and Norris (2002); Murphy
and Watson (2006); Shaaban et al (2008). Thus, ER.beta. can be
viewed in as a tumor suppressor gene in breast cancer. ER.beta. is
expressed in about 40% of TNBC cancers, and the therapeutic
potentials of targeting ER.beta. have not yet been investigated in
TNBC. Two recent advances have now made it feasible to move from
the preclinical models and into clinical research. The first
advance is a newly discovered mechanism of rallying ER.beta.'s
antitumor activity (Harris (2007); Hartman et al (2009); Heldring
et al. (2007); Thomas and Gustafsson (2011)), and the second is the
development of an oral formulation of an ER.beta. agonist (S-equol)
that has already been tested for clinical safety, pharmacodynamics
and tolerance in humans (Jackson et al. (2011a & b); Setchell
et al. (20005); Setchell (2002); Schwen et al. (2012).
[0015] Estrogen receptor (ER.beta.; nuclear or cytoplasmic) is
reported to be present in approximately half of TNBC (Marotti et
al. (2010); Reese et al. (2014)) as well as in melanoma (Marzagalli
et al., 2016). In addition to its tumor-intrinsic activity,
ER.beta. in host cells has also been implicated in melanoma tumor
inhibition (Cho et al. (2010)). In support of a tumor-extrinsic
antitumor activity of ER.beta., Cho et al. showed that syngeneic
murine melanoma cells grafted to recipient ER.beta.-KO animals grew
more robustly than those in WT recipient. In this study, the
authors showed that the growth of 3 different skin cancer cell
lines in their syngeneic murine hosts was sensitive to the activity
of the estrogen receptor. Therefore these transplantation studies,
used as models of skin cancer induction by SSUV irradiation,
strongly suggest that signaling by the ER.beta. is an endogenous
protective mechanism to inhibit skin cancer growth that is
partially mediated immunologically. The recognition of selective ER
modulators (SERMs) as agents able to elicit estrogenic effects in a
tissue-specific manner has expanded the potential population that
could benefit from ER ligand therapies. (Smith and O'Malley,
2018).
[0016] Thus, rallying ER.beta. antitumor activity through
ER.beta.-specific modification and/or ligand binding represents an
excellent therapeutic opportunity for TNBC and melanoma. However,
the therapeutic potentials of targeting ER.beta. have not been
extensively exploited, partly due to the paucity in the knowledge
of how to harness its antitumor activity. A phosphotyrosine residue
(Y36) in ER.beta., but not ER.alpha., has recently been identified
that is important for regulating the antitumor activity of ER.beta.
in TNBC cells (Yuan et al. (2014); Yuan et al. (2016)).
[0017] When this phosphotyrosine residue (pY36) was purposefully
mutated by adding a phenylalanine group (Y36F), activation of
ER.beta. target genes were decreased in a TNBC cell line
(MDA-MB-231). The Y36F mutation also obliterated the ability of
ER.beta. to inhibit tumor cell growth in vitro and in vivo. This
preclinical research therefore strongly supports the importance of
pY36 in the antitumor activity of ER.beta. (Yuan et al., 2014).
[0018] There is also evidence that pY36 status correlates with
survival of breast cancer patients. Research has been done using a
Prognostic Tissue Microarray (TMA) from the National Cancer
Institute (NCI), which consists of a large cohort of breast tumor
samples with a clinical follow-up record. Using a total of 726
readable IHC samples, patients with pY36-negative tumors were found
to have statistically significant shorter disease-free and overall
survival than those with pY36-positive tumors. Interestingly, the
association with survival was only seen in Stage II & III
disease, which raises the intriguing possibility that pY36 activity
may have an effect on disease progression from locally advanced to
metastatic breast cancer. Collectively, pY36 intensity appears to
have a stronger correlation with patient outcome than total
ER.beta., underscoring the clinical importance of this previously
unappreciated phosphotyrosine switch. See, FIG. 3.
[0019] S-equol, an ER.beta. agonist, was previously shown to
increase respiratory and maximal glycolysis fluxes in rat
hippocampal neurons, as well as cytochrome oxidase (COX) activity
and COX1 protein levels in brains from ovariectomized mice, and has
been studied in human subjects to assess its health impact and
safety (Yao et al. (2013); Jenks et al. (2002); Jackson et al.
(2011a & b); Usui et al., (2013)).
[0020] S-equol can be produced either chemically (i.e., chemical
synthesis) or by biotransformation (biosynthesis) through the
metabolism of daidzein, an isoflavone found in soy and red clover,
by gut bacteria. The structure of S-equol is shown below.
##STR00001##
[0021] Equol has a chiral center and therefore can exist in two
enantiomeric forms. S-equol, R-equol, racemic equol, and
non-racemic mixtures of equol (collectively "equol"); compositions
of equol; anhydrous crystalline polymorph of equol; processes for
the preparation of equol; and methods of using equol are described
in U.S. Pat. No. 8,716,497 (filed Sep. 10, 2012); U.S. Pat. No.
8,048,913 (filed Sep. 14, 2009); U.S. Pat. No. 7,960,432 (filed
Jul. 3, 2008); U.S. Pat. No. 7,396,855 (filed Jul. 24, 2003); U.S.
Pat. No. 8,263,790 (filed Jun. 1, 2011); U.S. Pat. No. 7,960,573
(filed May 4, 2009); U.S. Pat. No. 7,528,267 (filed Aug. 1, 2005);
U.S. Pat. No. 8,668,914 (filed Jul. 31, 2009); U.S. Pat. No.
8,580,846 (filed Aug. 18, 2006); U.S. Pat. No. 8,450,364 (filed
Apr. 9, 2012); and U.S. Pat. No. 8,153,684 (filed Oct. 2, 2009);
U.S. Pat. No. 9,408,824 (filed Mar. 5, 2014); and U.S. Pat. No.
9,914,718 (filed Oct. 14, 2015); each of which is hereby
incorporated by reference in its entirety.
[0022] Formulations comprising isoflavones and products derived
therefrom have been used in the past to treat disease. For example,
a mixture of equol, genistein, and daidzein, or a mixture of equol,
genistein, daidzein, and IBSO03569 have shown potential for
treating or preventing neurodegeneration and Alzheimer's disease.
See Zhao et al. (2009); U.S. Pat. No. 8,552,057; Yao et al. (2013)
(collectively "Brinton et al."). S-equol alone has also been
described for treating Alzheimer's Disease. See, U.S. application
Ser. No. 15/659,114 published as U.S. Patent Publication
2018/0028491, and International Patent Publication WO/2018/022604,
which are hereby incorporated by reference in their entireties.
[0023] However, there remains a need in the art for methods that
utilize S-equol for the treatment of other disease states that have
in the past been difficult to treat. In particular, there is a need
in the art for methods that utilize S-equol in the treatment of
melanoma.
SUMMARY OF THE INVENTION
[0024] The following brief summary is not intended to include all
features and aspects of the present invention, nor does it imply
that the invention must include all features and aspects discussed
in this summary.
[0025] The inventors have found that S-equol, preferably pure and
isolated S-equol, benefit melanoma patients. In particular, the
inventors have found that S-equol in combination with immunotherapy
is particularly effective in treating melanoma.
[0026] It is therefore an object of the invention to provide the
following: [0027] 1. A method for treating or preventing melanoma
by administering a pharmaceutically effective amount of a
formulation comprising S-equol to a subject in need thereof. [0028]
2. The method of Item 1, wherein the formulation comprises 10-200
mg S-equol. [0029] 3. The method of Item 1, wherein the formulation
comprises 50-150 mg S-equol. [0030] 4. The method of Item 1,
wherein the formulation comprises about 50 mg S-equol. [0031] 5.
The method of Item 1, wherein the formulation comprises about 150
mg S-equol. [0032] 6. The method of Item 1, wherein the formulation
is administered orally, intravenously, intraperitoneally, or
subcutaneously. [0033] 7. The method of Item 1, wherein said
subject is a human. [0034] 8. The method of Item 1, wherein the
S-equol is administered in combination with one or more other
cancer treatments. [0035] 9. The method of Item 8, wherein the
S-equol is administered in combination with an immunotherapeutic
agent. [0036] 10. The method of Item 9, wherein the
immunotherapeutic agent is an antibody. [0037] 11. The method of
Item 10, wherein the antibody is directed to programmed cell death
protein 1 (PD-1). [0038] 12. The method of Item 11, wherein the
antibody is directed to programmed death ligand 1 (PDL-1). [0039]
13. The method of Item 11, wherein the antibody is pembrolizumab.
[0040] 14. The method of Item 12, wherein the antibody is
atezolizumab. [0041] 15. The method of Item 12, wherein the
antibody is avelumab. [0042] 16. The method of Item 1, wherein the
formulation is essentially free of genistein, daidzein, and/or
IBSO03569. [0043] 17. The method of Item 1, wherein genistein,
daidzein, and/or IBSO03569 are not co-administered with S-equol.
[0044] 18. The method of Item 1, wherein the formulation is
essentially free of R-equol. [0045] 19. The method of Item 1,
wherein the S-equol is produced chemically. [0046] 20. The method
of Item 1, wherein the formulation is administered once per day.
[0047] 21. The method of Item 1, wherein the formulation is
administered twice per day. [0048] 22. The method of Item 1,
wherein the formulation is administered three times per day. [0049]
23. The method of Item 1, wherein the formulation is administered
four times per day.
[0050] The invention also relates to compositions comprising
S-equol as described for the methods herein.
[0051] The foregoing and other objects, features and advantages of
the invention will be apparent from the following more particular
description of preferred embodiments of the invention, as
illustrated in the accompanying drawings in which like reference
characters refer to the same parts throughout the different
views.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] FIG. 1. Schematic depiction of the action of CD8.sup.+
cells.
[0053] FIG. 2. Schematic depiction of the action of CD4.sup.+
cells.
[0054] FIG. 3. Association of pY36 with disease outcomes in breast
cancer. FIG. 3A shows the Kaplan-Meier estimate of disease-free and
overall survival in Stages II-III specimens correlating with IHC
intensity of pY36-ER.beta.. FIG. 3B shows the Kaplan-Meier estimate
of disease-free and overall survival in Stages II-III specimens
correlating with IHC intensity of (A) and total ER.beta. IHC.
[0055] FIG. 4. A schema for combination therapy. Mice were
administered one of: (1) vehicle+rat IgG2a; (2) S-equol+rat IgG2a;
(3) vehicle+anti-PD-1 antibody; or (4) S-equol+anti-PD-1 antibody.
Mice receiving anti-PD-1 antibody were given 250 .mu.g every 3
days, intraperitoneally, and mice receiving S-equol were given 50
mg/kg per day by oral gavage. Treatment started 7 days after tumor
challenge with B16 melanoma.
[0056] FIG. 5. A role of ER.beta. signaling in immune cells. FIG.
5A shows the schema for the chimera experiments. FIG. 5B shows
tumor growth in chimeric mice (WT>WT: n=10, KI>WT: n=4).
p=0.0003. FIG. 5C shows total CD8.sup.+ T cells from tumors. FIG.
5D shows interferon gamma (IFN-.gamma.) expressing CD8.sup.+ T
cells from tumors.
[0057] FIG. 6. Effect of S-equol in combination with an anti-PD-1
antibody on mice challenged with an B16 melanoma tumor cell line
over 12 days. Mice were administered anti-PD-1 antibody at 250
.mu.g every 3 days, intraperitoneally, and S-equol at 50 mg/kg per
day by oral gavage. FIG. 6 shows that separately, S-equol and
anti-PD-1 antibody reduce tumor volume compared to control, but
S-equol in combination with an anti-PD-1 antibody reduces tumor
volume compared to S-equol or anti-PD-1 antibody alone over 12 days
post-challenge.
[0058] FIG. 7. Effect of S-equol in combination with an anti-PD-1
antibody on mice challenged with an B16 melanoma tumor cell line
over 19 days. Mice were administered anti-PD-1 antibody at 250
.mu.g every 3 days, intraperitoneally, and S-equol at 50 mg/kg per
day by oral gavage. FIG. 7 shows that separately, S-equol and
anti-PD-1 antibody reduce tumor volume compared to control, but
S-equol in combination with an anti-PD-1 antibody reduces tumor
volume compared to S-equol or anti-PD-1 antibody alone over 19 days
post-challenge.
[0059] FIG. 8. Effect of S-equol in combination with an anti-PD-1
antibody on tumor weight in mice challenged with an B16 melanoma
tumor cell line. FIG. 8A shows that separately, S-equol and
anti-PD-1 antibody reduce tumor weight compared to control, but
S-equol in combination with an anti-PD-1 antibody reduces tumor
weight compared to S-equol or anti-PD-1 antibody alone. FIG. 8B
shows mouse total body weight for each treatment.
[0060] FIG. 9. Effect of S-equol in combination with an anti-PD-1
antibody and LY500307 on mice challenged with an B16 melanoma tumor
cell line over 16 days. Mice were administered anti-PD-1 antibody
at 250 .mu.g every 3 days, intraperitoneally, and S-equol at 50
mg/kg per day by oral gavage. FIG. 9A shows that separately,
S-equol and anti-PD-1 antibody reduce tumor volume compared to
control, but S-equol in combination with an anti-PD-1 antibody
reduces tumor volume compared to S-equol or anti-PD-1 antibody
alone over 12 days post-challenge. FIG. 9B shows results with
LY500307, a selective ER.beta. agonist (SERBA-1;
Erteberel.RTM.).
[0061] FIG. 10. Generalized protocol for human clinical trial with
S-equol.
DETAILED DESCRIPTION OF THE INVENTION
[0062] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by those
of ordinary skill in the art to which this invention belongs.
Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, the preferred methods and materials are
described. Generally, nomenclatures utilized in connection with,
and techniques of, cell and molecular biology and chemistry are
those well-known and commonly used in the art. Certain experimental
techniques, not specifically defined, are generally performed
according to conventional methods well known in the art and as
described in various general and more specific references that are
cited and discussed throughout the present specification. For
purposes of clarity, the following terms are defined below.
[0063] "Substantially pure" means about 90% pure, preferably 95%
pure, and more preferably 98% pure of any contaminating
proteins.
[0064] "Substantially free" means about less than about 10%,
preferably less than 5%, and more preferably less than 2% of any
contaminating proteins.
[0065] "Approximately" or "about" means within +/-10%, preferably
within +/-5%, more preferably within +/-2% of that which is being
measured.
[0066] The present invention relates to the prevention and/or
treatment of melanoma with S-equol.
[0067] Dosage amounts and administration schedules for S-equol will
depend on whether the S-equol is being administered
prophylactically to a patient at risk of developing melanoma, or
being administered as treatment for a patient already diagnosed
with melanoma. A person at risk for developing melanoma may be a
person with a family history of melanoma, and/or may have one or
more mutations in a CDKN2 A gene, MDm2 gene, RB1 gene, or MC1R
gene. In a patient already diagnosed with melanoma, dosages and
administration schedules may also vary depending on the stage of
the cancer (stage I, stage II, stage III, stage IV or stage V), the
number of lymph nodes involved, tumor size and the availability of
and/or decision to co-administer other therapies. Dosages and
administration schedules may also vary depending on various
molecular markers present on or in tumor cells, including, but not
limited to Hsp90, RGS1, osteopontin, HER3, ING4, ING3, NCOA3, MCM4,
MCM6, Ki67 and pY36. Diagnostic tests for markers which are
prognostic for the aggressiveness and/or likelihood of recurrence
of a tumor may be used as a factor or determining dosage regimens.
(Gogas et al. (2009), incorporated by reference in its entirety for
all purposes.)
[0068] S-equol can be administered one or more times per day at
1-400 mg per dose, more preferably 10-320 mg, more preferably
50-150 mg. Non-limiting examples include 2 mg, 5 mg, 10 mg, 15 mg,
20 mg, 40 mg, 50 mg, 80 mg, 100 mg, 150 mg, 160 mg, 200 mg, 250 mg,
300 mg 320 mg, etc. or approximately or about those doses. The dose
may be administered one, two, three or four times per day,
preferably twice per day (B.I.D.) The regimen can be continued
indefinitely, or for such time in intervals where markers are
examined and responsiveness to the dose is determined. Examples of
such intervals are two weeks, four weeks, two months, four months,
six months, etc. or about or approximately those intervals. No
upper limit, with respect to administration schedule, is
required.
[0069] The S-equol administered is preferably formulated for oral
administration; however, other routes of administration are also
contemplated, including rectal, optical, buccal (for example
sublingual), parenteral (for example subcutaneous, intramuscular,
intradermal and intravenous) and transdermal administration.
[0070] Compositions or formulations according to the present
invention can comprise one or more pharmaceutically-acceptable or
industrial standard fillers. The filler must not be deleterious to
a subject treated with the composition. The filler can be solid or
a liquid, or both. The filler can be formulated with the active
S-equol as a unit-dose, for example a tablet, which can typically
contain from about 10% to 80% by weight of S-equol. Compositions
can be prepared by any of the well known techniques of pharmacy,
for example admixing the components, optionally including
excipients, diluents (for example water) and auxiliaries as are
well known in the pharmaceutical field.
[0071] Compositions suitable for oral administration can be
presented in discrete units, such as capsules, cachets, lozenges,
or tablets, each containing a predetermined amount of the extract;
as a powder or granules; as a solution or a suspension in an
aqueous or non-aqueous liquid; or as an oil-in-water or
water-in-oil emulsion. Such compositions can be prepared by any
suitable method of pharmacy which includes the step of bringing
into association the active S-equol and one or more suitable
carriers (which can contain one or more accessory ingredients as
noted above). In general the compositions of the invention are
prepared by uniformly and intimately admixing the S-equol with a
liquid or finely divided solid carrier, or both, and then, if
necessary, shaping the resulting mixture. For example, a tablet can
be prepared by comprising or moulding a powder or granules
containing the extract, optionally with one or more accessory
ingredients. Compressed tablets can be prepared by compressing in a
suitable machine, the extracts in the form of a powder or granules
optionally mixed with a binder, lubricant, inert diluents, and/or
surface active/dispersing agent(s). Moulded tablets can be made by
moulding, in a suitable machine, the powdered compound moistened
with an inert liquid binder.
[0072] Suitable fillers, such as sugars, for example lactose,
saccharose, mannitol or sorbitol, cellulose preparations and/or
calcium phosphates, for example tricalcium phosphate or calcium
hydrogen phosphate, and also binders such as starch pastes using,
for example, corn, wheat, rice or potato starch, gelatin,
tragacanth, methylceullose and/or polyvinylpyrrolidone, and, if
desired, disintegrators, such as the above-mentioned starches, also
carboxymethyl starch, cross linked polyvinyl pyrrolidone, agar or
alginic acid or a salt thereof, such as sodium alginate. Excipients
can be flow conditioners and lubricants, for example silicic acid,
talc, stearic acid or salts thereof, such as magnesium or calcium
stearate, and/or polyethylene glycol. Dragee cores are provided
with suitable, optionally enteric, coatings, there being used,
inter alia, concentrated sugar solutions which can comprise gum
arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or
titanium dioxide, or coating solutions in suitable organic solvents
or solvent mixtures, or, for the preparation of enteric coatings,
solutions of suitable cellulose preparations, such as
microcrystalline cellulose, acetylcellulose phthalate or
hydroxypropylmethylcellulose phthalate. Dyes or pigments can be
added to the tablets or dragee coatings, for example for
identification purposes or to indicate different doses of active
ingredients.
[0073] Other orally administrable pharmaceutical compositions are
dry-filled capsules made, for example, of gelatin, and soft, sealed
capsules made of gelatin and a plasticiser, such as glycerol or
sorbitol. The dry-filled capsules can comprise the extracts in the
form of granules, for example in admixture with fillers, such as
lactose, binders, such as starches, and/or glicants, such as talc
or magnesium stearate, and, where appropriate, stabilizers. In soft
capsules, the extract is preferably dissolved or suspended in
suitable liquids, such as fatty oils, paraffin oil or liquid
polyethylene glycols, to which stabilizers can also be added.
[0074] According to one aspect of the invention, the compositions
comprising S-equol include those described in U.S. Pat. No.
7,960,432 (filed Jul. 3, 2008); U.S. Pat. No. 7,396,855 (filed Jul.
24, 2003); and U.S. Pat. No. 9,408,824 (filed Mar. 5, 2014)--the
disclosures of each are hereby incorporated by reference in their
entireties.
[0075] According to another aspect of the invention, S-equol can be
prepared chemically (i.e., chemical synthesis) according to the
processes described in U.S. Pat. No. 8,716,497 (filed Sep. 10,
2012); U.S. Pat. No. 8,263,790 (filed Jun. 1, 2011); U.S. Pat. No.
7,960,573 (filed May 4, 2009); U.S. Pat. No. 7,528,267 (filed Aug.
1, 2005) and U.S. Pat. No. 9,914,718 (filed Oct. 14, 2014)--the
disclosures of each are hereby incorporated by reference in their
entireties. For example, S-equol can be enantioselectively prepared
using an iridium catalyst with a chiral ligand. These methods of
enantioselectively preparing S-equol are incorporated by
reference.
[0076] According to another aspect of the invention, S-equol can be
a single anhydrous crystalline polymorph of S-equol, such as the
anhydrous crystalline polymorph of S-equol described in U.S. Pat.
No. 9,914,718 (application Ser. No. 14/883,617, filed Oct. 14,
2015)--the disclosure of which, including the chemical and physical
properties used to characterize the anhydrous crystalline polymorph
of S-equol, is incorporated by reference in their entireties. For
example, the anhydrous crystalline polymorph of S-equol described
in U.S. Patent Application Publication No. 2016/0102070 has the
following characteristic X-ray powder diffraction pattern
wavenumbers (cm.sup.-1): 3433, 3023, 3003, 2908, 2844, 1889, 1614,
1594, 1517, 1508, 1469, 1454, 1438, 1400, 1361, 1323, 1295, 1276,
1261, 1234, 1213, 1176, 1156, 1116, 1064, 1020, 935, 897, 865, 840,
825, 810, 769, 734, 631, 616, 547, 517, 480, and 461. The
characterizations of anhydrous crystalline polymorph of S-equol are
incorporated by reference.
[0077] The S-equol can be administered in combination with one or
more additional cancer treatments, including surgery, cytotoxic
chemotherapy, radiation, immunotherapy, cancer vaccines, inhibitors
of cellular pathways (protein or peptide, nucleic acid-based
[antisense oligonucleotides including DNA oligonucleotides,
antisense siRNA, shRNA] and/or hormonal (adjuvant endocrine)
therapy. Examples of cytotoxic chemotherapy suitable for treating
melanoma include, but are not limited to imiquimod cream
(Zyclara.RTM.), dacarbazine (DTIC) and temozolomide (Temodar.RTM.).
Examples of immunotherapy include but are not limited to nivolumab
(Opdivo.RTM.), pembrolizumab (Keytruda.RTM.), nivolumab
(Opdivo.RTM.), and ipilirurab (Yervoy.RTM.), imatinib
(Glevac.RTM.), nilotinib (Tasigna.RTM.) or interferon and
interleukin-2 (IL-2). Other options include injections of the T-VEC
vaccine (Imlygic.RTM.), or the Bacille Calmette-Guerin (BCG)
vaccine.
[0078] Appropriate dosages of immunotherapy monoclonal antibodies
or other cancer therapeutic are determined based on the indication,
and their determination is well within the skill in the art. Dosing
may be in mg, in mg/kg or mg/m.sup.2. See, Sachs et al. (2016),
which is incorporated by reference herein in its entirety for all
purposes, including examples of therapeutic antibodies, small
molecules and dosages. The S-equol and/or additional therapeutic
composition may be administered once, twice, three times or more
per day, depending on the dose, toxicity and indication. Dosing may
occur over several days, weeks or months, and may be continuous or
intermittent. Typical dosages for immunotherapeutics are 50-500
mg/day, more preferably 100-400 mg/day, more preferably 150-250
mg/day. In certain embodiments, the dose may be 150 mg/day or 150
mg twice per day, or 250 mg/day or 250 mg twice a day. In other
embodiments the dosage may be given based on patient weight, for
instance 0.5-10 mg/kg, more preferably about 1, 2 or 5 mg/kg.
Dosage may also be given as mg/m.sup.2, for example 50-500
mg/m.sup.2, more preferably 100-300 mg/m.sup.2, for example about
100, 150, 200, 250 or 300 mg/m.sup.2.
[0079] The dosages may be about or greater than the lower end of
any of the afore-stated ranges, or about or less than the upper end
of any of the afore-stated ranges.
[0080] Responsiveness to the S-equol+/-other cancer therapy can be
assessed by analyzing one or more cellular markers, by imaging
techniques, by cellular proliferation assays and by direct
examination of tumors. The analysis of cellular markers can be done
with immunohistochemical or immunocytochemical techniques or by
nucleic acid detection, such as hybridization techniques and
polymerase chain reaction (PCR). A preferred biomarker to assess
mitotic division and tumor growth is Ki67 (Beelen et al. (2012);
Urruticoechea et al. (2005)).
[0081] The following examples are provided to aid the understanding
of the present invention, the true scope of which is set forth in
the appended claims. It is understood that modifications can be
made in the procedures set forth without departing from the spirit
of the invention.
EXAMPLES
[0082] The processes of the present invention will be better
understood in connection with the following examples, which are
intended as an illustration only and without limiting the scope of
the invention. Various changes and modifications to the disclosed
embodiments will be apparent to those skilled in the art and such
changes and modifications including, without limitation, those
relating to the processes, formulations and/or methods of the
invention may be made without departing from the spirit of the
invention and the scope of the appended claims.
Example 1
[0083] To determine whether the newly identified phosphotyrosine
switch regulates ER.beta. tumor-extrinsic function, a whole-body
knock-in (K1) mouse model (C57BL/6) was established in which the
corresponding tyrosine residue of endogenous mouse ER.beta. is
mutated to phenylalanine (Y-F). Consistent with previously reported
ER.beta. knockout (KO) mice (Krege et al. (1998)), these
phosphorylation mutant KI mice had no overt developmental defects
and were grossly indistinguishable from their WT littermates
(unpublished data). Survey of ER.beta. expression in KO and KI mice
indicated that the Y-F mutant protein was expressed at levels
comparable to WT ER.beta. in multiple tissues, including bone
marrow and spleen (FIG. 4A). Syngeneic murine tumor cells of
various origins were then transplanted, including the MMTV-Wnt
mammary tumor cell line with a basal-like breast cancer profile
(Pfefferle et al. (2013)), into WT and KI recipient mice. In all
cases, tumor cells grew more robustly in ER.beta. KI recipient mice
than in their syngeneic WT counterparts (FIG. 4B, and data not
shown). These data clearly demonstrate that the phosphotyrosine
switch is important for ER.beta. tumor-extrinsic antitumor activity
in multiple tumor types.
Example 2
[0084] The following experiment sought to delineate further the
host ER.beta. signaling in tumor inhibition based on the role of
antitumor immunity and recent clinical advances in cancer
immunotherapies (Topalian et al. (2015); Chen and Flies (2013);
Sharma and Allison (2015)). A mouse chimera experiment was
conducted involving a bone marrow transplant. As illustrated in
FIG. 5A, WT recipient mice were first irradiated (10 Gy) to kill
endogenous bone marrow cells, followed by transplant with bone
marrow from syngeneic KI or WT donors. After confirming successful
chimerism in KI>WT and WT>WT mice, tumor cells were injected
8 weeks after bone marrow transplant. Tumor growth was
significantly greater in KI>WT chimeras (with KI immune cells)
versus WT>WT controls (FIG. 5B). This suggests that KI immune
cells poorly controlled tumor growth. Thus, these data link the
importance of tumor extrinsic ER.beta. antitumor activity to the
immune response.
Example 3
[0085] Next, tumor-infiltrating immune cell populations were
analyzed. Total numbers of tumor-infiltrating CD4.sup.+ and
CD8.sup.+ T cells were reduced in KI>WT versus WT>WT mice
(FIG. 5C and data not shown), further supporting the notion that
antitumor immunity in KI mice is compromised. Furthermore, the
prevalence of IFN.gamma.-producing CD8.sup.+ (antitumor) cells was
significantly lower in tumors from KI>WT versus WT>WT mice
(FIG. 5D), suggesting compromised cytotoxic potency of CD8+ T cells
in the absence of functional ER.beta. signaling. Additional
preliminary data not shown here indicate that activation of
dendritic cells, which prime antitumor T cells, was also
compromised in KI>WT chimeric mice, as evidenced by their
reduced MHC-II expression. Furthermore, effector T cells were less
activated (lower CD44/CD62L) and had other reduced effector
functions in KI>WT chimeras [e.g., lower tumor necrosis factor
alpha (TNF).alpha., interleukin (IL)-2, and perforin, data not
shown]. These data strongly suggest ER.beta.-dependent augmentation
of antitumor CD8.sup.+ T cell effector activity and improved
intra-tumor immune cell accumulation. Therefore, rallying ER.beta.
antitumor activity with clinically safe ER.beta. agonists such as
S-equol will improve efficacy of existing anticancer
immunotherapies and make them more effective to treat melanoma.
Example 4
[0086] Melanoma cell line B16 was tested to determine the effects
of a PD-1 inhibitor, S-equol and a combination of the two therapies
on tumor growth. This experimental design includes four arms: (1)
controls, (2) .alpha.PD-1, (3) S-equol, and (4) .alpha.PD-1+S-equol
(FIG. 4) by injecting into mice 5.times.10.sup.5 cells
subcutaneously. Antibodies were administered .alpha.-PD-1: 250
.mu.g/mice by i.p. injection every 3 days and S-equol was
administered at 50 mg/kg/day by oral gavage. Treatment began 7 day
after tumor challenge. Tumor growth trajectories were compared
within treated mice using a repeated measures linear mixed model.
The primary outcome was the logarithm of the tumor size, and the
test statistic is the treatment X time interaction. Tumors were
measured about 10 times. The number of recipient mice use used in
the studies (n=6 per group) was based on the above study. Results
(FIGS. 6-9) from this experiment demonstrate the ability of S-equol
to boost anticancer immunotherapy.
Example 5
[0087] In addition to measurements of tumor size and weight in each
arm, immunophenotyping is performed to gain more mechanistic
insight into the antitumor effects of mono- and combinational
therapies. Tumors, spleens, and draining lymph nodes are harvested
and weighed. Anti-coagulated blood was collected by cardiac
puncture. Tumors are paraffin-embedded for immunohistochemistry and
snap-frozen for mRNA and protein analysis by Luminex. Flow
cytometry is used (for phenotype/functions) and ViCell (for
quantification) to analyze immune cells from tumor infiltrates,
tumor-draining lymph nodes, and spleens. Analyses include CD3.sup.+
total T cells and CD4.sup.+ and CD8.sup.+ T cell subsets; effector
function (e.g., IFN-.gamma., TNF-.alpha., IL-2, perforin, CD107a);
activation (e.g., CD69, CD44, CD62L); and exhaustion (e.g., PD-1,
Tim3, Lag3). Antigen-presenting cells (CD11b.sup.+CD1c.sup.-
monocyte/macrophages, CD11b.sup.+CD11c+ dendritic cells),
NK1.1.sup.+NKp46.sup.+ NK cells, regulatory T cells (Treg,
CD3.sup.+CD4.sup.+CD25.sup.hiFoxp3.sup.+), and myeloid-derived
suppressor cells (MDSC, CD11b.sup.+Gr-1.sup.hi) are also analyzed.
CD8.sup.+ T cell receptor diversity were assessed with a commercial
kit (Adaptive Biotechnologies). Local cell proliferation is also
tested with Ki67 and BrdU stain.
Example 6
[0088] Further work in a preclinical animal model supports the
notion of using S-equol to treat melanoma. In immunocompromised
mouse experiments, using B16, a murine tumor cell line know to be
useful to study human melanoma metastasis and solid tumor
formation, growth of tumors is suppressed with S-equol treatment
compared to control. 0.5.times.10.sup.6 B16 cells are injected into
normal BL mice. When the tumor masses reach 50 to 80 mm.sup.3
(about one week after the inoculation), the mice are given daily
subcutaneous injections of S-equol (20 or 60 mg/kg per day) or PBS
as a vehicle control. Tumor development is followed by caliper
measurements along two orthogonal axes: length (L) and width (W)
and volume (V) is estimated by the formula V=[L.times.(W.sup.2)]/2.
Tumors harvested from mice are fixed in 10% neutral-buffered
formalin, dehydrated, embedded in paraffin, and sectioned at 3
.mu.m thickness. Representative tumor sections from vehicle control
and S-equol-treated mice are tested for Ki-67 expression to assess
cell proliferation, and for ER.beta. pY36. Statistical significance
in the experiments is assessed by two-tailed Student's t test. In
all assays, p<0.05 is considered statistically significant.
Example 7
[0089] A pilot biomarker clinical study is performed in 20 subjects
with documented melanoma to determine the effect of the ER.beta.
agonist S-equol on Ki67 an indicator of tumor cell proliferation.
Tumor cell proliferation is measured by immunohistological staining
for Ki-67. Ki-67 is encoded by the gene MKI67 gene and is required
to maintain individual mitotic chromosomes dispersed in the
cytoplasm following nuclear envelope disassembly. Measuring the
fraction of Ki-67-positive cells in a tumor is known to be a
reliable parameter for assessing cancer patient prognosis. Subjects
with TNBC are known to have very high levels of Ki67.
[0090] Patients enrolled in the study receive a history, physical,
laboratory assessment, radiological imaging, and diagnostic
biopsies. Immunohistochemistry (IHC) of Ki67 (proliferation
marker), total ER.beta., and pY36-ER.beta. (phosphorylated
ER.beta.), B7H1 (also known as PDL-1) and MIA and S 100B are
conducted using core needle biopsy tissue samples prior to
treatment. Patients are given oral S-equol 50 mg, twice daily for
approximately two weeks (10-21 days) prior to the scheduled
oncologic surgeries or start of primary systemic therapy. Tumor
samples are obtained from the surgery or the two-week repeat
biopsy. Post-treatment IHC evaluation of Ki67, total ER.beta.,
phosphorylated ER.beta. (pY36) and MIA and S 100B are obtained.
Ki67 reduction is compared pre- and post-treatment with S-equol.
S-equol causes a measurable decrease in Ki67, indicating its
efficacy in this tumor type. A c-DNA microarray platform is also
used to discover downstream transcriptional targets of ER.beta.
resulting from activation by S-equol.
[0091] Patients eligible for the study have newly diagnosed,
previously untreated, melanoma. All stages of the disease are
eligible. They take S-equol during the time they undergo standard
preoperative evaluation or pre-therapeutic work-up for advanced
disease, such as staging procedures or central line placement. If
primary surgery is performed, tissue leftover after standard
diagnostic evaluation is used for the two-week biomarker
assessment. If the patient does not have primary surgery at the end
of the two-week S-equol treatment, a second set of biopsies is
obtained before the start of any standard systemic therapy. See,
FIG. 10.
[0092] A standard procedure for acquiring core biopsy is utilized
for determining melanoma prior to enrollment. Whenever possible,
the tumor tissues used for IHC analyses are acquired at the same
time as the diagnostic core biopsy. The tissue samples are sent for
staining of total and pY36, ER.beta. and Ki67, and analyzed.
Whole-Genome DASL HT platform from Illumina, which is designed for
gene expression profiling with extremely low input of RNA (50 pg),
is used to interrogate paired FFPE tissue obtained before and after
exposure to S-equol.
[0093] Statistical analysis is conducted which shows that
ER.beta.-expressing melanoma responds to S-equol as manifested by
measurable decline in Ki67. The primary analysis estimates the
geometric mean change of the Ki67 expression from baseline to two
weeks. This is performed using a one-sample t-test of the pre/post
differences in the log-transformed data, and summarized by the 95%
confidence interval. All computations are performed with SAS v9.2+
(Cary, N.C.) or R v2.15+ (Vienna, Austria).
Example 8
[0094] A follow-up clinical study enrolls newly diagnosed melanoma
patients, and patients are treated and assessed as discussed in
Example 7, except that the dose of S-equol is increased to 150 mg
twice daily. Treatment with S-equol is for 10-21 days. Ki67 is
analyzed as described in Example 7.
REFERENCES
[0095] The patent and scientific literature referred to herein
establishes the knowledge that is available to those with skill in
the art. All United States patents and published or unpublished
United States patent applications cited herein are incorporated by
reference. All published foreign patents and patent applications
cited herein are hereby incorporated by reference. All other
published references, documents, manuscripts and scientific
literature cited herein are hereby incorporated by reference.
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