VALPROIC ACID SALTS

[0001] This application claims priority to US provisional patent application 61/054,691, filed May 20, 2008, the entire contents of which are incorporated by reference.

1. FIELD OF THE INVENTION

[0002] The present invention relates to therapeutically acceptable salts of valproic acid, such as tamoxifen valproic acid salt, compositions comprising salts of valproic acid, and methods of treating cancer with valproic acid salts. Compositions according to the invention include pharmaceutical formulations of valproic acid salts. The invention therefore provides methods of treatment and pharmaceutical compositions.

2. BACKGROUND OF THE INVENTION

[0003] Although many breast cancer therapies exist, there is a need to develop therapeutics that are safe and effective, and which circumvent resistance against hormonal and other therapies in breast tumors, that do not cause increases in other types of cancer, which extend the disease-free survival of patients, and may represent advantageous formulations. For example, while the majority of patients with ERα-positive breast tumors initially respond favorably to antiestrogen therapy with tamoxifen or fulvestrant, or to estrogen ablation therapy with aromatase inhibitors, most tumors eventually acquire resistance to these hormonal therapies despite maintaining ERa expression (Clarke, R., et al, J Steroid Biochem MoI Biol, 2001. 76(1-5): p. 71-84; Osborne, C.K., N Engl J Med., 1998. 339(22): p. 1609-18; Ring, A. and M. Dowsett, Endoer Relat Cancer., 2004. 11(4): p. 643-58). In addition, tamoxifen therapy has the undesirable side effect of stimulating proliferation of uterine endometrial cells, putting women at a higher risk for developing uterine adenocarcinoma (Fisher, B., et al., B-14. J Natl Cancer Inst, 1994. 86(7): p. 527- 37). One therapeutic strategy is to combine hormonal therapies that target ERα-driven proliferation with agents that target separate biochemical pathways to determine if the combination would provide enhanced and more long-lived efficacy. Although several randomized trials have combined antiestrogens simultaneously with traditional adjuvant chemotherapy, these trials have produced disappointing results, perhaps because hormonal therapy can antagonize the effectiveness of chemotherapy, leading to the result that these combinations are less effective when combined than when the individual

therapeutic compounds are administered separately (Gelber, R.D., et al., Lancet, 1996. 347(9008): p. 1066-71; Rivkin, S.E., et al., J Clin Oncol, 1994. 12(10): p. 2078-85). Such antagonist action of hormonal therapy is seen in cell culture, and may result both from antiestrogen effects on cell cycling and independent actions (Woods, K.E., J.K. Randolph, and D.A. Gewirtz, Biochem Pharmacol, 1994. 47(8): p. 1449-52; De Soto, J.A., et al., Anticancer Res, 2002. 22(2A): p. 1007-9; Osborne, C.K., L, Kitten, and CI. Arteaga, J. Clin Oncol, 1989. 7(6): p. 710-7).

[0004] A more effective strategy, therefore, may be to combine hormonal therapy with a second therapy whose target is different from standard chemotherapy, so that the combined effects are not less effective than any of the individual compounds administered separately. Studies combining various therapies have shown some promise, though generally there is confusion in the literature (Johnston, S. R., Clin Cancer Res, 2006. 12(3 Pt 2): p. 1061s-1068s). For example, a recent trial of the combination of the aromatase inhibitor letrozole with temsirolimus, an inhibitor of mTOR, the downstream effector of IGF signaling, was terminated for lack of benefit {Termination of phase 3 clinical program with oral temsirolimus in women with metastatic breast cancer. Press release, Wyeth Pharmaceuticals, 2006).

[0005] Histone deacetylase (HDAC) inhibitors are a structurally diverse group of pharmacological agents that inhibit proliferation, induce differentiation and/or apoptosis in a wide range of cancer cells and hold much promise as anti-neoplastic agents (Villar- Garea, A. and M. Esteller, Int J Cancer, 2004. 112(2): p. 171-8; Marks, P., et al., Nat Rev Cancer, 2001. 1(3): p. 194-202). Hyperacetylation is associated with a transcriptionally permissive environment and HDAC inhibitors, although they affect only a small number of target genes, activate genes involved in cell cycle arrest, apoptosis and differentiation (Glaser, K.B., et al., MoI Cancer Ther, 2003. 2(2): p. 151-63; Richon, V.M., et al., Proc Natl Acad Sci USA, 2000. 97(18): p. 10014-9; Munster, P.N., et al., Cancer Res., 2001. 61(23): p. 8492-7). Furthermore, HDAC inhibitors increase the efficiency of several anticancer drugs that target the DNA (Kim, M.S., et al., Cancer Res, 2003. 63(21): p. 7291-300; Castro-Galache, M.D., et al., Int J Cancer, 2003. 104(5): p. 579-86). It has been suggested that valproic acid acts an inhibitor of histone deacetylases (see Groner et al., US 2005/0038113, February 17, 2005).

[0006] The clinical effects of combining HDAC inhibitors with other therapies, such as antiestrogen therapy in ERα-positive breast tumors are uncertain; an increase in

antiestrogen driven ERa activity at the transcriptional level could potentially result in partial agonist activity at the proliferative level (see, for e.g., Munster, P.N., et al., Cancer Res., 2001. 61(23): p. 8492-7; Vigushin, D.M., et al., Clin Cancer Res., 2001. 7(4): p. 971-6; Margueron, R., et al., J Endocrinol, 2003. 179(1): p. 41-53 Webb, P., P. Nguyen, and P.J. Kushner, J Biol Chem, 2003. 278(9): p. 6912-20; Jang, E.R., et al., Oncogene 2004. 23(9): p. 1724-36; Margueron, R., et al, J. MoI. Endocrinol, 2004, 32(2): p. 583- 94). In addition, there are reports suggesting against the combination of HDAC inhibitors with hormonal therapy (e.g., Jansen, M. et al, 2004. Proc Natl Acad Sci U S A 101 :7199 - 20).

[0007] There is a need, therefore, to develop new therapeutic strategies and formulations for the treatment and prevention of cancer, and in particular, for the treatment and prevention of breast cancer.

3. SUMMARY OF THE INVENTION

[0008] The present inventors have investigated whether valproic acid (VPA) and its salts combine effectively with hormonal therapy, including antiestrogens, aromatase inhibitors, and selective estrogen receptor modulators, for treatment of cancer, such as human ERa -positive breast cancer cells.

[0009] The inventors have discovered that certain combinations of VPA and its salts combine effectively with therapeutic agents for convenient formulations, and that the combinations do not reduce the activity of any single component, or that the combinations are more effective or more conveniently administered than either of the components separately. The findings indicate that VPA may combine effectively with the actions of other therapeutic agents such as antiestrogens and aromatase inhibitors, and unlike compounds traditionally used in combination in the treatment and prevention of breast cancer, these combinations either do not reduce the effectiveness compared to that of the individual components, or are more conveniently administered than separate formulations, or both. As an added benefit, VPA may counter the pro-proliferative action of agents such as tamoxifen on uterine cells. Furthermore, VPA may be effective in combining with agents such as tamoxifen in cells rendered tamoxifen-resistant by overexpression of HER2/neu.

[0010] Thus, the present inventors have found that certain combinations of VPA and other therapeutic compounds combine effectively with each other for the treatment and

prevention of cancer, and in particular, for treating or preventing the progression of breast cancer. The present invention provides methods of treating and preventing estrogen receptor positive breast cancer, as well as pharmaceutical compositions comprising the compounds used in the combination therapies.

[0011] The present invention encompasses methods of treatment for (including management of, amelioration of symptoms of, preventing the progression of, and preventing the recurrence of) breast cancer, using certain combination therapies, as well as the pharmaceutical compositions comprising these combination therapies. The invention is based, in part, on the recognition that certain combinations of compounds combine effectively with each other, and may be superior to other combinations of compounds, as well as improving the tolerance of, and/or reducing the side effects caused by at least one of the compounds in the combination. Subjects are mammalian, and preferably are human, and more preferably are human females.

[0012] In one aspect, the present invention provides a pharmaceutically acceptable salt of valproic acid, wherein the salt includes a valproate ion and a counterion derived from an additional therapeutic agent. In one embodiment, the counterion derived from an additional therapeutic agent is a counterion derived from an antiestrogen, an aromatase inhibitor, an estrogen ablation therapy, or a selective estrogen receptor modulator. Estrogen ablation therapy may include an aromatase inhibitor. According to the invention, an aromatase inhibitor can be, but is not limited to, exemestane, letrozole, or anastrozole. In one aspect, the therapeutic agent is selected from the group consisting of tamoxifen, afϊmoxifene (4-hydroxytamoxifen), endoxifen, letrozole, anastrozole, raloxifene, toremifene, 4-hydroxytoremifene, ormeloxifene, arzoxifene, bazedoxifene, clomifene, lasofoxifene, fulvestrant, miproxifene, metabolites thereof, and mixtures thereof.

[0013] In one embodiment according to the invention, the salt of valproic acid is selected from tamoxifen valproic acid salt, 4-hydroxytamoxifen valproic acid salt, endoxifen valproic acid salt, letrozole valproic acid salt, anastrozole valproic acid salt, and raloxifene valproic acid salt. Variously, the therapeutic agent is selected from the group consisting of tamoxifen, one or more metabolites of tamoxifen, and mixtures thereof. In one embodiment, the salt is tamoxifen valproic acid salt.

[0014] In one embodiment, the salt according to the invention is further combined with one or more pharmaceutically acceptable carriers, diluents, excipients, adjuvants, or mixtures thereof. For example, such additional components may include buffers, stabilizers, co-solvents, or the like.

[0015] In one aspect, the invention encompasses compositions comprising a salt of valproic acid as described above, further comprising valproic acid, an additional valproate salt (which is different from the salt of valproic acid as described above), and mixtures thereof. In one aspect, the composition is in the form of a solution of the additional therapeutic agent in valproic acid, yielding a valproate salt of the additional therapeutic agent dissolved in valproic acid. Such solutions may be obtained by dissolving the free base of the additional therapeutic agent in valproic acid, or by adding for example a different salt form of the additional therapeutic agent to an excess of valproic acid such that valproate ion replaces any other counterions. As a solution, valproic acid may be the solvent, or may exist with additional cosolvents, either aqueous or non-aqueous. In one embodiment, the composition is a solution of tamoxifen in valproic acid, i.e., there is an amount of tamoxifen and an excess molar amount of valproic acid.

[0016] In one embodiment, the invention is a composition comprising i) valproic acid, a pharmaceutically acceptable salt thereof, or mixtures thereof; and ii) a pharmaceutically acceptable salt of valproic acid, wherein the salt includes a valproate ion and a counterion derived from an additional therapeutic agent, wherein the additional therapeutic agent is selected from an antiestrogen, an aromatase inhibitor, and a selective estrogen receptor modulator; and iii) one or more pharmaceutically acceptable carriers, diluents, excipients, or mixtures thereof. In combinations of i) and ii), it should be understood that when i) includes a valproic acid salt, the salt of i) is not the same as the salt of ii). As an example, i) may be valproic acid, sodium valproate, or mixtures thereof. Additional therapeutic agents may be selected from tamoxifen, afimoxifene (4- hydroxytamoxifen), endoxifen, letrozole, anastrozole, raloxifene, toremifene, A- hydroxytoremifene, ormeloxifene, arzoxifene, bazedoxifene, clomifene, lasofoxifene, fulvestrant, miproxifene, metabolites thereof, and mixtures thereof. Variously, the component ii) is selected from tamoxifen valproic acid salt, 4-hydroxytamoxifen valproic acid salt, endoxifen valproic acid salt, letrozole valproic acid salt, anastrozole valproic acid salt, and raloxifene valproic acid salt. In one embodiment, the component ii) is tamoxifen valproic acid salt.

[0017] In a composition according to the invention, valproic acid, a pharmaceutically acceptable salt thereof, or mixtures thereof (the VPA component of i) above) may be present in amounts of about 50 to about 1000 mg; and component ii) (the valproic acid salt of an additional therapeutic agent as described above) may be present in amounts of about 1 to about 50 mg. For any valproic acid salt that meets the requirements of component ii), it should be understood that the amount of such a valproic acid salt should be considered as part of ii) rather than contributing to both i) and ii). In one embodiment, the amount of VPA component is about 200 to about 300 mg, while component ii) is present in about 5 to about 20 mg. In one embodiment, the amount of VPA component is about 250 mg, and the amount of ii) component is about 10 mg. Alternatively, the amount of VPA component is about 400 to about 600 mg, while component ii) is present in about 5 to about 20 mg. In one embodiment, the amount of VPA component is about 500 mg, and the amount of component ii) is about 10 mg. In one embodiment, the daily dose of VPA component is from about 15 mg/kg to about 60 mg/kg. In one embodiment, the daily dose of VPA component is sufficient to achieve about 300 to about 867 micromolar in patient serum. In another embodiment, the daily dose of VPA component is sufficient to achieve about 300 to about 1000 micromolar in patient serum, and in another embodiment, the daily dose of VPA component is sufficient to achieve about 500 to about 1000 micromolar in patient serum. In some embodiments, the dose of component ii) is from about 10 mg/day to about 50 mg/day. In another embodiment, the dose of component ii) is about 20 mg/day. In one embodiment in which a patient has metastatic breast cancer, the dose of component ii) is from about 20 mg/day to about 40 mg/day.

[0018] In one embodiment, a composition according to the invention is a non-solid formulation comprising a concentration in 5 mL of composition of about 50 to about 1000 mg of VPA component and about 1 to about 50 mg of component ii), with the understanding that valproate salts meeting the requirements of ii) are included in the amount of component ii) but not necessarily included in the amount of VPA component. In one embodiment, the amount of VPA component per 5 mL of composition is about 200 to about 300 mg or about 400 to about 600 mg, and the amount of component ii) is about 5 to about 20 mg. As an example, the amount of VPA component may be about 250 or about 500 mg and the amount of component ii) is about 10 mg. Where a dosage unit

other than 5 rnL is desired, the concentrations may be adjusted accordingly to reflect the desired dosage unit.

[0019] In one embodiment, the ratio of valproic acid to additional therapeutic agent ranges from 1 to 1 up to 500 to 1 or greater. Alternative embodiments range from 10- 400 to 1, 20-300 to 1, or 30-200 to 1. For example, a ratio of valproic acid to additional therapeutic agent of 45-180 to 1 is envisioned for a 60 kg patient. In one embodiment, the ratio of valproic acid to tamoxifen is 45-180 to 1. In one embodiment, the ratio of valproic acid to tamoxifen ranges from 22-180 to 1 for a metastatic breast cancer patient of 60 kg.

[0020] In one embodiment, the composition of the invention is formulated in a form selected from the group consisting of a solution, paste, ointment, cream, patch, lotion, gel, oral gel, spray, aerosol, emulsion, microemulsion, suspension, powder, liniment, salve, mist, drops, gum, pill, tablet, capsule, microcapsule, granules, microgranules, cachet, gelcap, syrup, injection solution, suppository, enema, depot, encapsulated powder, encapsulated granules, and mixtures thereof. In one embodiment, the composition is formulated as a gelcap. The composition may be formulated for immediate release, slow release, controlled release, sustained release, pulsatile release, or differential release of VPA component and additional therapeutic agent salt.

[0021] The invention encompasses the use of salts and compositions according to the invention in methods of treating cancer in a mammal in need thereof comprising administering a salt or composition according to the invention to such a mammal. In one embodiment, the mammal is a human, and the cancer is a solid tumor. In one aspect, the mammal is a human, and the cancer is breast cancer. In one embodiment, the cancer is tamoxifen-resistant breast cancer. In one aspect, the present invention provides methods of treating and methods of preventing estrogen receptor positive breast cancer, comprising administering to a subject a therapeutically effective amount of a valproic acid or salt thereof in combination with a course of hormonal therapy in the same formulation.

[0022] The present invention also encompasses methods and compositions for preventing breast cancer, particularly in subjects who are at risk for breast cancer that is greater than the average risk for breast cancer. Risk factors considered in preventing breast cancer in subjects include family history of breast cancer (relatives with breast

cancer), genetic markers for breast cancer such as BRCAl and BRCA2, age at menarche, age at first live birth, the number of breast biopsies, presence of atypical hyperplasia on breast biopsy, population rates of breast cancer and death from other causes. The present invention also provides methods and compositions for preventing the progression of breast cancer to a later stage for those who already have breast cancer or precancerous indicators, as well as preventing the recurrence of breast cancer for those in remission from breast cancer.

[0023] In some embodiments, the invention contemplates methods of preventing the progression of DCIS to breast cancer, and methods of preventing the progression of atypical hyperplasia to breast cancer. In some preferred embodiments, the invention encompasses treating or preventing estrogen receptor positive breast cancer.

[0024] In yet another embodiment, the present invention encompasses treating DCIS and, in another embodiment, the present invention encompasses treating atypical hyperplasia.

[0025] The present invention also encompasses methods of treating and methods of preventing breast cancer comprising administering to a subject suffering therefrom a therapeutically effective amount of a composition as indicated above, further comprising a therapeutically effective amount of one or more of an IGF-I receptor inhibitor, an EGFR inhibitor, or an mTOR inhibitor.

[0026] The present invention provides pharmaceutical compositions comprising or consisting essentially of the combinations of compounds described herein, as well as kits comprising the combinations.

[0027] The present invention further contemplates that the combination therapies as described herein can also reduce or eliminate other side effects of treatment, at least in part because lower doses of compounds can be used in treatment or prevention protocols. For example, treatment with certain therapeutic agents such as tamoxifen or raloxifene may be compromised by an increased risk in uterine cancer. In one embodiment, a distinct advantage of the present invention is that administration of a VPA component in combination with a therapeutic agent such as tamoxifen or raloxifene may reduce or eliminate the risk of uterine cancer. In some embodiments, this risk is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or it is virtually or wholly eliminated.

[0028] It is within the scope of the invention to treat breast cancer that is tamoxifen resistant, as well as to treat breast cancer that overexpresses Her2/Neu. The invention also contemplates treating subjects with breast cancer for whom previous therapy has failed, or for whom the cancer is recurring. In some embodiments of the invention, the invention is to treat subjects with breast cancer who are post-menopausal, and in some embodiments, the invention contemplates treating subjects who are genetically predisposed to breast cancer or otherwise at increased risk. The invention also encompasses methods of treating subjects to prevent progression of breast cancer, and in some embodiments, the invention encompasses treating or preventing breast cancer in patients with pre-cancerous growths or benign tumors. It is within the scope of the invention to treat subjects that are in remission from breast cancer, and to treat subjects with breast cancer that have previously undergone treatment.

4. BRIEF DESCRIPTION OF THE FIGURES

[0029] FIG. 1 shows the results of treatment of MCF-7 cells with a formulation of anastrozole dissolved in valproic acid.

5. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present inventors have found that certain combinations of VPA and another therapeutic agent combine effectively with each other in a pharmaceutical composition for the treatment and prevention of cancer such as breast cancer, and for preventing the progression of cancer such as breast cancer, or for convenience of formulation and administration in treating patients. The present invention provides methods of treating and preventing estrogen receptor positive breast cancer, as well as pharmaceutical compositions comprising the compounds used in the combination therapies disclosed herein.

[0031] In some embodiments, the individual compounds in the combination therapies combine effectively with each other, and in other embodiments, the individual compounds in the combination therapies synergize with each other, while in other embodiments, a benefit is found in the stability or ease of formulating or administering the composition to patients.

5.1 Definitions

[0032] As used herein, the term "cancer" refers to a disease involving cells that have the potential to metastasize to distal sites and exhibit phenotypic traits that differ from those of non-cancer cells. Cancer cells acquire a characteristic set of functional capabilities during their development, albeit through various mechanisms. Such capabilities include evading apoptosis, self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion/metastasis, limitless replicative potential, and sustained angiogenesis. The term "cancer cell" is meant to encompass both pre -malignant and malignant cancer cells.

[0033] "Estrogen receptor positive breast cancer" refers to breast cancers that are in the positive or intermediate range for the estrogen receptor protein. For example, when estrogen receptor protein can be measured as femtomoles per milligram of cytosol protein. In this assay, values above 10 are positive, values from 3 to 10 are intermediate, and values less than 3 are negative. Other assays known in the art can be used to determine if the breast cancer is estrogen receptor positive, in particular assays based on antibodies to estrogen receptors alpha and beta and their use in biochemical or histological assays.

[0034] The terms "histone deacetylase inhibitor" and "inhibitor of histone deacetylase" mean a compound which is capable of interacting with a histone deacetylase and inhibiting its enzymatic activity. "Inhibiting histone deacetylase enzymatic activity" means reducing the ability of a histone deacetylase to remove an acetyl group from a histone. (see, e.g., Minucci et al, Nature 6:38-51 (2006). In some preferred embodiments, such reduction of histone deacetylase activity is at least about 50%, more preferably at least about 75%, and still more preferably at least about 90%. In other preferred embodiments, histone deacetylase activity is reduced by at least 95% and more preferably by at least 99%. Assays for determining inhibition are described in Phiel, C. J., et al., J Biol Chem., 2001. 276(39): p. 36734-41 and Gottlicher, M., et al., Embo J., 2001. 20(24): p. 6969-78. Preferably, such inhibition is specific, i.e., the histone deacetylase inhibitor reduces the ability of a histone deacetylase to remove an acetyl group from a histone at a concentration that is lower than the concentration of the inhibitor that is required to produce another, unrelated biological effect. Preferably, the concentration of the inhibitor required for histone deacetylase inhibitory activity is at least 2-fold lower, more preferably at least 5 -fold lower, even more preferably at least 10-fold lower, and

most preferably at least 20-fold lower than the concentration required to produce an unrelated biological effect.

[0035] As used herein, the terms "active ingredient" and "therapeutic agent" include compounds and compositions having a clinically beneficial or prophylactic effect on cancer including breast cancer. This does not include inactive ingredients such as pharmaceutical carriers, excipients, and the like.

[0036] "Mammalian target of rapamycin protein inhibitor" or "mTOR inhibitor" includes drugs such as rapamycin, temsirolimus, and everolimus that selectively inhibit the mammalian target of rapamycin (mTOR). The mTOR inhibitors contemplated in the methods and compositions of the present invention can be any known to one of skill in the art. The mTOR inhibitor may be for example, temsirolimus, everolimus, rapamycin and rapamycin derivatives, including those rapamycin derivatives disclosed in U.S. Patent Application No. 20040147541 to Lane, which is hereby incorporated by reference in its entirety.

[0037] "IGF-I receptor inhibitor" refers to drugs such as picrophodophyllin and podophyllotoxin that selectively inhibit the IGF-I receptor. The IGF-IR inhibitors contemplated in the methods and compositions of the present invention can be any known to one of skill in the art. The IGF-IR inhibitor may be for example, picrophodophyllin, podophyllotoxin, podophyllotoxin derivatives, including those disclosed in U.S. Patent Application No. 20070123491 to Axelson, EGCG, cyclolignans such as those disclosed in U.S. Patent Application No. 2004/0186169 to Larsson, and IGF-IR inhibitors such as those disclosed in U.S. Patent Application No. 20060193772 to Ochiai. Each of the above patent applications is hereby incorporated by reference in its entirety.

[0038] "EGF receptor inhibitor" of "EGFR inhibitor" refers to drugs such as gefitinib and erlotinib that selectively inhibit the EGF receptor. The EGFR inhibitors contemplated in the methods and compositions of the present invention can be any known to one of skill in the art. The EGFR inhibitor may be for example, gefitinib, erlotinib, cetuximab, imatinib, genistein, genistin, quercetin, equol, staurosporine, aeroplysinin, indocarbazole, lavendustin, piceatannol, kaempferol, daidzein, erbstatin, and tyrphostins.

[0039] "Insufficient to fully prevent production of estrogen" refers to the inability of an aromatase inhibitor to fully prevent a tumor cell from converting an estrogen precursor into a functional estrogen that can stimulate tumor proliferation.

[0040] "Less than estrogen receptor-saturating amounts" refers to amounts of fulvestrant less than 100 fold molar excess to the amounts of estradiol or less than 10 nanomolar in patient circulation.

[0041] "Hormonal therapy" refers to drugs or treatments that block the effect of, or reduce the levels of hormones, and in particular which block the effect of estrogen or lower estrogen levels, including anti-estrogen therapy and estrogen ablation therapy.

[0042] As used herein, the terms "prevent," "preventing" and "prevention" refer to the prevention of the recurrence, worsening, or spread of a disease in a subject resulting from the administration of a prophylactic or therapeutic agent. The terms may also refer to reducing the incidence of an initial diagnosis or relapse in a general or specific population, or a reduction in severity of symptoms.

[0043] The terms "overexpress," "overexpression" or "overexpressed" interchangeably refer to a protein or nucleic acid (RNA) that is translated or transcribed at a detectably greater level, usually in a cancer cell, in comparison to a normal cell. The term includes overexpression due to transcription, post transcriptional processing, translation, post-translational processing, cellular localization (e.g., organelle, cytoplasm, nucleus, cell surface), and RNA and protein stability, as compared to a normal cell. Overexpression can be detected using conventional techniques for detecting mRNA (i.e., RT-PCR, PCR, hybridization, microarray) or proteins (i.e., ELISA, immunohistochemical techniques). Overexpression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a normal cell. In certain instances, overexpression is 1-fold, 2-fold, 3 -fold, 4-fold or higher levels of transcription or translation in comparison to a normal cell.

[0044] As used herein, the term "in combination" refers to the use of more than one prophylactic and/or therapeutic agents. The use of the term "in combination" does not restrict the order in which prophylactic and/or therapeutic agents are administered to a subject with cancer, especially breast cancer. A first prophylactic or therapeutic agent can be administered prior to (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, hours, 96

hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second prophylactic or therapeutic agent to a subject which had, has, or is susceptible to cancer, especially breast cancer. The prophylactic or therapeutic agents are administered to a subject in a sequence and within a time interval such that the agent of the invention can act together with the other agent to provide an increased benefit than if they were administered otherwise. Any additional prophylactic or therapeutic agent can be administered in any order with the other additional prophylactic or therapeutic agents. Alternatively, combination may refer to physical or chemical admixture.

[0045] As used herein, the term "combine effectively" refers to a combination of therapies (e.g., a combination of prophylactic or therapeutic agents) which is more effective than any single agent administered alone. Combining effectively may also refer to combinations of therapies that are not less effective than any single agent or even less effective than any single agent, but which also eliminate or reduce the adverse effects of one or more of the agents, such as eliminating or reducing the risk of uterine cancer associated with one or more of the agents.

[0046] As used herein, the term "synergistic" refers to a combination of therapies (e.g., a combination of prophylactic or therapeutic agents) which is more effective than the additive effects of any two or more single agents. A synergistic effect of a combination of therapies permits the use of lower dosages of one or more of the therapies and/or less frequent administration of said therapies (e.g., agents) to a subject with a disease or disorder, in particular, cancer, or a condition or symptom associated therewith. The ability to utilize lower dosages of therapies and/or to administer said therapies less frequently reduces the toxicity associated with the administration of said therapies to a subject without reducing the efficacy of said therapies in the prevention, management, or treatment of a disease or disorder, in particular, cancer or a condition or symptom associated therewith. In addition, a synergistic effect can result in improved efficacy of therapies in the prevention, management, or treatment of a disease or disorder, in particular, cancer or a condition or symptom associated therewith. Finally, the synergistic effect of a combination of therapies may avoid or reduce adverse or unwanted side effects associated with the use of any single therapy.

[0047] As used herein, the phrase "side effects" encompasses unwanted and adverse effects of a prophylactic or therapeutic agent. Adverse effects are always unwanted, but

unwanted effects are not necessarily adverse. An adverse effect from a prophylactic or therapeutic agent might be harmful or uncomfortable or risky. Side effects can refer specifically to an increase in uterine cell proliferation, as well as to an increase in the frequency of uterine cancer and an increase in the risk of developing uterine cancer. Side effects from chemotherapy include, but are not limited to, gastrointestinal toxicity such as, but not limited to, early and late-forming diarrhea and flatulence, nausea, vomiting, anorexia, leukopenia, anemia, neutropenia, asthenia, abdominal cramping, fever, pain, loss of body weight, dehydration, alopecia, dyspnea, insomnia, dizziness, mucositis, xerostomia, and kidney failure, as well as constipation, nerve and muscle effects, temporary or permanent damage to kidneys and bladder, flu-like symptoms, fluid retention, and temporary or permanent infertility. Side effects from radiation therapy include but are not limited to fatigue, dry mouth, and loss of appetite. Side effects from biological therapies/immunotherapies include but are not limited to rashes or swellings at the site of administration, flu-like symptoms such as fever, chills and fatigue, digestive tract problems and allergic reactions. Side effects from hormonal therapies include but are not limited to nausea, fertility problems, depression, loss of appetite, eye problems, headache, and weight fluctuation. Additional undesired effects typically experienced by patients are numerous and known in the art. Many are described in the Physicians' Desk Reference (56 ^th ed., 2002).

[0048] "Without attendant risk in increase of uterine cancer" refers to a lowered or eliminated risk of developing uterine cancer as compared to patients who have an increased risk for developing uterine cancer due to a course of anti-estrogen therapy.

[0049] By "therapeutically effective amount or dose" or "therapeutically sufficient amount or dose" or "effective or sufficient amount or dose" herein is meant a dose that produces therapeutic effects for which it is administered, in the context of the combination therapy in which it is administered. Often, the therapeutically effective or sufficient amount or dose of the compounds comprising the pharmaceutical compositions of the invention will be lower when administered in the specific combinations, than the doses that would be therapeutically effective or sufficient when the compounds are administered separately. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations

(1999); and Remington. The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). In some embodiments, a therapeutically effective amount refers to that amount of the therapeutic agent sufficient to destroy, modify, control or remove primary, regional or metastatic cancer tissue. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the spread of cancer. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of cancer. Further, a therapeutically effective amount with respect to a therapeutic agent of the invention means that amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of cancer. In sensitized cells, the therapeutically effective dose can often be lower than the conventional therapeutically effective dose for non-sensitized cells. In some embodiments, a therapeutically effective amount refers to the amount of a therapeutic agent that, e.g., reduces the proliferation of cancer cells, increases the death of cancer cells or, reduces the size of a tumor or spread of a tumor in a subject. Preferably, a therapeutically effective amount of a therapeutic agent reduces the size of a tumor or the spread of a tumor in a subject by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% relative to a control such as PBS. In some embodiments, a therapeutically effective amount refers to the amount of a therapeutic agent that increases survival by 1 month, 2 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more. In some embodiments, a therapeutically effective amount refers to the amount of a therapeutic agent that prevents the progression from DCIS or atypical hyperplasia to breast cancer.

[0050] Certain terms such as "salt" and "solvent" are used as is conventional in the chemical arts and in the practice of medicinal chemistry. In one sense, salts are ionic compounds composed of cations (positively charged ions) and anions (negative ions) so that the product is electrically neutral (without a net charge). In one aspect, a salt may be understood as the product formed from the neutralization reaction of acids and bases, or the product of a base and an acid anhydride, or the product of an acid and a base anhydride, or the like. A salt may also be the product of replacing one anion with another or one cation with another. Salts can also form if solutions of different salts are mixed,

their ions recombine, and the new salt is insoluble and precipitates. However, the manner of formation is not determinative. A solvent may be understood as a liquid or gas that dissolves a solid, liquid, or gaseous solute, resulting in a solution.

[0051] By "bioequivalent" or "bioequivalence" is meant a term in pharmacokinetics used to assess the expected in vivo biological properties of an active pharmaceutical ingredient. The United States Food and Drug Administration (FDA) has defined bioequivalence as, "the absence of a significant difference in the rate and extent to which the active ingredient or active moiety in pharmaceutical equivalents or pharmaceutical alternatives becomes available at the site of drug action when administered at the same molar dose under similar conditions in an appropriately designed study" (FDA, "Bioavailability and Bioequivalence Studies for Orally Administered Drug Products - General Considerations," Rockville, MD, 2003). The FDA considers two products bioequivalent if the 90% CI of the relative mean Cmax, AUC(O-t) and AUC(O-∞) of the test to reference should be within 80.00% to 125.00% in the fasting state. In one embodiment, two compounds are said to be bioequivalent if they would be expected to have similar or nearly identical pharmacokinetic profiles. In one embodiment, a bioequivalent comparison of test to reference formulations requires administration after an appropriate meal at a specified time before taking the drug, a so-called "fed" or "food- effect" study.

5.2 Dosages

[0052] In therapeutic use for the treatment of cancer, the salts according to the invention either alone in combination with valproic acid as utilized in the pharmaceutical method of the invention are administered at the initial dosage of about 0.001 mg/kg to about 1000 mg/kg daily. A daily dose range of about 0.01 mg/kg to about 500 mg/kg, or about 0.1 mg/kg to about 200 mg/kg, or about 1 mg/kg to about 100 mg/kg, or about 10 mg/kg to about 50 mg/kg, can be used. In a composition according to the invention, valproic acid, a pharmaceutically acceptable salt thereof, or mixtures thereof (the VPA component of i) above) may be present in amounts of about 50 to about 1000 mg; and component ii) (the valproic acid salt of an additional therapeutic agent as described above) may be present in amounts of about 1 to about 50 mg. For any valproic acid salt that meets the requirements of component ii), it should be understood that the amount of such a valproic acid salt should be considered as part of ii) rather than contributing to both i) and ii). In one embodiment, the amount of VPA component is about 200 to about

300 mg, while component ii) is present in about 5 to about 20 mg. In one embodiment, the amount of VPA component is about 250 mg, and the amount of ii) component is about 10 mg. Alternatively, the amount of VPA component is about 400 to about 600 mg, while component ii) is present in about 5 to about 20 mg. In one embodiment, the amount of VPA component is about 500 mg, and the amount of component ii) is about 10 mg. In one embodiment, the daily dose of VPA component is from about 15 mg/kg to about 60 mg/kg. In one embodiment, the daily dose of VPA component is sufficient to achieve about 300 to about 867 micromolar in patient serum. In another embodiment, the daily dose of VPA component is sufficient to achieve about 300 to about 1000 micromolar in patient serum, and in another embodiment, the daily dose of VPA component is sufficient to achieve about 500 to about 1000 micromolar in patient serum. In some embodiments, the dose of component ii) is from about 10 mg/day to about 50 mg/day. In another embodiment, the dose of component ii) is about 20 mg/day. In one embodiment in which a patient has metastatic breast cancer, the dose of component ii) is from about 20 mg/day to about 40 mg/day.

[0053] In one embodiment, a composition according to the invention is a non-solid formulation comprising a concentration in 5 mL of composition of about 50 to about 1000 mg of VPA component and about 1 to about 50 mg of component ii), with the understanding that valproate salts meeting the requirements of ii) are included in the amount of component ii) but not necessarily included in the amount of VPA component. In one embodiment, the amount of VPA component per 5 mL of composition is about 200 to about 300 mg or about 400 to about 600 mg, and the amount of component ii) is about 5 to about 20 mg. As an example, the amount of VPA component may be about 250 or about 500 mg and the amount of component ii) is about 10 mg. Where a dosage unit other than 5 mL is desired, the concentrations may be adjusted accordingly to reflect the desired dosage unit.

[0054] However, the dosages of valproic acid component and/or salt of additional therapeutic agent may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. For example, dosages can be empirically determined considering the type and stage of cancer diagnosed in a particular patient. The doses administered to a patient, in the context of the present invention should be sufficient for a beneficial therapeutic response in the patient over time. The size of the doses also will be determined by the existence, nature, and

extent of any adverse side-effects that accompany the administration of a particular compound or combination in a particular patient. Determination of the proper dosages for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosages are increased by small increments until the optimum effect under circumstances is reached. For convenience, the total daily dosages may be divided and administered in portions during the day, if desired. Doses of compounds or combinations can be given daily, or on alternate days, as determined by the treating physician.

[0055] In one aspect, the present invention provides a method of treating and methods of preventing estrogen receptor positive breast cancer, comprising administering to a subject a therapeutically effective amount of a composition comprising a VPA component in combination with a hormonal therapy component.

[0056] In one embodiment, the daily dose of valproic acid is from about 15 mg/kg to about 60 mg/kg. In one embodiment, the dose of valproic acid is sufficient to achieve from about 300 to about 1000 micromolar in patient serum. In another embodiment, the dose of valproic acid is sufficient to achieve from about 300 to about 867 micromolar in patient serum. In another embodiment, the dose of valproic acid is sufficient to achieve from about 500 to about 1000 micromolar in patient serum.

[0057] Hormonal agents are a group of drugs that regulate the growth and development of their target organs. Most of the hormonal agents used in the treatment of breast cancer are sex steroids and their derivatives and analogs thereof, such as estrogens, androgens, and progestins. These hormonal agents may serve as antagonists of receptors for the sex steroids to down regulate receptor expression and transcription of genes. Such hormonal therapy agent include, but are not limited to synthetic estrogens (e.g. diethylstilbestrol), antiestrogens (e.g. tamoxifen, fulvestrant, fluoxymesterol, raloxifene, and toremifene), antiandrogens (bicalutamide, nilutamide, flutamide), aromatase inhibitors (e.g., aminoglutethimide, anastrozole, letrozole, and tetrazole), ketoconazole, goserelin acetate, leuprolide, megestrol acetate and mifepristone.

[0058] Any form of hormonal therapy known to one of skill in the art for the treatment of breast cancer is contemplated as useful in the combination therapies of the present invention. In some preferred embodiments, the hormonal therapy is anti-estrogen therapy, which can be, but is not limited to tamoxifen, raloxifene, fulvestrant, and

toremifene. In some embodiments, the dose of tamoxifen salt according to the invention is from about 10 mg/day to about 50 mg/day. In another embodiment, the dose of tamoxifen salt is about 20 mg/day. In one embodiment in which a patient has metastatic breast cancer, the dose of tamoxifen salt is from about 20 mg/day to about 40 mg/day.

[0059] In some embodiments, the valproic acid component and additional therapeutic agent salt component are administered in combination, with ratios of those compounds which preserve the recommended daily doses of the compounds. In some embodiments, compounds are administered in combination, with ratios of those compounds which preserve the ranges of doses as described herein. In one embodiment, the ratio of valproic acid to additional therapeutic agent ranges from 1 to 1 up to 500 to 1 or greater. Alternative embodiments range from 10-400 to 1, 20-300 to 1, or 30-200 to 1. For example, a ratio of valproic acid to additional therapeutic agent of 45-180 to 1 is envisioned for a 60 kg patient. In one embodiment, the ratio of valproic acid to tamoxifen is 45-180 to 1. In one embodiment, the ratio of valproic acid to tamoxifen ranges from 22-180 to 1 for a metastatic breast cancer patient of 60 kg. Such ratios may be by weight/weight or on a molar basis. Alternatively, ratios may be according to bioequivalence (i.e., an amount of tamoxifen valproic acid salt which is bioequivalent to tamoxifen hydrochloride, or the like).

[0060] In other preferred embodiments, the hormonal therapy can be estrogen ablation therapy, including an aromatase inhibitor. According to the invention, the aromatase inhibitor can be, but is not limited to exemestane, letrozole, fadrozole, retrozole, raloxifene, and anastrozole. In one embodiment, the dose of letrozole salt is from about 1 mg/day to about 5 mg/day. In another embodiment, the dose of letrozole salt is about 2.5 mg/day. In one embodiment, the dose of exemestane salt is from about 10 mg/day to about 40 mg/day. In another embodiment, the dose of exemestane salt is about 25 mg/day. In yet another embodiment, the dose of anastrozole salt is from about 0.5 mg/day to about 3 mg/day. In another embodiment, the dose of anastrozole salt is about 1 mg/day. In yet another embodiment, the dose of raloxifene salt is from about 0.5 mg/day to about 3 mg/day.

[0061] Any therapy (e.g., chemotherapies, radiation therapies, hormonal therapies, and/or biological therapies/immunotherapies) which is known to be useful, or which has been used or is currently being used for the prevention, treatment, management or amelioration of cancer or one or more symptoms thereof can be used in accordance with

the invention, and may be combined with any of the compositions described herein, and may encompass the other active ingredient described for some of the combination therapies herein.

[0062] In some embodiments, additional anti-cancer agents contemplated in the methods and compositions of the present invention, which can be administered in combination with the compositions of the present invention include, but are not limited to doxorubicin, epirubicin, the combination of doxorubicin and cyclophosphamide (AC), the combination of cyclophosphamide, doxorubicin and 5-fluorouracil (CAF), the combination of cyclophosphamide, epirubicin and 5-fluorouracil (CEF), herceptin, tamoxifen, the combination of tamoxifen and cytotoxic chemotherapy, taxanes (such as docetaxel and paclitaxel). In a further embodiment, the combinations of the invention can be administered with taxanes plus standard doxorubicin and cyclophosphamide for adjuvant treatment of node-positive, localized breast cancer. In one embodiment, the dose of doxorubicin hydrochloride (i.v.) is 60-75 mg/m ² on day 1 of treatment. In another embodiment, the dose of epirubicin (i.v.) is 100-120 mg/m ² on day 1 of each cycle or divided equally and given on days 1-8 of the treatment cycle. In yet another embodiment, the dose of docetaxel (i.v.) is 60-100 mg/m ² over 1 hour. In another embodiment, the dose of paclitaxel (i.v.) is 175 mg/m ² over 3 hours.

5.3 Pharmaceutical Formulations

[0063] In one aspect, the present invention provides a pharmaceutically acceptable salt of valproic acid, wherein the salt includes a valproate ion and a counterion derived from an additional therapeutic agent. In one embodiment, the counterion derived from an additional therapeutic agent is a counterion derived from an antiestrogen, an aromatase inhibitor, an estrogen ablation therapy, or a selective estrogen receptor modulator. Estrogen ablation therapy may include an aromatase inhibitor. According to the invention, an aromatase inhibitor can be, but is not limited to, exemestane, letrozole, or anastrozole. In one aspect, the therapeutic agent is selected from the group consisting of tamoxifen, afϊmoxifene (4-hydroxytamoxifen), endoxifen, letrozole, anastrozole, raloxifene, toremifene, 4-hydroxytoremifene, ormeloxifene, arzoxifene, bazedoxifene, clomifene, lasofoxifene, fulvestrant, miproxifene, metabolites thereof, and mixtures thereof. In one embodiment according to the invention, the salt of valproic acid is selected from tamoxifen valproic acid salt, 4-hydroxytamoxifen valproic acid salt, endoxifen valproic acid salt, letrozole valproic acid salt, anastrozole valproic acid salt,

and raloxifene valproic acid salt. Variously, the therapeutic agent is selected from the group consisting of tamoxifen, one or more metabolites of tamoxifen, and mixtures thereof. In one embodiment, the salt is tamoxifen valproic acid salt.

[0064] In one embodiment, the invention is a composition comprising i) valproic acid, a pharmaceutically acceptable salt thereof, or mixtures thereof; and ii) a pharmaceutically acceptable salt of valproic acid, wherein the salt includes a valproate ion and a counterion derived from an additional therapeutic agent, wherein the additional therapeutic agent is selected from an antiestrogen, an aromatase inhibitor, and a selective estrogen receptor modulator; and iii) one or more pharmaceutically acceptable carriers, diluents, excipients, or mixtures thereof. For example, the composition of the invention may comprise i) valproic acid and sodium valproate; ii) tamoxifen valproic acid salt; and iii) carriers and the like to form a gelcap.

[0065] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington 's Pharmaceutical Sciences, 20 ^l ed., 2003).

[0066] The formulations for the salts and combinations encompassed by the methods and compositions of the present invention include valproic acid and pharmaceutically acceptable salts of valproic acid. Pharmaceutically acceptable non-toxic salts include the base addition salts (formed with free carboxyl groups) which may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino- ethanol, histidine, procaine, and the like.

[0067] Certain components of the invention may be formulated into pharmaceutical compositions as neutral or ionic forms. Pharmaceutically acceptable non-toxic salts include the base addition salts (formed with free carboxyl or other anionic groups) which may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine, procaine, and the like. Certain components may also be formed as acid addition salts with any free cationic groups and will generally be formed with inorganic acids such as, for example, hydrochloric, sulfuric,

or phosphoric acids, or organic acids such as acetic, p-toluenesulfonic, methanesulfonic acid, oxalic, tartaric, mandelic, and the like. Compositions of the invention may comprise amine salts formed by the protonation of an amino group with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like. Compositions of the invention may also include amine salts formed by the protonation of an amino group with suitable organic acids, such as p-toluenesulfonic acid, acetic acid, and the like. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. The use of buffers and pH adjusters is envisioned to adjust the pH for reasons of stability, biocompatibility, ease of handling, or the like.

[0068] Additional excipients which are contemplated for use in the practice of the present invention are those available to those of ordinary skill in the art, for example, those found in the United States Pharmacopeia Vol. XXII and National Formulary Vol. XVII, U.S. Pharmacopeia Convention, Inc., Rockville, Md. (1989), the relevant contents of which is incorporated herein by reference.

[0069] In a preferred embodiment, a composition of the invention is a pharmaceutical composition. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of

solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a prophylactically or therapeutically effective amount of a prophylactic or therapeutic agent preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. In a preferred embodiment, the pharmaceutical compositions are sterile and in suitable form for administration to a subject, preferably an animal subject, more preferably a mammalian subject, and most preferably a human subject.

[0070] In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering one or more prophylactic or therapeutic agents, care must be taken to use materials to which the prophylactic or therapeutic agents do not absorb.

[0071] In another embodiment, the composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, pp. 317-327; see generally above.).

[0072] In yet another embodiment, the composition can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321 :574). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the antibodies of the invention or fragments thereof (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, FIa. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y. (1984); Ranger and Peppas, 1983, J.,

Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 7 1 :105); U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; International Publication No. WO 99/15154; and International Publication No. WO 99/20253. Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In a preferred embodiment, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In yet another embodiment, a controlled or sustained release system can be placed in proximity of the therapeutic target, i.e., the lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

[0073] Controlled release systems are discussed in the review by Langer (1990, Science 249:1527-1533). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more antibodies of the invention or fragments thereof. See, e.g., U.S. Pat. No. 4,526,938, International publication No. WO 91/05548, International publication No. WO 96/20698, Ning et al., 1996, "Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel," Radiotherapy & Oncology 39:179-189, Song et al., 1995, "Antibody Mediated Lung Targeting of Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science & Technology 50:372-397, Cleek et al., 1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application," Pro. Int'l. Symp. Control. ReI. Bioact. Mater. 24:853-854, and Lam et al., 1997, "Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery," Proc. Int'l. Symp. Control ReI. Bioact. Mater. 24:759-760, each of which is incorporated herein by reference in their entirety.

[0074] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of suitable routes of administration include, but are not limited to, parenteral (e.g., intravenous, intramuscular, intradermal,

intra-tumoral, intra-synovial, and subcutaneous), oral (e.g., ingestion, inhalation), intranasal, transdermal (topical), transmucosal, intra-tumoral, intra-synovial, vaginal, and rectal administration. In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intra-tumoral, intra synovial, intranasal or topical administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. In a preferred embodiment, the composition of the invention is formulated for oral administration. Alternatively, the composition of the invention may be formulated for parenteral administration.

[0075] If the compositions of the invention are to be administered topically, the compositions can be formulated in the form of, e.g., a toothpaste, ointment, cream, transdermal patch, lotion, gel, oral gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 4 ^th ed., Lea & Febiger, Philadelphia, Pa. (1985). For non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity preferably greater than water are typically employed. Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, such as, for example, osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as FREON), or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well-known in the art.

[0076] If the compositions of the invention are to be administered intranasally, the compositions can be formulated in an aerosol form, spray, mist or in the form of drops. In particular, prophylactic or therapeutic agents for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from

pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0077] If the compositions of the invention are to be administered orally, the compositions can be formulated orally in the form of, e.g., gum, tablets, capsules, cachets, gelcaps, solutions, suspensions and the like. Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well-known in the art. Encapsulation and microencapsulation are envisioned. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). Soft gelatin capsules may be prepared in which capsules contain a mixture of the active ingredients and vegetable oil or non-aqueous, water miscible materials such as, for example, polyethylene glycol and the like. Hard gelatin capsules may contain granules of the active ingredients in combination with a solid, pulverulent carrier, such as, for example, lactose, saccharose, sorbitol, mannitol, potato starch, corn starch, amylopectin, cellulose derivatives, or gelatin. The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release or sustained release of a prophylactic or therapeutic agent(s).

[0078] The compositions of the invention may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Encapsulation and microencapsulation is envisioned.

[0079] The compositions of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0080] In addition to the formulations described previously, the compositions of the invention may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compositions may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0081] Generally, the ingredients of compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0082] In addition to being formulated for compatibility for the method of administration, compositions according to the invention may be formulated for chemical stability. By "chemical stability" is meant that the formulation shows less than 10% degradation of active pharmaceutical ingredients as measured by HPLC following formulation and packaging in a sealed container and storage for one month at about 25

degrees Celsius. Alternatively, chemical stability may be measured at about 40 degrees Celsius. Alternatively, stability may be measured by a means other than HPLC according to FDA requirements, or the like. In various embodiments, a formulated and packaged composition shows less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.1% degradation of active pharmaceutical ingredients under the tested conditions. In one embodiment, a pharmaceutical composition comprising tamoxifen valproic acid salt with valproic acid (or valproic acid and sodium valproate) is formulated and packaged in a sealed container and exhibits chemical stability as measured by HPLC in storage at 40 degrees Celsius for one month.

[0083] Alternatively, chemical compatibility may be evaluated. For example, the chemical compatibility of the additional therapeutic agent (such as tamoxifen) may be evaluated when combined with valproic acid, valproic acid salts, or combinations thereof. Stability with respect to pH may be evaluated for acid lability, or stability with respect to oxidation may be evaluated for oxidative lability. Chemical compatibility may be evaluated prior to final formulation as a pharmaceutical in order to evaluate the need for particular formulating agents to increase stability upon storage of an intermediate or final formulation.

[0084] Where chemical compatibility between valproic acid or its salts and the additional therapeutic agent is less than desired, compositions according to the invention may be formulated to provide a chemical or physical barrier between the valproic acid and the salt of the additional therapeutic agent. For example, one component may be formed into a core (i.e., small beads, pellets, tablets, capsules, or the like) by conventional pharmaceutical procedures for further processing. The cores containing an active ingredient may be separated from one or more additional layers containing the non- compatible component. Such additional layers may be an enteric coating polymer(s) containing free carboxyl groups, which otherwise is capable of causing degradation/discoloration of the active component during the coating process or during storage. A subcoating layer (i.e., a separating/barrier layer), may also be present, which may also serve as a pH-buffering zone to contain sufficient buffer capacity. The pH- or oxidative- buffering properties of the separating layer can be further strengthened by introducing in the layer substances chosen from a group of compounds usually used in such formulations. The separating layer usually consists of one or more water soluble inert layers, optionally containing pH-buffering substances. The material for a separating

layer may be chosen among the pharmaceutically acceptable water soluble, inert compounds or polymers used for film-coating applications such as, for example, sugar, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, hydroxypropyl cellulose, hydroxymethyl cellulose, hydroxypropyl methylcellulose, or the like. The thickness of a separating layer may be determined according to the skilled artisan. Such formulations may also be envisioned for differential release of the valproic acid component and the valproic acid salt of the additional therapeutic agent. See, e.g., Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 4 ^th ed., Lea & Febiger, Philadelphia, Pa. (1985) and subsequent editions, or, Remington: The Science and Practice of Pharmacy, 21 ^st edition.

5.4 Kits and Articles of Manufacture

[0085] The present invention provides pharmaceutical packs or kits that can be used in the above methods. The invention provides a pharmaceutical pack or kit comprising one or more containers filled with individual components (in pharmaceutical formulations) of the combination therapies described herein. The pharmaceutical pack or kit may further comprise one or more other prophylactic or therapeutic agents useful for the treatment of a disease or disorder. The kit may further comprise one or more other prophylactic or therapeutic agents, or active ingredients useful for the treatment of cancer in one or more containers. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0086] The present invention also encompasses a finished packaged and labeled pharmaceutical product. This article of manufacture includes the appropriate unit dosage form in an appropriate vessel or container such as a glass vial or other container that is hermetically sealed. In the case of dosage forms suitable for parenteral administration the active ingredient is sterile and suitable for administration as a particulate free solution. In other words, the invention encompasses both parenteral solutions and lyophilized powders, each being sterile, and the latter being suitable for reconstitution prior to injection. Alternatively, the unit dosage form may be a solid suitable for oral, transdermal, intratumoral, intra-synovial, topical or mucosal delivery.

[0087] In a specific embodiment, the unit dosage form is suitable for oral, intravenous, intramuscular, intratumoral, intra-synovial, or subcutaneous delivery. Thus, the invention encompasses solutions, preferably sterile, suitable for each delivery route.

[0088] As with any pharmaceutical product, the packaging material and container are designed to protect the stability of the product during storage and shipment. Further, the products of the invention include instructions for use or other informational material that advise the physician, technician or patient on how to appropriately prevent or treat the disease or disorder in question. In other words, the article of manufacture includes instruction means indicating or suggesting a dosing regimen including, but not limited to, actual doses, monitoring procedures (such as methods for monitoring, tumor cell counts, marker concentration, and tumor size) and other monitoring information.

[0089] More specifically, the invention provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, sprayer, insufflator, intravenous (i.v.) bag, envelope and the like; and at least one unit dosage form of a pharmaceutical agent contained within said packaging material.

5.5 Treatment of Cancer / Therapeutic or Prophylactic Utility

[0090] It is within the scope of the present invention to treat many different types of subjects or patients, though preferably, the subject is a mammal. Preferred mammals include primates such as humans and chimpanzees, domestic animals such, as horses, cows, pigs, etc. and pets such as dogs and cats. Most preferably, the invention encompasses treating humans, and in particular, human females. The pharmaceutical compositions described herein may be used for the treatment of cancer, particularly for breast cancer. The pharmaceutical compositions and methods of the present invention can be used to treat an individual with any type and/or stage of breast cancer. There are several types of breast cancer and there are several stages of breast cancer, all of which are contemplated as treated by the methods and compositions of the present invention.

[0091] The present invention can be used to treat a patient with any type of breast cancer. Breast cancers may include carcinoma in situ, infiltrating (or invasive) ductal carcinoma, infiltrating (or invasive) lobular carcinoma, medullary carcinoma, colloid carcinoma, tubular carcinoma, and inflammatory carcinoma.

[0092] In addition to the different types of breast cancer, there are also different stages of breast cancer, referred to as stages 0-IV. The system most often used to describe

the growth and spread of breast cancer is the TNM staging system, also known as the American Joint Committee on Cancer (AJCC) system. In TNM staging, information about the tumor, nearby lymph nodes, and distant organ metastases is combined and a stage is assigned to specific TNM groupings. The grouped stages are described using Roman numerals from I to IV. The clinical stage is determined by results from physical examination and tests. The pathologic stage includes the findings of the pathologist after surgery. Most of the time, pathologic stage is the most important stage because usually the cancer isn't known to have spread to lymph nodes until the pathologist examines them under the microscope. In the TNM staging system, T stands for the size of the cancer (measured in centimeters; 2.54 centimeters 1 inch); N stands for spread to lymph nodes in the area of the breast, and M is for metastasis (spread to distant organs of the body).

[0093] The T category describes the original (primary) tumor. Tis: Tis is used only for carcinoma in situ or noninvasive breast cancer such as ductal carcinoma in situ, (DCIS) or lobular carcinoma in situ (LCIS). Tl : The cancer is 2 cm in diameter (about 3/4 inch) or smaller. T2: The cancer is more than 2 cm but not more than 5 cm in diameter. T3: The cancer is more than 5 cm in diameter. T4: The cancer is any size and has spread to the chest wall, the skin, or lymphatics.

[0094] The N category is based on which of the lymph nodes near the breast, if any, are affected by the cancer. NO: The cancer has not spread to lymph nodes. Nl : The cancer has spread to lymph nodes under the arm on the same side as the breast cancer. Lymph nodes have not yet attached to one another or to the surrounding tissue. N2: The cancer has spread to lymph nodes under the arm on the same side as the breast cancer and are attached to one another or to the surrounding tissue or enlarged. Or, the cancer can be seen to have spread to the internal mammary lymph nodes (next to the sternum), but not to the lymph nodes under the arm. N3 : The cancer has spread to lymph nodes above or just below the collarbone on the same side as the cancer, and may or may not have spread to lymph nodes under the arm. Or, the cancer has spread to internal mammary lymph nodes and lymph nodes under the arm, both on the same side as the cancer.

[0095] M categories: The M category depends on whether the cancer has spread to any distant tissues and organs. MO: No distant cancer spread. Ml : Cancer has spread to distant organs.

[0096] There are different types of staging. Clinical staging estimates how much cancer there is based on the results of the physical exam, imaging tests (x-rays, CT scans, etc.) and sometimes biopsies of affected areas. For certain cancers the results of other tests, such as blood tests, are also used in staging. Pathologic staging can only be done on patients who have had surgery to remove or explore the extent of the cancer. It combines the results of clinical staging (physical exam, imaging tests, etc.) with the results from the surgery. In some cases, the pathologic stage may be different from the clinical stage (for example, if the surgery shows the cancer is more extensive than it was previously thought to be). Restaging is sometimes used to determine the extent of the disease if a cancer recurs (comes back) after treatment.

[0097] In one embodiment, the methods and compositions of the present invention are used to treat patients with stage I breast cancer. In one embodiment, the methods and compositions of the present invention are used to treat patients with stage II breast cancer. In one embodiment, the methods and compositions of the present invention are used to treat patients with stage III breast cancer. In one embodiment, the methods and compositions of the present invention are used to treat patients with stage IV breast cancer, i.e. patients with metastatic cancer.

[0098] In another embodiment, the patient having breast cancer has already failed other treatment regimens such as chemotherapy.

[0099] In one embodiment, the methods and pharmaceutical compositions of the present invention may be used to prevent the development of a cancer, particularly in an individual at higher risk than average to develop such cancer than other individuals, or to treat a patient afflicted with breast cancer.

[00100] There are a number of ways to assess an individual's risk for breast cancer, and any means of risk assessment is contemplated by the present invention as determining which subjects are at risk for breast cancer and can undergo treatment via the methods and compositions of the present invention. The invention contemplates treatment for individuals with a higher than average lifetime risk for breast cancer, the average being about one in eight women in the U.S.

[00101] The invention provides methods treating asymptomatic patients who have a likelihood of benefiting from therapeutic treatment of breast cancer. The asymptomatic patients can comprise patients in any of the many high risk groups for breast cancer. The

high risk groups can include e.g. patients with a family history of breast cancer, patients of increasing age (e.g., patients 40 years of age or older), menopausal patients, patients having at least one high risk parity factor (e.g. early start of menses, late onset of menopause, no pregnancies, or late-age pregnancy), patients having high risk gene status (e.g. patients testing positive for a mutation in BRCAl or BRCA2 genes, or others, as described below), patients having at least one previous breast biopsy (benign or otherwise), patients having a previous diagnosis of breast cancer, and patients having any other risk factor for breast cancer. Other risk factors are continually being defined and can include such considerations, as geographic location (e.g. where women living in a particular region have been found to have a higher incidence of breast cancer). Diet is also thought to play a role in breast cancer risk; specifically women who include more fat in their diet may be more likely to develop breast cancer (see Kniget et al. Cancer Epidemiol Biomarkers Prev 8(2): 123-8, 1999).

[00102] The Gail model is a common means of determining risk for breast cancer, and was developed based on the Breast Cancer Detection Demonstration Project (see Gail, M. et al, J Natl Cancer Inst., 1989. 81 : p. 1879-86). The risk factors used in the Gail model are age, age at menarche, age at first live birth, number of previous breast biopsies, number of first-degree relatives with breast cancer. These risk factors are broadly consistent with those selected from other large population-based studies. A revised Gail model also incorporates race, presence of atypical hyperplasia on breast biopsy, and 1987 population rates of breast cancer and death from other causes.

[00103] Another commonly used prediction model is the Claus model, based on the Cancer and Steroid Hormone Study (see Claus E. et al., Cancer, 1994. 73: 643-51) and incorporates more extensive information about family history. The Claus model provides individual estimates of breast-cancer risk according to decade from 29-79 years of age. It takes into account factors such as the number of first-degree and number of second- degree relatives with breast cancer, as well as different combinations of different degree relatives with breast cancer.

[00104] The invention also contemplates treatment for early stages of cancer, for recurrent cancer, and for those in remission from cancer.

[00105] The present invention also encompasses treatment for subjects with markers for breast cancer, including, but not limited to having mutations or other alterations in the

genes, BRCAl, BRCA2, P53, P65, ATM, or pS2, or a changed ratio of the expression of the genes HOXB 13 and IL17BR, amplification of the AIBl/pCIP coactivator gene, overproduction of HER2 protein and/or gene, and alterations in levels of hormones, such as estrogen and progesterone, or their receptors.

[00106] Markers can also include neoplastic ductal epithelial cells, transforming growth factor-β, carcinoma embryonic antigen (CEA), prostate specific antigen (PSA), Erb B2 antigen, gross cystic disease fluid protein- 15 (GCDFP- 15), lactose dehydrogenase (LDH), measured in the ductal fluid, or a chromosomal abnormality in the ductal epithelial cells. Where the marker is neoplastic ductal epithelial cells, the cells can be at a stage including hyperplasia, atypical hyperplasia, low grade ductal carcinoma in situ (LG- DCIS), high grade ductal carcinoma in situ (HG-DCIS) or invasive carcinoma. The present invention encompasses providing the pharmaceutical compositions described herein to treat subjects with any of the described markers, and also to prevent the progression from DCIS and from atypical hyperplasia to breast cancer.

[00107] The combinations of the invention may be administered either prior to or following surgical removal of primary tumors and/or treatment such as administration of radiotherapy or conventional chemotherapeutic drugs.

[00108] The methods and compositions of the present invention may be used advantageously in combination with any other treatment regimen for breast cancer. Treatments for breast cancer are well known in the art and continue to be developed. Treatments include but are not limited to surgery, including axillary dissection, sentinel lymph node biopsy, reconstructive surgery, surgery to relieve symptoms of advanced cancer, lumpectomy (also called breast conservation therapy), partial (segmental) mastectomy, simple or total mastectomy, modified radical mastectomy, and radical mastectomy; immunotherapy, e.g. using Herceptin™ (trastuzumab), an anti-HER2 humanized monoclonal antibody developed to block the HER2 receptor; bone marrow transplantation; peripheral blood stem cell therapy; bisphosphonates; additional chemotherapy agents; radiation therapy; acupressure; and acupuncture. Any combination of therapies may be used in conjunction with the present invention.

[00109] The methods and compositions comprising the combination therapies described herein may also be used to reduce the proliferation of cancer cells, increase the death of cancer cells or, reduces the size of a tumor or spread of a tumor in a subject. It is

contemplated by the present invention that the combination therapies described herein may reduce the size of a tumor or the spread of a tumor in a subject by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% relative to a control such as PBS. In some embodiments, the combination therapies described herein may increase survival by 1 month, 2 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more, it may render the subject disease-free, or it may prevent the progression from DCIS or atypical hyperplasia to breast cancer.

[00110] Toxicity and efficacy of the prophylactic and/or therapeutic treatments and protocols of the instant invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD ₅₀ ZED ₅₀ . Prophylactic and/or therapeutic agents that exhibit large therapeutic indices are preferred. While prophylactic and/or therapeutic agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[00111] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage of the prophylactic and/or therapeutic agents for use in humans. The dosage of such agents lies preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC ₅₀ (i.e., the concentration of the test compound that achieves a half- maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[00112] In order to determine therapeutic or prophylactic utility, it is encompassed by the present invention to use any of the assays described herein, including those described and illustrated in the Examples section below, as well as those known in the art. Also encompassed by the invention to determine therapeutic or prophylactic utility are any relevant cancer, and more specifically, breast cancer animal models. For example, one may utilize a an MCF-7 xenograft model, or a modified MCF-7 xenograft model (Hale L.V. et al., 1997, Lab Anim Sci.,47(l):82-85). Further encompassed by the invention, pending safety and efficacy, are clinical trials to assess the combinations of the present invention.

6. EXAMPLES

[00113] Unless otherwise specified in the examples below, methods were carried out in the following way.

[00114] Example 1 : A solution of tamoxifen in an organic solvent is treated with an equimolar quantity of valproic acid. The solvent is removed under reduced pressure to afford tamoxifen valproic acid salt, or tamoxifen valproate.

[00115] Example 2: In a 2 mL Eppendorf vial, 13.9 mg of tamoxifen (free base, Sigma) was dissolved in 700 microliters valproic acid (Aldrich) by addition of the valproic acid to the solid tamoxifen. To achieve full dissolution, the suspension was agitated on a vortex mixer for 5 min. The resulting clear solution of tamoxifen valproic acid salt in valproic acid was stored at room temperature or at -4° C and was stable by visual inspection.

[00116] Example 3: Tamoxifen valproic acid salt (0.0001-50 % w/w) is formulated with valproic acid and sodium valproate. This mixture may be milled to afford particles of a specific size or blended with suitable excipients and pressed into tablets.

[00117] Example 4: A solution of 4-hydroxytamoxifen in an organic solvent is treated with an equimolar quantity of valproic acid. The solvent is removed under reduced pressure to afford 4-hydroxytamoxifen valproic acid salt.

[00118] Example 5: 4-Hydroxytamoxifen (0.0001-50 % w/w) is dissolved in valproic acid to afford a solution of 4-hydroxytamoxifen valproic acid salt in valproic acid.

[00119] Example 6: 4-Hydroxytamoxifen valproic acid salt (0.0001-50 % w/w) is formulated with valproic acid and sodium valproate. This mixture may be milled to

afford particles of a specific size or blended with suitable excipients and pressed into tablets.

[00120] Example 7: A solution of endoxifen in an organic solvent is treated with an equimolar quantity of valproic acid. The solvent is removed under reduced pressure to afford endoxifen valproic acid salt.

[00121] Example 8: Endoxifen (0.0001-50 % w/w) is dissolved in valproic acid to afford a solution of endoxifen valproic acid salt in valproic acid.

[00122] Example 9: Endoxifen valproic acid salt (0.0001-50 % w/w) is formulated with valproic acid and sodium valproate. This mixture may be milled to afford particles of a specific size or blended with suitable excipients and pressed into tablets.

[00123] Example 10: A solution of letrozole in an organic solvent is treated with an equimolar quantity of valproic acid. The solvent is removed under reduced pressure to afford letrozole valproic acid salt.

[00124] Example 11 : Letrozole (0.0001-50 % w/w) is dissolved in valproic acid to afford a solution of letrozole valproic acid salt in valproic acid.

[00125] Example 12: Letrozole valproic acid salt (0.0001-50 % w/w) is formulated with valproic acid and sodium valproate. This mixture may be milled to afford particles of a specific size or blended with suitable excipients and pressed into tablets.

[00126] Example 13: Inhibition of breast cancer proliferation in a dose-responsive manner: MCF-7 cells engineered to overexpress aromatase were seeded in hormone- depleted media, and the next day treated with the doses and combinations of anastrozole (anas) and valproic acid (VPA), in the presence of 10 nM androstenediol (adiol), as indicated in Figure 1. Cells were harvested on day 7 and proliferation was assayed using a fluorescent DNA-binding dye. Bars represent fluorescence from triplicate wells, and error bars represent SEM.

[00127] Example 14: Compatibility of tamoxifen and valproic acid with excipients for oral formulation

[00128] Materials were obtained from commercial sources (Tamoxifen, Sigma Lot 117K1079; Valproic acid, Sigma Lot 036K0678; Gelatin, Type B, Sigma Lot 53H0271; Titanium dioxide, Sigma Lot 087K3723; and Glycerin, Spectrum Lot WG0604).

[00129] Each material was weighed (±0.01 g) according to the following Table in tared 20-mL scintillation vials (four vials per Group), and mixed thoroughly to yield a homogenous mixture in each vial, except Group 2 was a "semi-homogeneous" mixture, and Group 5 was not homogeneous.

[00130] Table of Formulations

[00131] Within each Group of four vials, one vial of each Group was kept at room temperature (control), 2 vials at 6O ⁰ C (48 hours and 2 weeks) and one vial at 4O ⁰ C / 75% relative humidity (2 weeks). Each vial was assayed for tamoxifen content by HPLC.

[00132] HPLC assay was performed as follows: Column- Varian Inertsil Phenyl-3, 250 x 4.6-mm, 5-μm. Mobile Phase: Dissolved 1.08 g sodium 1-octanesulfonate in 320 mL water, added 2 mL glacial acetic acid and diluted the final volume to 1.0 L with methanol. Flow rate: 1.5 mL/min. UV detection: 254 nm. Injection volume: 25 μL. See USP 31 for Tamoxifen Citrate Tablets. For preparation of a standard solution, to tamoxifen API (10 mg in a 50 mL volumetric flask) was added mobile phase with mixing to dissolve, and additional mobile phase was added with mixing to yield a 0.2 mg/mL solution, assuming API purity at 100.0%. For preparation of testing sample, mobile phase was added to each vial, with mixing to dissolve, and additional mobile phase was added with mixing to yield about 0.2 mg/mL solution. For Groups 2 and 5, 20 mL mobile phase was added, the mixture was sonicated for 30 minutes, centrifuged, and the supernatant was diluted for HPLC analysis. RSD of 6 replicated injections of standard: NMT 3.0%.

[00133] Table of Results

[00134] Tamoxifen was stable up to 2 weeks at 60 ⁰ C or in the tamoxifen/valproic acid mixture at 1 :9 ratio. In the presence of other excipients (gelatin, glycerin, TiO ₂ ), it was difficult to yield a homogeneous mixture by manual mixing. It is expected tamoxifen is also compatible with these excipients.

[00135] Example 15: Compatibility of anastrozole and valproic acid with excipients for oral formulation

[00136] Materials were obtained from commercial sources (Anastrozole, LKT Labs Lot 2593407; Valproic acid, Sigma Lot 036K0678; Gelatin, Type B, Sigma Lot 53H0271; Titanium dioxide, Sigma Lot 087K3723; and Glycerin, Spectrum Lot WG0604).

[00137] Each material was weighed (±0.01 g) according to the following Table in tared 20-mL scintillation vials with screw caps (four vials per Group), and mixed thoroughly to yield a homogenous mixture in each vial, except Group 4 was a "semi-homogeneous" mixture.

[00138] Table of Formulations

[00139] Within each Group of four vials, one vial of each Group was kept at room temperature (control), 2 vials at 6O ⁰ C (3 days and 2 weeks) and one vial at 4O ⁰ C / 75% relative humidity (2 weeks). Each vial was assayed for anastrozole content by HPLC.

[00140] HPLC assay can be performed by reverse phase HPLC. Samples of Groups 2 and 4 were discolored and did not dissolve readily in mobile phase during assay preparation. Each sample was sonicated 2 x 30 min, followed by centrifugation prior to HPLC injection.

[00141] Table of Results

[00142] Anastrozole in the presence or absence of valproic acid was stable after 2 weeks at 6O ⁰ C or 4O ⁰ C / 75% relative humidity. However, anastrozole in Groups 2 and 4 exhibited evidence of discoloration and poor solubility during sample preparation.

[00143] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.