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.

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.

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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.

Images