WO2007056243A2

WO2007056243A2 - Methods of treating cancers with saha and fluorouracil and other combination therapies

Info

Publication number: WO2007056243A2
Application number: PCT/US2006/043127
Authority: WO
Inventors: Stanley Frankel; Marwan Fakih
Original assignee: Merck & Co. Inc.; Roswell Park Cancer Institute
Priority date: 2005-11-04
Filing date: 2006-11-03
Publication date: 2007-05-18
Also published as: WO2007056243A3

Abstract

The present invention relates to a method of treating cancer in a subject in need thereof, by administering to a subject in need thereof a first amount of a histone deacetylase (HDAC) inhibitor such as suberoylanilide hydroxamic acid (SAHA) or a pharmaceutically acceptable salt or hydrate thereof, and a second amount of one or more anti-cancer agents such as Fluorouracil, Leucovorin, and Oxaliplatin (e.g., combined as FOLFOX) and optionally an amount of another anti-cancer agent. The HDAC inhibitor and the anti-cancer agents may be administered to comprise therapeutically effective amounts. In various aspects, the effect of the HDAC inhibitor and the anti-cancer agents may be additive or synergistic.

Description

METHODS OF TREATING CANCERS WITH SAHA AND FLUOROURACIL AND OTHER COMBINATION THERAPIES

FIELD QF THE INVENTION The present invention relates to a method of treating cancer (e.g., colon, colorectal, gastric, or esophageal cancer) by administering a histone deacetylase (HDAC) inhibitor such as suberoylanilide hydroxamic acid (SAHA) in combination with one or more anti-cancer agents such as Fluorouracil, Leucovorin, and Oxaliplatin (e.g., combined as FOLFOX). The combined amounts together can comprise a therapeutically effective amount.

BACKGROUND OF THE INVENTION

Cancer is a disorder in which a population of cells has become, in varying degrees, unresponsive to the control mechanisms that normally govern proliferation and differentiation. Therapeutic agents used in clinical cancer therapy can be categorized into several groups, including, alkylating agents, antibiotic agents, antimetabolic agents, biologic agents, hormonal agents, and plant-derived agents.

Cancer therapy is also being attempted by the induction of terminal differentiation of the neoplastic cells (M. B., Roberts, A. B., and Driscoll, J. S. (1985) in Cancer: Principles and Practice of Oncology, eds. Hellman, S., Rosenberg, S. A., and DeVita, V. T., Jr., Ed. 2, (J. B. Lippincott, Philadelphia), P. 49). In cell culture models, differentiation has been reported by exposure of cells to a variety of stimuli, including: cyclic AMP and retinoic acid (Breitman, T. R., Selonick, S. E., and Collins, S. J. (1980) Proc. Natl. Acad. ScL USA 77: 2936-2940; Olsson, I. L. and Breitman, T. R. (1982) Cancer Res. 42: 3924-3927), aclarubicin and other anthracyclines (Schwartz, E. L. and Sartorelli, A. C. (1982) Cancer Res. 42: 2651- 2655). There is abundant evidence that neoplastic transformation does not necessarily destroy the potential of cancer cells to differentiate (Sporn et al; Marks, P. A., Sheffery, M., and Rifkind, R. A. (1987) Cancer Res. 47: 659; Sachs, L. (1978) Nature (Lond.) 21 A: 535). There are many examples of tumor cells which do not respond to the normal regulators of proliferation and appear to be blocked in the expression of their differentiation program, and yet can be induced to differentiate and cease replicating. A variety of agents can induce various transformed cell lines and primary human tumor explants to express more differentiated characteristics. Histone deacetylase inhibitors such as suberoylanilide hydroxamide acid (SAHA), belong to this class of agents that have the ability to induce tumor cell growth arrest, differentiation, and/or apoptosis (Richon, V.M., Webb, Y., Merger, R., et al. (1996) PNAS 93:5705-8). These compounds are targeted towards mechanisms inherent to the ability of a neoplastic cell to become malignant, as they do not appear to have toxicity in doses effective for inhibition of tumor growth in animals (Cohen, L.A., Amin, S., Marks, P.A., Rifkind, R.A., Desai, D., and Richon, V.M. (1999) Anticancer Research 19:4999- 5006). There are several lines of evidence that histone acetylation and deacetylation are mechanisms by which transcriptional regulation in a cell is achieved (Grunstein, M. (1997) Nature 389:349-52). These effects are thought to occur through changes in the structure of chromatin by altering the affinity of histone proteins for coiled DNA in the nucleosome. There are five types of histones that have been identified (designated Hl, H2A, H2B,

H3 and H4). Histones H2A, H2B, H3, and H4 are found in the nucleosomes and Hl is a linker located between nucleosomes. Each nucleosome contains two of each histone type ■ within its core, except for Hl, which is present singly in the outer portion of the nucleosome structure. It is believed that when the histone proteins are hypoacetylated, there is a greater affinity of the histone to the DNA phosphate backbone. This affinity causes DNA to be tightly bound to the histone and renders the DNA inaccessible to transcriptional regulatory elements and machinery. The regulation of acetylated states occurs through the balance of activity between two enzyme complexes, histone acetyl transferase (HAT) and histone deacetylase (HDAC). The hypoacetylated state is thought to inhibit transcription of associated DNA. This hypoacetylated state is catalyzed by large multiprotein complexes that include HDAC enzymes. In particular, HDACs have been shown to catalyze the removal of acetyl groups from the chromatin core histones.

Colorectal cancer is the fourth most commonly diagnosed cancer and the second leading cause of cancer-related deaths in the United States (American Cancer Society. Cancer Facts and Figures 2004; Hurwitz, 2005, Oncologist 10:320-322). There is a low genetic predisposition to cancer of the large bowel, but familial and hereditary cancers (e.g., familial polyposis, Lynch syndrome) are described. About 70% of colorectal cancers occur in the rectum and sigmoid, and about 95% are adenocarcinomas. Almost a third of patients already have metastatic disease at diagnosis, and half the patients diagnosed and resected with early-stage disease subsequently develop metastases (Macdonald JS, 1999, CA Cancer J. Clin. 49:202-219). Nearly all patients with metastases die of their disease. For many decades, the only effective first-line treatment for metastatic colorectal cancer remained Fluorouracil (see, e.g., Hurwitz, 2005, Oncologist 10:320-322). Subsequently, coadministration of Fluorouracil with Leucovorin increased response rates and time to progression, but only marginally improved overall survival (Thirion P, et al. 2004, J. Clin. Oncol. 22:3766-3775; Budd GT, et al., 1987, J. Clin. Oncol. 5:272-277). Various attempts have been made to optimize this regimen using different doses and schedules, but these have failed to produce real clinical benefit (see, e.g., Hurwitz, 2005, Oncologist 10:320-322). The addition of Oxaliplatin or Irinotecan to Fluorouracil in the first line treatment of metastatic colorectal cancer has produced some improvement in time to progression and survival (Goldberg RM, et al. 2004, J Clin Oncol. 22:23-30; Douillard JY, et ah, 2000, Lancet 355:1041-7), however, the median survival time stands at less then two years. Thus, there is an urgent need to discover suitable methods for the treatment of cancer, including combination treatments that result in decreased side effects and that are effective at treating and controlling malignancies.

SUMMARY OF THE INVENTION The present invention is based on the discovery that histone deacetylase (HDAC) inhibitors, for example suberoylanilide hydroxamic acid (SAHA), can be used in combination with one or more anti-cancer agents such as Fluorouracil, Leucovorin, and Oxaliplatin (e.g., combined as FOLFOX®) to provide therapeutic efficacy.

The invention relates to a method for treating cancer or other disease comprising administering to a subject in need thereof an amount of an HDAC inhibitor, e.g., SAHA, and an amount of one or more additional anti-cancer agents such as Fluorouracil, Leucovorin, and Oxaliplatin (e.g., FOLFOX) and optionally an amount of another anti-cancer agent.

The invention further relates to pharmaceutical combinations useful for the treatment of cancer or other disease comprising an amount of an HDAC inhibitor, e.g., SAHA, and an amount of one or more anti-cancer agents such as Fluorouracil, Leucovorin, and Oxaliplatin (e.g., FOLFOX) and optionally an amount of another anti-cancer agent.

The invention further relates to the use of an amount of an HDAC inhibitor, e.g., SAHA, and an amount of one or more anti-cancer agents such as Fluorouracil, Leucovorin, and Oxaliplatin (e.g., FOLFOX), and optionally an amount of another anti-cancer agent, for the manufacture of one or more medicaments for treating cancer or other disease. The invention further relates to methods for selectively inducing terminal differentiation, cell growth arrest, and/or apoptosis of neoplastic cells, thereby inhibiting proliferation of such cells in a subject by administering to the subject an amount of an HDAC inhibitor, e.g., SAHA, and an amount of one or more anti-cancer agents such as Fluorouracil, Leucovorin, and Oxalip latin (e.g., FOLFOX) and optionally an amount of another anti-cancer agent., wherein the HDAC inhibitor and one or more anti-cancer agents are administered in amounts effective to induce terminal differentiation, cell growth arrest, or apoptosis of the cells.

In particular embodiments of this invention, the combined treatments together comprise a therapeutically effective amount. In addition, the combination of the HDAC inhibitor and one or more anti-cancer agents can provide additive or synergistic therapeutic effects. In further embodiments, the HDAC inhibitors suitable for use in the present invention include but are not limited to hydroxamic acid derivatives, e.g., SAHA, Short Chain Fatty Acids (SCFAs), cyclic tetrapeptides, benzamide derivatives, or electrophilic ketone derivatives.

In further embodiments, the treatment procedures are performed sequentially in any order, alternating in any order, simultaneously, or any combination thereof. In particular, the administration of an HDAC inhibitor, e.g., SAHA, and the administration of one or more anti-cancer agents such as Fluorouracil, Leucovorin, and Oxaliplatin (e.g., FOLFOX), can be performed concurrently, consecutively, or e.g., alternating concurrent and consecutive administration. In further embodiments, the HDAC inhibitor, e.g., SAHA, is administered in combination with any one or more of an additional HDAC inhibitor, an alkylating agent (e.g., Oxaliplatin), an antibiotic agent, an antimetabolic agent (e.g., Fluorouracil), a hormonal agent, a plant-derived agent, an anti-angiogenic agent, a differentiation inducing agent, a cell growth arrest inducing agent, an apoptosis inducing agent, a cytotoxic agent, a biologic agent, a gene therapy agent, an adjunctive agent (e.g., Leucovorin), or any combination thereof (e.g., FOLFOX).

In further embodiments, the HDAC inhibitor is SAHA, which can be administered in combination with any one or more of another HDAC inhibitor, an alkylating agent (e.g., Oxaliplatin), an antibiotic agent, an antimetabolic agent (e.g., Fluorouracil), a hormonal agent, a plant-derived agent, an anti-angiogenic agent, a differentiation inducing agent, a cell growth arrest inducing agent, an apoptosis inducing agent, a cytotoxic agent, a biologic agent, a gene therapy agent, an adjunctive agent (e.g., Leucovorin), or any combination thereof (e.g., FOLFOX). In further embodiments, the combination therapy of the invention is used to treat diseases characterized by cellular hyperproliferation (e.g., cancers, such as colon, colorectal, gastric, or esophageal cancers), solid tumors, or any combination thereof.

In further embodiments, the combination therapy is used to treat diseases such as adenocarcinoma, or advanced or metastatic adenocarcinoma.

In particular embodiments, SAHA is administered in combination with one or more of Fluorouracil, Leucovorin, and Oxaliplatin (for example, FOLFOX), and optionally an amount of another anti-cancer agent, e.g., for colorectal cancer.

Other features and advantages of the invention will be apparent from and are encompassed by the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features, and advantages of the invention will be apparent from the more particular description of embodiments of the invention, as illustrated in the accompanying drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

FIG. IA depicts expression of thymidylate synthase in a liver metastases biopsy from a patient prior to treatment as described in Example 8.

FIG. IB depicts expression of thymidylate synthase in a liver metastases biopsy from a patient after four days of treatment with SAHA as described in Example 8. ( We do not seem to have figures on file)

DETAILED DESCRIPTION OF THE INVENTION

It has been unexpectedly discovered that a combination treatment procedure that includes administration of an HDAC inhibitor, as described herein, and one or more anticancer agents, as described herein, can provide improved therapeutic effects.

Besides the aim to increase the therapeutic efficacy, another purpose of combination treatment is the potential decrease of the doses of the individual components in the resulting combinations in order to decrease unwanted or harmful side effects caused by higher doses of the individual components.

The invention further relates to a method of treating cancer or other disease, in a subject in need thereof, by administering to a subject in need thereof an amount of suberoylanilide hydroxamic acid (SAHA), or a pharmaceutically acceptable salt or hydrate thereof, in a treatment procedure, and an amount of one or more anti-cancer agents (e.g., alkylating agents, antinietabolic agents, and adjunctive agents, such as Fluorouracil, Oxaliplatin, and Leucovorin, e.g., FOLFOX) and optionally an amount of another anti-cancer agent, in another treatment procedure, wherein the amounts can comprise a therapeutically effective amount. The effect of SAHA and the anti-cancer agent can be, e.g., additive or synergistic. In the preferred embodiment of the present invention, the cancer treated with the combination of SAHA and an amount of one or more anti-cancer agent is progressive metastatic or unresectable colorectal cancer.

In one aspect, the method comprises administering to a patient in need thereof a first amount of a histone deacetylase inhibitor, e.g., SAHA, or a pharmaceutically acceptable salt or hydrate thereof, in a first treatment procedure, and another amount of one or more anticancer agents such as Fluorouracil, Leucovorin, and Oxaliplatin (e.g., FOLFOX) ) and optionally an amount of another anti-cancer agent. The invention further relates to pharmaceutical combinations useful for the treatment of cancer or other disease. In one aspect, the pharmaceutical combination comprises a first amount of an HDAC inhibitor, e.g., SAHA, or a pharmaceutically acceptable salt or hydrate thereof, and another amount of one or more anti-cancer agents, e.g., Fluorouracil, Leucovorin, and Oxaliplatin (e.g., FOLFOX) ) and optionally an amount of another anti-cancer agent. The first and second amounts and optionally the third amount can comprise a therapeutically effective amount. The invention further relates to the use of an amount of an HDAC inhibitor and an amount of an anti-cancer agent for the manufacture of a medicament for treatment of cancer or other disease. In one aspect, the medicament comprises a first amount of an HDAC inhibitor, e.g., SAHA or a pharmaceutically acceptable salt or hydrate thereof, and another amount of one or more anti-cancer agents such as Fluorouracil, Leucovorin, Oxaliplatin (e.g., FOLFOX) ) and optionally an amount of another anti-cancer agent.

The combination therapy of the invention provides a therapeutic advantage in view of the differential toxicity associated with the two treatment modalities. For example, treatment with HDAC inhibitors can lead to a particular toxicity that is not seen with the anti-cancer agent, and vice versa. As such, this differential toxicity can permit each treatment to be administered at a dose at which said toxicities do not exist or are minimal, such that together the combination therapy provides a therapeutic dose while avoiding the toxicities of each of the constituents of the combination agents. Furthermore, when the therapeutic effects achieved as a result of the combination treatment are enhanced or synergistic, for example, significantly better than additive therapeutic effects, the doses of each of the agents can be reduced even further, thus lowering the associated toxicities to an even greater extent.

Definitions The term "treating" in its various grammatical forms in relation to the present invention refers to preventing (e.g., chemoprevention), curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a disease state, disease progression, disease causative agent (e.g., bacteria or viruses) or other abnormal condition. For example, treatment may involve alleviating a symptom (i.e., not necessary all symptoms) of a disease or attenuating the progression of a disease. Because some of the inventive methods involve the physical removal of the etiological agent, the artisan will recognize that they are equally effective in situations where the inventive compound is administered prior to, or simultaneous with, exposure to the etiological agent (prophylactic treatment) and situations where the inventive compounds are administered after (even well after) exposure to the etiological agent.

Treatment of cancer, as used herein, refers to partially or totally inhibiting, delaying or preventing the progression of cancer including cancer metastasis; inhibiting, delaying or preventing the recurrence of cancer including cancer metastasis; or preventing the onset or development of cancer (e.g., chemoprevention) in a mammal, for example, a human. In addition, the method of the present invention is intended for the treatment (e.g., chemoprevention) of human patients with cancer. However, it is also likely that the method would be effective in the treatment of cancer in other mammals.

The "anti-cancer agents" of the invention encompass those described herein, including any pharmaceutically acceptable salts or hydrates of such agents, or any free acids, free bases, or other free forms of such agents, and as non-limiting examples: A) Polar compounds (Marks et al. (1987); Friend, C, Scher, W., Holland, J. W., and Sato, T. (1971) Proc. Natl. Acad. ScL (USA) 68: 378-382; Tanaka, M., Levy, J., Terada, M., Breslow, R., Rifkind, R. A., and Marks, P. A. (1975) Proc. Natl. Acad. ScL (USA) 72: 1003-1006; Reuben, R. C, Wife, R. L., Breslow, R., Rifkind, R. A., and Marks, P. A. (1976) Proc. Natl. Acad. Sd. (USA) 73: 862-866); B) Derivatives of vitamin D and retinoic acid (Abe, E., Miyaura, C, Sakagami, H., Takeda, M., Konno, K., Yamazaki, T., Yoshika, S., and Suda, T. (1981) Proc. Natl. Acad. Sci. (USA) 78: 4990-4994; Schwartz, E. L., Snoddy, J. R., Kreutter, D., Rasmussen, H., and Sartorelli, A. C. (1983) Proc. Am. Assoc. Cancer Res. 24: 18; Tanenaga, K., Hozumi, M., and Sakagami, Y. (1980) Cancer Res. 40: 914-919); C) Steroid hormones (Lotem, J. and Sachs, L. (1975) Int. J. Cancer 15: 731-740); D) Growth factors (Sachs, L. (1978) Nature (Lond.) 214: 535, Metcalf, D. (1985) Science, 229: 16-22); E) Proteases (Scher, W., Scher, B. M., and Waxman, S. (1983) Exp. Hematol. 11: 490-498; Scher, W., Scher, B. M., and Waxman, S. (1982) Biochem. & Biophys. Res. Comm. 109: 348-354); F) Tumor promoters (Huberman, E. and Callaham, M. F. (1979) Proc. Natl. Acad. Sd. (USA) 16: 1293-1297; Lottem, J. and Sachs, L. (1979) Proc. Natl. Acad. Sd. (USA) 16: 5158-5162); and G) Inhibitors of DNA or RNA synthesis (Schwartz, E. L. and Sartorelli, A. C. (1982) Cancer Res. 42: 2651-2655, Terada, M., Epner, E., Nudel, U., Salmon, J., Fibach, E., Rifkind, R. A., and Marks, P. A. (1978) Proc. Natl. Acad. Sd. (USA) 75: 2795-2799; Morin, M. J. and Sartorelli, A. C. (1984) Cancer Res. 44: 2807-2812; Schwartz, E. L., Brown, B. J., Nierenberg, M., Marsh, J. C, and Sartorelli, A. C. (1983) Cancer Res. 43: 2725-2730; Sugano, H., Furusawa, M., Kawaguchi, T., and Ikawa, Y. (1973) Bibl. Hematol. 39: 943-954; Ebert, P. S., Wars, L, and Buell, D. N. (1976) Cancer Res. 36: 1809-1813; Hayashi, M., Okabe, J., and Hozumi, M. (1979) Gann 70: 235-238).

As used herein, the term "therapeutically effective amount" is intended to qualify the combined amount of treatments in the combination therapy. The combined amount will achieve the desired biological response. In the present invention, the desired biological response is partial or total inhibition, delay or prevention of the progression of cancer including cancer metastasis; inhibition, delay or prevention of the recurrence of cancer including cancer metastasis; or the prevention of the onset or development of cancer (e.g., chemoprevention) in a mammal, for example a human.

As used herein, the terms "combination treatment", "combination therapy", "combined treatment" or "combinatorial treatment", used interchangeably, refer to a treatment of an individual with at least two different therapeutic agents. According to one aspect of the invention, the individual is treated with a first therapeutic agent, e.g., SAHA or another HDAC inhibitor as described herein. The second therapeutic agent may be another HDAC inhibitor, or may be any other clinically established anti-cancer agent (such as an alkylating agent, antimetabolic agent, or adjunctive agent) as defined herein. A combinatorial treatment may include a third or even further therapeutic agent. The combination treatments may be carried out consecutively or concurrently.

An "adjunctive agent" refers to any compound used to enhance the effectiveness of an anti-cancer agent or to prevent or treat conditions associated with an anti-cancer agent such as low blood counts, neutropenia, anemia, thrombocytopenia, hypercalcemia, mucositis, bruising, bleeding, toxicity, fatigue, pain, nausea, and vomiting.

As recited herein, "HDAC inhibitor" (e.g., SAHA) encompasses any synthetic, recombinant, or naturally-occurring inhibitors, including any pharmaceutical salts or hydrates of such inhibitors, and any free acids, free bases, or other free forms of such inhibitors. "Hydroxamic acid derivative," as used herein, refers to the class of histone deacetylase inhibitors that are hydroxamic acid derivatives. Specific examples of inhibitors are provided herein.

"Patient" or "subject" as the terms are used herein, refer to the recipient of the treatment. Mammalian and non-mammalian patients are included. In a specific embodiment, the patient is a mammal, such as a human, canine, murine, feline, bovine, ovine, swine, or caprine. In a particular embodiment, the patient is a human.

The terms "intermittent" or "intermittently" as used herein means stopping and starting at either regular or irregular intervals. The term "hydrate" includes but is not limited to hemihydrate, monohydrate, dihydrate, trihydrate, and the like.

Histone Deacetylases and Histone Deacetylase Inhibitors

Histone deacetylases (HDACs) include enzymes that catalyze the removal of acetyl groups from lysine residues in the amino terminal tails of the nucleosomal core histones. As such, HDACs together with histone acetyl transferases (HATs) regulate the acetylation status of histones. Histone acetylation affects gene expression and inhibitors of HDACs, such as the hydroxamic acid-based hybrid polar compound suberoylanilide hydroxamic acid (SAHA) induce growth arrest, differentiation, and/or apoptosis of transformed cells in vitro and inhibit tumor growth in vivo.

HDACs can be divided into three classes based on structural homology. Class I HDACs (HDACs 1, 2, 3, and 8) bear similarity to the yeast RPD3 protein, are located in the nucleus and are found in complexes associated with transcriptional co-repressors. Class II HDACs (HDACs 4, 5, 6, 7, and 9) are similar to the yeast HDAl protein, and have both nuclear and cytoplasmic subcellular localization. Both Class I and II HDACs are inhibited by hydroxamic acid-based HDAC inhibitors such as SAHA. Class III HDACs form a structurally distant class of NAD dependent enzymes that are related to the yeast SIR2 proteins and are not inhibited by hydroxamic acid-based HDAC inhibitors. Histone deacetylase inhibitors, also called HDAC inhibitors, are compounds that are capable of inhibiting the deacetylation of histones in vivo, in vitro, or both. As such, HDAC inhibitors inhibit the activity of at least one histone deacetylase. As a result of inhibiting the deacetylation of at least one histone, an increase in acetylated histone occurs. The accumulation of acetylated histone provides a suitable biological marker for assessing the activity of HDAC inhibitors. Therefore, procedures that can assay for the accumulation of acetylated histones can be used to determine the HDAC inhibitory activity of compounds of interest. It is understood that compounds that can inhibit histone deacetylase activity can also bind to other substrates and as such can inhibit other biologically active molecules such as enzymes. It is also to be understood that the compounds of the present invention are capable of inhibiting any of the histone deacetylases set forth above, or any other histone deacetylases.

For example, in patients receiving HDAC inhibitors, the accumulation of acetylated histones in peripheral mononuclear cells as well as in tissue treated with HDAC inhibitors can be determined against a suitable control.

HDAC inhibitory activity of a particular compound can be determined in vitro using, for example, an enzymatic assay which shows inhibition of at least one histone deacetylase. Further, determination of the accumulation of acetylated histones in cells treated with a particular composition can be determinative of the HDAC inhibitory activity of a compound. Assays for the accumulation of acetylated histones are well known in the literature.

See, for example, Marks, P.A. et al., J. Natl. Cancer Inst., 92:1210-1215, 2000, Butler, L.M. et al, Cancer Res. 60:5165-5170 (2000), Richon, V.M. et al., Proc. Natl. Acad. ScL, USA, 95:3003-3007, 1998, and Yoshida, M. et al, J. Biol. Chem., 265:17174-17179, 1990.

For example, an enzymatic assay to determine the activity of an HDAC inhibitor compound can be conducted as follows. Briefly, the effect of an HDAC inhibitor compound on affinity purified human epitope-tagged (e.g., Flag) HDACl can be assayed by incubating the enzyme preparation in the absence of substrate on ice for about 20 minutes with the indicated amount of inhibitor compound. Substrate (e.g., [³H]acetyl-labeled murine erythroleukemia cell-derived histone) can be added and the sample can be incubated for 20 minutes at 37⁰C in a total volume of 30 μL. The reaction can then be stopped and released acetate can be extracted and the amount of radioactivity release determined by scintillation counting. An alternative assay useful for determining the activity of an HDAC inhibitor compound is the HDAC Fluorescent Activity Assay; Drug Discovery Kit-AK-500 available from BIOMOL® Research Laboratories, Inc., Plymouth Meeting, PA.

In vivo studies can be conducted as follows. Animals, for example, mice, can be injected intraperitoneally with an HDAC inhibitor compound. Selected tissues, for example, brain, spleen, liver etc, can be isolated at predetermined times, post administration. Histones can be isolated from tissues essentially as described (see, e.g., Yoshida et ah, J. Biol. Chem. 265:17174-17179, 1990). Equal amounts of histones (about 1 μg) can be electrophoresed on 15% SDS-polyacrylamide gels and can be transferred to Hybond-P filters (Amersham). Filters can be blocked with 3% milk and can be probed with a rabbit purified polyclonal anti- acetylated histone H4 antibody (αAc-H4) and anti-acetylated histone H3 antibody (αAc-H3) (Upstate Biotechnology, Inc.). Levels of acetylated histone can be visualized using a horseradish peroxidase-conjugated goat anti-rabbit antibody (1:5000) and the SuperSignal chemiluminescent substrate (Pierce). As a loading control for the histone protein, parallel gels can be run and stained with Coomassie Blue (CB). Hydroxamic acid-based HDAC inhibitors have also been shown to up regulate the expression of the p21wAFi gene. The p21wAFi protein is induced within 2 hours of culture with HDAC inhibitors in a variety of transformed cells using standard methods. The induction of the P21_WA_FI gene is associated with accumulation of acetylated histones in the chromatin region of this gene. Induction of P21W_AFI can therefore be recognized as involved in the Gl cell cycle arrest caused by HDAC inhibitors in transformed cells.

U.S. Patent Numbers 5,369,108, 5,932,616, 5,700,811, 6,087,367 and 6,511,990, issued to some of the present inventors, disclose compounds useful for selectively inducing terminal differentiation of neoplastic cells, which compounds have two polar end groups separated by a flexible chain of methylene groups or a by a rigid phenyl group, wherein one or both of the polar end groups is a large hydrophobic group. Some of the compounds have an additional large hydrophobic group at the same end of the molecule as the first hydrophobic group which further increases differentiation activity about 100 fold in an enzymatic assay and about 50 fold in a cell differentiation assay. Methods of synthesizing the compounds used in the methods and pharmaceutical compositions of this invention are fully described the aforementioned patents, the entire contents of which are incorporated herein by reference.

Thus, the present invention includes within its broad scope compositions comprising HDAC inhibitors which are 1) hydroxamic acid derivatives; 2) Short-Chain Fatty Acids (SCFAs); 3) cyclic tetrapeptides; 4) benzamides; 5) electrophilic ketones; and/or any other class of compounds capable of inhibiting histone deacetylases, for use in inhibiting histone deacetylase, inducing terminal differentiation, cell growth arrest and/or apoptosis in neoplastic cells, and/or inducing differentiation, cell growth arrest and/or apoptosis of tumor cells in a tumor.

Non-limiting examples of such HDAC inhibitors are set forth below. It is understood that the present invention includes any salts, crystal structures, amorphous structures, hydrates, derivatives, metabolites, stereoisomers, structural isomers, prodrugs, and any free acids, free bases, or other free forms of the HDAC inhibitors described herein. A. Hydroxamic Acid Derivatives such as Suberoylanilide hydroxamic acid (SAHA)

(Richon et al, Proc. Natl. Acad. Sd. USA 95,3003-3007 (1998)); m-Carboxycinnamic acid bishydroxamide (CBHA) (Richon et al, supra); Pyroxamide; Trichostatin analogues such as Trichostatin A (TSA) and Trichostatin C (Koghe et al. 1998. Biochem. Pharmacol. 56: 1359- 1364); Salicylbishydroxamic acid (Andrews et al., InternationalJ. Parasitology 30,761-768 (2000)); Suberoyl bishydroxamic acid (SBHA) (U.S. Patent No. 5,608,108); Azelaic bishydroxamic acid (ABHA) (Andrews et al., supra); Azelaic-l-hydroxamate-9-anilide (AAHA) (Qiu et al, MoI. Biol. Cell 11, 2069-2083 (2000)); 6-(3-Chlorophenylureido) carpoic hydroxamic acid (3C1-UCHA); Oxamflatin [(2E)-5-[3-[(phenylsufonyl) aminol phenyl]-pent-2-en-4-ynohydroxamic acid] (Kim et al. Oncogene, 18: 2461 2470 (1999)); A- 161906, Scriptaid (Su et al 2000 Cancer Research, 60: 3137-3142); PXD-101 (Prolifix); LAQ-824; CHAP; MW2796 (Andrews et al., supra); MW2996 (Andrews et al., supra); or any of the hydroxamic acids disclosed in U.S. Patent Numbers 5,369,108, 5,932,616, 5,700,811, 6,087,367, and 6,511,990.

B. Cyclic Tetrapeptides such as Trapoxin A (TPX)-cyclic tetrapeptide (cyclo-(L- phenylalanyl-L-phenylalanyl-D-pipecolinyl-L-2-amino-8-oxo-9, 10-epoxy decanoyl)) (Kijima et al., J. Biol. Chem. 268, 22429-22435 (1993)); FR901228 (FK228, depsipeptide) (Nakajima et al, Ex. Cell Res. 241,126-133 (1998)); FR225497 cyclic tetrapeptide (H. Mori et al, PCT Application WO 00/08048 (17 February 2000)); Apicidin cyclic tetrapeptide [cyclo(N-O- methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L^-amino-S-oxodecanoyl)] (Darkin- Rattray et al, Proc. Natl. Acad. Sd. L^4 93,13143-13147 (1996)); Apicidin Ia, Apicidin Ib, Apicidin Ic, Apicidin Ha, and Apicidin lib (P. Dulski et al, PCT Application WO 97/11366); CHAP, HC-toxin cyclic tetrapeptide (Bosch et al, Plant Cell 1, 1941-1950 (1995)); WF27082 cyclic tetrapeptide (PCT Application WO 98/48825); and Chlamydocin (Bosch et al, supra).

C. Short chain fatty acid (SCFA) derivatives such as: Sodium Butyrate (Cousens et al, J. Biol. Chem. 254,1716-1723 (1979)); Isovalerate (McBain et al, Biochem. Pharm. 53: 1357-1368 (1997)); Valerate (McBain et al, supra); 4-Phenylbutyrate (4-PBA) (Lea and Tulsyan, Anticancer Research, 15,879-873 (1995)); Phenylbutyrate (PB) (Wang et al, Cancer Research, 59, 2766-2799 (1999)); Propionate (McBain et al, supra); Butyramide (Lea and Tulsyan, supra); Isobutyramide (Lea and Tulsyan, supra); Phenylacetate (Lea and Tulsyan, supra); 3-Bromopropionate (Lea and Tulsyan, supra); Tributyrin (Guan et al, Cancer Research, 60,749-755 (2000)); Valproic acid, Valproate, and Pivanex™. D. Benzamide derivatives such as CI-994; MS-275 [N- (2-aminophenyl)-4-[N-

(pyridin-3-yl methoxycarbonyl) aminomethyl] benzamide] (Saito et al, Proc. Natl. Acad. ScL USA 96, 4592-4597 (1999)); and 3'-amino derivative of MS-275 (Saito et al, supra).

E. Electrophilic ketone derivatives such as Trifluoromethyl ketones (Frey et al, Bioorganic & Med. Chem. Lett. (2002), 12, 3443-3447; U.S. 6,511,990) and α-keto amides such as N-methyl- α-ketoamides.

F. Other HDAC Inhibitors such as natural products, psammaplins, and Depudecin (Kwon et al. 1998. PNAS 95: 3356-3361).

Hydroxamic acid based HDAC inhibitors include suberoylanilide hydroxamic acid (SAHA), m-carboxycinnamic acid bishydroxamate (CBHA) and pyroxamide. SAHA has been shown to bind directly in the catalytic pocket of the histone deacetylase enzyme. SAHA induces cell cycle arrest, differentiation, and/or apoptosis of transformed cells in culture and inhibits tumor growth in rodents. SAHA is effective at inducing these effects in both solid tumors and hematological cancers. It has been shown that SAHA is effective at inhibiting tumor growth in animals with no toxicity to the animal. The SAHA-induced inhibition of tumor growth is associated with an accumulation of acetylated histones in the tumor. SAHA is effective at inhibiting the development and continued growth of carcinogen-induced (N- methylnitrosourea) mammary tumors in rats. SAHA was administered to the rats in their diet over the 130 days of the study. Thus, SAHA is a nontoxic, orally active antitumor agent whose mechanism of action involves the inhibition of histone deacetylase activity. HDAC inhibitors include those disclosed in U.S. Patent Numbers 5,369,108,

5,932,616, 5,700,811, 6,087,367, and 6,511,990, issued to some of the present inventors disclose compounds, the entire contents of which are incorporated herein by reference, non- limiting examples of which are set forth below: Specific HDAC inhibitors include suberoylanilide hydroxamic acid (SAHA; N- Hydroxy-iV'-phenyl octanediamide), which is represented by the following structural formula:

• Other examples of such compounds and other HDAC inhibitors can be found in U.S.

Patent No. 5,369,108, issued on November 29, 1994, U.S. Patent No. 5,700,811, issued on December 23, 1997, U.S. Patent No. 5,773,474, issued on June 30, 1998, U.S. Patent No. 5,932,616, issued on August 3, 1999 and U.S. Patent No. 6,511,990, issued January 28, 2003, all to Breslow et al; U.S. Patent No. 5,055,608, issued on October 8, 1991, U.S. Patent No. 5,175,191, issued on December 29, 1992 and U.S. Patent No. 5,608,108, issued on March 4, 1997, all to Marks et al; as well as Yoshida, M., et al, Bioassays 17, 423-430 (1995); Saito, A., et al, PNAS USA 96, 4592-4597, (1999); Furamai R. et al, PNAS USA 98 (1), 87-92 (2001); Komatsu, Y., et al, Cancer Res. 61(11), 4459-4466 (2001); Su, G.H., et al, Cancer Res. 60, 3137-3142 (2000); Lee, B.I. et al, Cancer Res. 61(3), 931-934; Suzuki, T., et al, J. Med. Chem. 42(15), 3001-3003 (1999); published PCT Application WO

01/18171 published on March 15, 2001 to Sloan-Kettering Institute for Cancer Research and The Trustees of Columbia University; published PCT Application WO 02/246144 to Hoffmann-La Roche; published PCT Application WO 02/22577 to Novartis; published PCT Application WO 02/30879 to Prolifix; published PCT Applications WO 01/38322 (published May 31, 2001); WO 01/70675 (published on September 27, 2001); and WO 00/71703

(published on November 30, 2000) all to Methylgene, Inc.; published PCT Application WO 00/21979 published on October 8, 1999 to Fujisawa Pharmaceutical Co., Ltd.; published PCT Application WO 98/40080 published on March 11, 1998 to Beacon Laboratories, L.L.C.; and Curtin M. (Current patent status of HDAC inhibitors Expert Opin. Ther. Patents (2002) 12(9): 1375-1384 and references cited therein).

SAHA or any of the other HDACs can be synthesized according to the methods outlined in the Experimental Details Section, or according to the method set forth in U.S. Patent Nos. 5,369,108, 5,700,811, 5,932,616 and 6,511,990, the contents of which are incorporated by reference in their entirety, or according to any other method known to a person skilled in the art.

Specific non-limiting examples of HDAC inhibitors are provided in the Table below. It should be noted that the present invention encompasses any compounds which are . ^■ structurally similar to the compounds represented below, and which are capable of inhibiting histone deacetylases.

Stereochemistry

Many organic compounds exist in optically active forms having the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are non- superimposable mirror images of one another. A specific stereoisomer can also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture.

Many of the compounds described herein can have one or more chiral centers and therefore can exist in different enantiomeric forms. If desired, a chiral carbon can be designated with an asterisk (*). When bonds to the chiral carbon are depicted as straight lines in the formulas of the invention, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within the formula. As is used in the art, when it is desired to specify the absolute configuration about a chiral carbon, one of the bonds to the chiral carbon can be depicted as a wedge (bonds to atoms above the plane) and the other can be depicted as a series or wedge of short parallel lines is (bonds to atoms below the plane). The Cahn-Inglod-Prelog system can be used to assign the (R) or (S) configuration to a chiral carbon.

When the HDAC inhibitors of the present invention contain one chiral center, the compounds exist in two enantiomeric forms and the present invention includes both enantiomers and mixtures of enantiomers, such as the specific 50:50 mixture referred to as a racemic mixtures. The enantiomers can be resolved by methods known to those skilled in the art, for example by formation of diastereoisomeric salts which may be separated, for example, by crystallization (see, CRC Handbook of Optical Resolutions via Diastereomeric Salt Formation by David Kozma (CRC Press, 2001)); formation of diastereoisomeric derivatives or complexes which may be separated, for example, by crystallization, gas-liquid or liquid chromatography; selective reaction of one enantiomer with an enantiomer-speciflc reagent, for example enzymatic esterification; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support for example silica with a bound chiral ligand or in the presence of a chiral solvent. It will be appreciated that where the desired enantiomer is converted into another chemical entity by one of the separation procedures described above, a further step is required to liberate the desired enantiomeric form. Alternatively, specific enantiomers may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer into the other by asymmetric transformation.

Designation of a specific absolute configuration at a chiral carbon of the compounds of the invention is understood to mean that the designated enantiomeric form of the compounds is in enantiomeric excess (ee) or in other words is substantially free from the other enantiomer. For example, the (R) forms of the compounds are substantially free from the (S) forms of the compounds and are, thus, in enantiomeric excess of the (S) forms. Conversely, (S) forms of the compounds are substantially free of (R) forms of the compounds and are, thus, in enantiomeric excess of the (R) forms. Enantiomeric excess, as used herein, is the presence of a particular enantiomer at greater than 50%. For example, the enantiomeric excess can be about 60% or more, such as about 70% or more, for example about 80% or more, such as about 90% or more. In a particular embodiment when a specific absolute configuration is designated, the enantiomeric excess of depicted compounds is at least about 90%. In a more particular embodiment, the enantiomeric excess of the compounds is at least about 95%, such as at least about 97.5%, for example, at least 99% enantiomeric excess.

When a compound of the present invention has two or more chiral carbons it can have more than two optical isomers and can exist in diastereoisomeric forms. For example, when there are two chiral carbons, the compound can have up to 4 optical isomers and 2 pairs of enantiomers ((S,S)/(R,R) and (R,S)/(S,R)). The pairs of enantiomers (e.g., (S,S)/(R,R)) are mirror image stereoisomers of one another. The stereoisomers which are not mirror-images (e.g., (S, S) and (R,S)) are diastereomers. The diastereoisomeric pairs may be separated by methods known to those skilled in the art, for example chromatography or crystallization and the individual enantiomers within each pair may be separated as described above. The present invention includes each diastereoisomer of such compounds and mixtures thereof.

As used herein, "a," "an," and "the" include singular and plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an active agent" or "a pharmacologically active agent" includes a single active agent as well a two or more different active agents in combination, reference to "a carrier" includes mixtures of two or more carriers as well as a single carrier, and the like.

This invention is also intended to encompass prodrugs of the HDAC inhibitors disclosed herein. A prodrug of any of the compounds can be made using well known pharmacological techniques.

This invention, in addition to the above listed compounds, is intended to encompass the use of homologs and analogs of such compounds. In this context, homo logs are molecules having substantial structural similarities to the above-described compounds and analogs are molecules having substantial biological similarities regardless of structural similarities.

Alkylating Agents

Examples of alkylating agents include, but are not limited to, bischloroethylamines (nitrogen mustards, e.g., Chlorambucil, Cyclophosphamide, Ifosfamide, Mechlorethamine, Melphalan, uracil mustard), aziridines (e.g., Thiotepa), alkyl alkone sulfonates (e.g.,

Busulfan), nitrosoureas (e.g., Carmustine, Lomustine, Streptozocin), nonclassic alkylating agents (e.g., Altretamine, Dacarbazine, and Procarbazine), platinum compounds (e.g., Carboplastin and Cisplatin). These compounds react with phosphate, amino, hydroxyl, sulfihydryl, carboxyl, and imidazole groups. Oxaliplatin (e.g., Eloxatin™, Sanofi-Synthelabo, Inc., New York, NY) is an organoplatinum complex in which the platinum atom is complexed with 1,2- diaminocyclohexane (DACH) and with an oxalate ligand as a leaving group. Oxaliplatin undergoes nonenzymatic conversion in physiologic solutions to active derivatives which form inter- and intrastrand platinum-DNA crosslinks. Crosslinks are formed between the N7 positions of two adjacent guanines (GG), adjacent adenine-guanines (AG), and guanines separated by an intervening nucleotide (GNG). These crosslinks inhibit DNA replication and transcription in cancer and non-cancer cells. The chemical name for Oxaliplatin is of cis- [(li?,2i?)-l,2-cyclohexanediamine-N,N'] [oxalato(2-)-0,0'] platinum, as represented by the structure:

Under physiological conditions, these drugs ionize and produce positively charged ions that attach to susceptible nucleic acids and proteins, leading to cell cycle arrest and/or cell death. The alkylating agents exert their activity independently of a specific phase of the cell cycle. The nitrogen mustards and alkyl alkone sulfonates are most effective against cells in the Gl or M phase. Nitrosoureas, nitrogen mustards, and aziridines impair progression from the Gl and S phases to the M phases. Chabner and Collins eds. (1990) "Cancer Chemotherapy: Principles and Practice", Philadelphia: JB Lippincott.

The alkylating agents are active against wide variety of neoplastic diseases, with significant activity in the treatment of leukemias and lymphomas as well as solid tumors. Clinically this group of drugs is routinely used in the treatment of acute and chronic leukemias; Hodgkin's disease; non-Hodgkin's lymphoma; multiple myeloma; primary brain tumors; carcinomas of the breast, ovaries, testes, lungs, bladder, cervix, head and neck; and malignant melanoma.

Antimetabolic Agents

Antimetabolic agents (i.e., antimetabolites) are a group of drugs that interfere with metabolic processes vital to the physiology and proliferation of cancer cells. Actively proliferating cancer cells require continuous synthesis of large quantities of nucleic acids, proteins, lipids, and other vital cellular constituents.

Many of the antimetabolites inhibit the synthesis of purine or pyrimidine nucleosides or inhibit the enzymes of DNA replication. Some antimetabolites also interfere with the synthesis of ribonucleosides and RNA and/or amino acid metabolism and protein synthesis as well. By interfering with the synthesis of vital cellular constituents, antimetabolites can delay or arrest the growth of cancer cells. Antimitotic agents are included in this group. Examples of antimetabolic agents include, but are not limited to, Fluorouracil (5-FU), Floxuridine (5- FUdR), Methotrexate, Leucovorin, Hydroxyurea, Thioguanine (6-TG), Mercaptopurine (6- MP), Cytarabine, Pentostatin, Fludarabine Phosphate, Cladribine (2-CDA), Asparaginase, and Gemcitabine.

Fluorouracil (e.g., Fluorouracil Injection, Gensia Sicor Pharmaceuticals, Inc., Irvine, CA; Adracil®, SP Pharmaceuticals Albuquerque, NM; 5-FU) is a fluorinated pyrimidine. The metabolism of Fluorouracil in the anabolic pathway may block the methylation reaction of deoxyuridylic acid to thymidylic acid. In this manner, Fluorouracil can interfere with the synthesis of DNA and to a lesser extent inhibit the formation of ribonucleic acid (RNA). Since DNA and RNA are essential for cell division and growth, the effect of fluorouracil may be to create a thymine deficiency which provokes unbalanced growth and death of the cell. The effects of DNA and RNA inhibition are most marked on those cells which grow more rapidly and which take up fluorouracil at a more rapid rate. The chemical formula for Fluorouracil is 5-fluoro-2,4 (lH,3H)-pyrimidinedione, as represented by the structure:

Antimetabolic agents have widely used to treat several common forms of cancer including carcinomas of colon, rectum, breast, liver, stomach and pancreas, malignant melanoma, acute and chronic leukemia, and hair cell leukemia.

Additional Therapies

Recent developments have introduced additional therapies for the treatment of cancer, in addition to the traditional cytotoxic and hormonal therapies. For example, many forms of gene therapy are undergoing preclinical or clinical trials, ϋi addition, approaches are currently under development that are based on the inhibition of tumor vascularization (i.e., angiogenesis). These approaches can be used to cut off a tumor from the nutrition and oxygen supply provided by a newly built tumor vascular system. Li addition, cancer therapy is also being attempted by the induction of terminal differentiation of the neoplastic cells. Suitable differentiation agents include the compounds disclosed in any one or more of the following references, the contents of which are incorporated by reference herein.

A) Polar compounds (Marks et al. (1987); , Friend, C, Scher, W., Holland, J. W., and Sato, T. (1971) Proc. Natl. Acad. ScL (USA) 68: 378-382; Tanaka, M., Levy, J., Terada, M., Breslow, R., Rifkind, R. A., and Marks, P. A. (1975) Proc. Natl. Acad. ScL (USA) 72: 1003- 1006; Reuben, R. C, Wife, R. L., Breslow, R., Rifkind, R. A., and Marks, P. A. (1976) Proc. Natl. Acad. Sci. (USA) 73: 862-866); B) Derivatives of vitamin D and retinoic acid (Abe, E., Miyaura, C, Sakagami, H., Takeda, M., Konno, K., Yamazaki, T., Yoshika, S., and Suda, T.

(1981) Proc. Natl. Acad. Sci. (USA) 78: 4990-4994; Schwartz, E. L., Snoddy, J. R., Kreutter, D., Rasmussen, H., and Sartorelli, A. C. (1983) Proc. Am. Assoc. Cancer Res. 24: 18;

Tanenaga, K., Hozumi, M., and Sakagami, Y. (1980) Cancer Res. 40: 914-919); C) Steroid hormones (Lotem, J. and Sachs, L. (1975) Int. J. Cancer 15: 731-740); D) Growth factors (Sachs, L. (1978) Nature (Lond.) 21 A: 535, Metcalf, D. (1985) Science, 229: 16-22); E) Proteases (Scher, W., Scher, B. M., and Waxman, S. (1983) Exp. Hematol. 11: 490-498; Scher, W., Scher, B. M., and Waxman, S. (1982) Biochem. & Biophys. Res. Comm. 109: 348- 354); F) Tumor promoters (Huberman, E. and Callaham, M. F. (1979) Proc. Natl. Acad. Sci. (USA) 76: 1293-1297; Lottem, J. and Sachs, L. (1979) Proc. Natl. Acad. Sci. (USA) 76: 5158- 5162); and G) Inhibitors of DNA or RNA synthesis (Schwartz, E. L. and Sartorelli, A. C.

(1982) Cancer Res. 42: 2651-2655, Terada, M., Epner, E., Nudel, U., Salmon, J., Fibach, E., Rifkind, R. A., and Marks, P. A. (1978) Proc. Natl. Acad. Sci. (USA) 75: 2795-2799; Morin,

M. J. and Sartorelli, A. C. (1984) Cancer Res. 44: 2807-2812; Schwartz, E. L., Brown, B. J., Nierenberg, M., Marsh, J. C, and Sartorelli, A. C. (1983) Cancer Res. 43: 2725-2730;

Sugano, H., Furusawa, M., Kawaguchi, T., and Ikawa, Y. (1973) Bibl. Hematol. 39: 943-954; Ebert, P. S., Wars, L, and Buell, D. N. (1976) Cancer Res. 36: 1809-1813; Hayashi, M., Okabe, J., and Hozumi, M. (1979) Gann 70: 235-238),

Other agents may also be useful for use with the present invention, for example, for adjunct therapies. Such adjunctive agents can be used to enhance the effectiveness of anticancer agents or to prevent or treat conditions associated with anti-cancer agents such as low blood counts, neutropenia, anemia, thrombocytopenia, hypercalcemia, mucositis, bruising, bleeding, toxicity, fatigue, pain, nausea, and vomiting. Agents include epoetin alpha (e.g., Procrit®, Epogen®) for stimulating red blood cell production, G-CSF (granulocyte colony- stimulating factor; filgrastim, e.g., Neupogen®) for stimulating neutrophil production, GM- CSF (granulocyte-macrophage colony-stimulating factor) for stimulating production of several white blood cells, including macrophages, and IL-11 (interleukin-11 , e.g., Neumega®) for stimulating production of platelets.

Leucovorin (e.g., Leucovorin calcium, Roxane Laboratories, Inc., Columbus, OH; also called folinic acid, calcium folinate, citrovorum factor) is useful as an antidote to drugs which act as folic acid antagonists. Leucovorin calcium is used to reduce the toxicity and counteract the effects of impaired methotrexate elimination and of inadvertent overdose of folic acid antagonists. Following administration, Leucovorin is absorbed and enters the general body pool of reduced folates. The increase in plasma and serum folate activity seen after administration of Leucovorin is predominantly due to 5-methyltetrahydrofolate. Leucovorin does not require reduction by the enzyme dihydro folate reductase in order to participate in reactions utilizing folates. Leucovorin can also be used to potentiate the activity of Fluorouracil. Leucovorin calcium is the calcium salt of N-[4-[[(2-amino-5-formyl- 1 ,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid, as represented by the structure:

The use of all of these approaches in combination with HDAC inhibitors, e.g. SAHA, is within the scope of the present invention.

Administration of the HDAC Inhibitor Routes of Administration

The HDAC inhibitor (e.g. SAHA), can be administered by any known administration method known to a person skilled in the art. Examples of routes of administration include but are not limited to oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, topical, sublingual, intramuscular, rectal, transbuccal, intranasal, liposomal, via inhalation, vaginal, intraoccular, via local delivery by catheter or stent, subcutaneous, intraadiposal, intraarticular, intrathecal, or in a slow release (e.g., sustained release) dosage form. SAHA or any one of the HDAC inhibitors can be administered in accordance with any dose and dosing schedule that, together with the effect of the anti-cancer agent, achieves a dose effective to treat disease. Of course, the route of administration of SAHA or any one of the other HDAC inhibitors can be independent of the route of administration of the anti-cancer agent. A particular route of administration for SAHA is oral administration. Thus, in accordance with this embodiment, SAHA is administered orally, and the second agent (anti-cancer agent) is administered orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingualis intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery by catheter or stent, subcutaneously, intraadiposally, intraarticularly, intrathecally, or in a slow release (e.g., sustained release) dosage form. As examples, the HDAC inhibitors of the invention can be administered in such oral forms as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. Likewise, the HDAC inhibitors can be administered by intravenous (e.g., bolus or infusion), intraperitoneal, subcutaneous, intramuscular, or other routes using forms well known to those of ordinary skill in the pharmaceutical arts. A particular route of administration of the HDAC inhibitor is oral administration.

The HDAC inhibitors can also be administered in the form of a depot injection or implant preparation, which may be formulated in such a manner as to permit a sustained release of the active ingredient. The active ingredient can be compressed into pellets or small cylinders and implanted subcutaneously or intramuscularly as depot injections or implants. Implants may employ inert materials such as biodegradable polymers or synthetic silicones, for example, Silastic, silicone rubber or other polymers manufactured by the Dow-Corning Corporation.

The HDAC inhibitor can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines. Liposomal preparations of anti-cancer agents may also be used in the methods of the invention. Liposome versions of anti-cancer agents may be used to increase tolerance to the agents. The HDAC inhibitors can also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled.

The HDAC inhibitors can also be prepared with soluble polymers as targetable drug carriers. Such polymers can include polyvinlypyrrolidone, pyran copolymer, polyhydroxy- propyl-methacrylamide-phenol, polyhydroxyethyl-aspartarnide-phenol, or polyethyleneoxide- polylysine substituted with palmitoyl residues. Furthermore, the HDAC inhibitors can be prepared with biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross linked or amphipathic block copolymers of hydrogels.

In a specific embodiment, the HDAC inhibitor, e.g. SAHA, is administered orally in a gelatin capsule, which can comprise excipients such as microcrystalline cellulose, croscarmellose sodium and magnesium stearate. A further embodiment includes 200 mg of solid SAHA with 89.5 mg of microcrystalline cellulose, 9 mg of sodium croscarmellose, and 1.5 mg of magnesium stearate contained in a gelatin capsule.

Dosages and Dosage Schedules The dosage regimen utilizing the HDAC inhibitors can be selected in accordance with a variety of factors including type, species, age, weight, sex and the type of disease being treated; the severity (i.e., stage) of the disease to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. A dosage regimen can be used, for example, to prevent, inhibit (fully or partially), or arrest the progress of the disease.

In accordance with the invention, an HDAC inhibitor (e.g., SAHA or a pharmaceutically acceptable salt or hydrate thereof) can be administered by continuous or intermittent dosages. For example, intermittent administration of an HDAC inhibitor may be administration one to six days per week or it may mean administration in cycles (e.g. daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week) or it may mean administration on alternate days. The compositions may be administered in cycles, with rest periods in between the cycles (e.g. treatment for two to eight weeks with a rest period of up to a week between treatments).

For example, SAHA or any one of the HDAC inhibitors can be administered in a total daily dose of up to 800 mg. The HDAC inhibitor can be administered once daily (QD), or divided into multiple daily doses such as twice daily (BID), and three times daily (TID). The HDAC inhibitor can be administered at a total daily dosage of up to 800 mg, e.g., up to 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, or 800 mg, which can be administered in one daily dose or can be divided into multiple daily doses as described above. In specific aspects, the administration is oral.

In one embodiment, the composition is administered once daily at a dose at or about 200-600 mg. In another embodiment, the composition is administered twice daily at a dose at or about 200-400 mg. In another embodiment, the composition is administered twice daily at a dose at or about 200-400 mg intermittently, for example three, four or five days per week. In one embodiment, the daily dose is 200 mg which can be administered once-daily, twice- daily or three-times daily. In one embodiment, the daily dose is 300 mg which can be administered once-daily, twice-daily or three-times daily. In one embodiment, the daily dose is 400 mg which can be administered once-daily, twice-daily, or three-times daily.

SAHA or any one of the HDAC inhibitors can be administered in accordance with any dose and dosing schedule that, together with the effect of the anti-cancer agent, achieves a dose effective to treat cancer. The HDAC inhibitors can be administered in a total daily dose that may vary from patient to patient, and may be administered at varying dosage schedules. For example, SAHA or any of the HDAC inhibitors can be administered to the patient at a total daily dosage of between 25-4000 mg/m². In particular, SAHA or any one of the HDAC inhibitors can be administered in a total daily dose of up to 800 mg, especially by oral administration, once, twice, or three times daily, continuously (every day) or intermittently (e.g., 3-5 days a week). In addition, the administration can be continuous, i.e., every day, or intermittently.

A particular treatment protocol comprises continuous administration (i.e., every day), once, twice or three times daily at a total daily dose in the range at or about 200 mg to at or about 600 mg. Another treatment protocol comprises intermittent administration of between three to five days a week, once, twice, or three times daily at a total daily dose in the range at or about 200 mg to at or about 600 mg.

In one particular embodiment, the HDAC inhibitor is administered continuously once daily at a dose of 400 mg or twice daily at a dose of 200 mg. In another particular embodiment, the HDAC inhibitor is administered intermittently three days a week, once daily at a dose of 400 mg or twice daily at a dose of 200 mg. In another particular embodiment, the HDAC inhibitor is administered intermittently four days a week, once daily at a dose of 400 mg or twice daily at a dose of 200 mg. In another particular embodiment, the HDAC inhibitor is administered intermittently five days a week, once daily at a dose of 400 mg or twice daily at a dose of 200 mg.

In one particular embodiment, the HDAC inhibitor is administered continuously once daily at a dose of 600 mg, twice daily at a dose of 300 mg, or three times daily at a dose of 200 mg. In another particular embodiment, the HDAC inhibitor is administered intermittently three days a week, once daily at a dose of 600 mg, twice daily at a dose of 300 mg, or three times daily at a dose of 200 mg. In another particular embodiment, the HDAC inhibitor is administered intermittently four days a week, once daily at a dose of 600 mg, twice daily at a dose of 300 mg, or three times daily at a dose of 200 mg. In another particular embodiment, the HDAC inhibitor is administered intermittently five days a week, once daily at a dose of 600 mg, twice daily at a dose of 300 mg, or three times daily at a dose of200 mg.

In addition, the HDAC inhibitor may be administered according to any of the schedules described above, consecutively for a few weeks, followed by a rest period. For example, the HDAC inhibitor may be administered according to any one of the schedules described above from two to eight weeks, followed by a rest period of one week, e.g., for administration twice daily at a dose of 300 mg for three to five days a week. In another particular embodiment, the HDAC inhibitor is administered three times daily for two consecutive weeks, followed by one week of rest.

In one embodiment, the composition is administered continuously (i.e., daily) or intermittently (e.g., at least 3 days per week) with a once daily dose at or about 300 mg, at or about 400 mg, at or about 500 mg, at or about 600 mg, at or about 700 mg, or at or about 800 mg. hi another embodiment, the composition is administered once daily at a dose at or about 300 mg, at or about 400 mg, at or about 500 mg, at or about 600 mg, at or about 700 mg, or at or about 800 mg for at least one period of 7 out of 21 days (e.g., 7 consecutive days or Days 1-7 in a 21 day cycle). hi another embodiment, the composition is administered once daily at a dose at or about 400 mg, at or about 500 mg, or at or about 600 mg for at least one period of 14 out of 21 days (e.g., 14 consecutive days or Days 1-14 in a 21 day cycle).

In another embodiment, the composition is administered once daily at a dose at or about 300 mg or at or about 400 mg for at least one period of 14 out of 28 days (e.g., 14 consecutive days or Days 1-I4 of a 28 day cycle). m another embodiment, the composition is administered once daily at a dose at or about 400 mg, for example, for at least one period of 21 out of 28 days (e.g., 21 consecutive days or Days 1-21 in a 28 day cycle). In another embodiment, the composition is administered continuously (i.e., daily) or intermittently (e.g., at least 3 days per week) with a twice daily dose at or about 200 mg, at or about 250 mg, at or about 300 mg, or at or about 400 mg (per dose). In another embodiment, the composition is administered twice daily at a dose at or about 200 mg, at or about 250 mg, or at or about 300 mg (per dose) for at least one period of

3 out of 7 days (e.g., 3 consecutive days with dosage followed by 4 consecutive days without dosage). In another embodiment, the composition is administered twice daily at a dose at or about 200 mg, at or about 250 mg, or at or about 300 mg (per dose) for at least one period of

4 out of 7 days (e.g., 4 consecutive days with dosage followed by 3 consecutive days without dosage).

In another embodiment, the composition is administered twice daily at a dose at or about 200 mg, at or about 250 mg, or at or about 300 mg (per dose) for at least one period of

5 out of 7 days (e.g., 5 consecutive days with dosage followed by 2 consecutive days without dosage).

In another embodiment, the composition is administered twice daily at a dose at or about 200 mg, at or about 250 mg, or at or about 300 mg (per dose) for at least one period of 3 out of 7 days in a cycle of 21 days (e.g., 3 consecutive days or Days 1-3 for up to 3 weeks in a 21 day cycle).

3 out of 7 days in a cycle of 28 days (e.g., 3 consecutive days or Days 1-3 for up to 4 weeks in a 28 day cycle).

4 out of 7 days in a cycle of 21 days (e.g., 4 consecutive days or Days 1-4 for up to 3 weeks in a 21 day cycle). In another embodiment, the composition is administered twice daily at a dose at or about 200 mg, at or about 250 mg, or at or about 300 mg (per dose) for at least one period of

5 out of 7 days in a cycle of 21 days (e.g., 5 consecutive days or Days 1-5 for up to 3 weeks in a 21 day cycle).

In another embodiment, the composition is administered twice daily at a dose at or about 200 mg, at or about 250 mg, or at or about 300 mg (per dose), for example, for one period of 3 out of 7 days in a cycle of 21 days (e.g., 3 consecutive days or Days 1-3 in a 21 day cycle). In another embodiment, the composition is administered twice daily at a dose at or about 200 mg, at or about 250 mg, or at or about 300 mg (per dose), for example, for at least two periods of 3 out of 7 days in a cycle of 21 days (e.g., 3 consecutive days or Days 1-3 and Days 8-10 for Week 1 and Week 2 of a 21 day cycle). In another embodiment, the composition is administered twice daily at a dose at or about 200 mg, at or about 250 mg, or at or about 300 mg (per dose), for example, for at least three periods of 3 out of 7 days in a cycle of 21 days (e.g., 3 consecutive days or Days 1-3, Days 8-10, and Days 15-17 for Week 1, Week 2, and Week 3 of a 21 day cycle).

In another embodiment, the composition is administered twice daily at a dose at or about 200 mg, at or about 250 mg, or at or about 300 mg (per dose) for at least four periods of 3 out of 7 days in a cycle of 28 days (e.g., 3 consecutive days or Days 1-3, Days 8-10, Days 15-17, and Days 22-24 for Week 1, Week 2, Week 3, and Week 4 in a 28 day cycle). In another embodiment, the composition is administered twice daily at a dose at or about 100 mg, at or about 200 mg, at or about 300 mg, or at or about 400 mg (per dose), for example, for at least one period of 7 out of 14 days (e.g., 7 consecutive days or Days 1-7 in a 14 day cycle).

In another embodiment, the composition is administered twice daily at a dose at or about 200 mg, at or about 300 mg, or at or about 400 mg (per dose), for example, for at least one period of 11 out of 21 days (e.g., 11 consecutive days or Days 1-11 in a 21 day cycle). In another embodiment, the composition is administered once daily at a dose at or about 200 mg, at or about 300 mg, or at or about 400 mg (per dose), for example, for at least one period of 10 out of 21 days (e.g., 10 consecutive days or Days 1-10 in a 21 day cycle), hi another embodiment, the composition is administered twice daily at a dose at or about 200 mg, at or about 300 mg, or at or about 400 mg (per dose), for example, for at least one period of 10 out of 21 days (e.g., 10 consecutive days or Days 1-10 in a 21 day cycle), hi another embodiment, the composition is administered twice daily at a dose at or about 200 mg, at or about 300 mg, or at or about 400 mg (per dose), for example, for at least one period of 14 out of 21 days (e.g., 14 consecutive days or Days 1-14 in a 21 day cycle).

Intravenously or subcutaneously, the patient can receive the HDAC inhibitor in quantities sufficient to deliver at or about 3-1500 mg/m² per day, for example, at or about 3, 30, 60, 90, 180, 300, 600, 900, 1200 or 1500 mg/m² per day. Such quantities maybe administered in a number of suitable ways, e.g. large volumes of low concentrations of HDAC inhibitor during one extended period of time or several times a day. The quantities can be administered for one or more consecutive days, intermittent days, or a combination thereof per week (7 day period). Alternatively, low volumes of high concentrations of HDAC inhibitor during a short period of time, e.g. once a day for one or more days either consecutively, intermittently, or a combination thereof per week (7 day period). For example, a dose of 300 mg/m per day can be administered for 5 consecutive days for a total of 1500 mg/m² per treatment. In another dosing regimen, the number of consecutive days can also be 5, with treatment lasting for 2 or 3 consecutive weeks for a total of 3000 mg/m² and 4500 mg/m² total treatment.

Typically, an intravenous formulation may be prepared which contains a concentration of HDAC inhibitor at or about 1.0 mg/mL to at or about 10 mg/mL, e.g. 2.0 mg/mL, 3.0 mg/mL, 4.0 mg/mL, 5.0 mg/mL, 6.0 mg/mL, 7.0 mg/mL, 8.0 mg/mL, 9.0 mg/mL and 10 mg/mL and administered in amounts to achieve the doses described above. In one example, a sufficient volume of intravenous formulation can be administered to a patient in a day such that the total dose for the day is at or about 300 to at or about 1500 mg/m². Subcutaneous formulations can be prepared according to procedures well known in the art at a pH in the range between about 5 and about 12, which include suitable buffers and isotonicity agents, as described below. They can be formulated to deliver a daily dose of HDAC inhibitor in one or more daily subcutaneous administrations, e.g., one, two or three times each day. The HDAC inhibitors can also be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, or course, be continuous rather than intermittent throughout the dosage regime. It is apparent to a person skilled in the art that any one or more of the specific dosages and dosage schedules of the HDAC inhibitors are also applicable to any one or more of the anti-cancer agents to be used in the combination treatment. Moreover, the specific dosage and dosage schedule of the anti-cancer agent can further vary, and the optimal dose, dosing schedule, and route of administration can be determined based upon the specific anti-cancer agent that is being used. Further, the various modes of administration, dosages, and dosing schedules described herein merely set forth specific embodiments and should not be construed as limiting the broad scope of the invention. Any permutations, variations, and combinations of the dosages and dosing schedules are included within the scope of the present invention.

Administration of Anti-Cancer Agents Any of the specific dosages and dosage schedules of the HDAC inhibitors is also applicable to any of the anti-cancer agents to be used in the combination treatment. Moreover, the specific dosage and dosage schedule of the anti-cancer agent can further vary, and the optimal dose, dosing schedule, and route of administration can be determined based upon the specific anti-cancer agent that is being used. Of course, the route of administration of SAHA or any one of the other HDAC inhibitors can be independent of the route of administration of the anti-cancer agent. A particular route of administration for SAHA is oral administration. Thus, in accordance with this embodiment, SAHA is administered orally, and the other anti-cancer agent can be administered orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery by catheter or stent, subcutaneously, intraadiposally, intraarticularly, intrathecally, or in a slow release (e.g., sustained release) dosage form.

In addition, the HDAC inhibitor and anti-cancer agent may be administered by the same mode of administration, i.e., both agents can be administered orally, by IV, etc. However, it is also within the scope of the present invention to administer the HDAC inhibitor by one mode of administration, e.g. oral, and to administer the anti-cancer agent by another mode of administration, e.g. IV, or by any one or more other administration modes described herein.

Commonly used anti-cancer agents and daily dosages usually administered include but are not restricted to:

Antimetabolites: Methotrexate: 20-40 mg/m² i.v.

Methotrexate: 4-6 mg/m² p.o.

Methotrexate: 12000 mg/m² high dose therapy

6-Mercaptopurine: 100 mg/m²

6- Thioguanine: 1-2 x 80 mg/m² p.o.

Pentostatin 4 mg/m² i.v.

Fludarabinphosphate: 25 nig/m i.v.

Cladribine: 0.14 mg/kg BW i.v. 5-Fluorouracil 500-2600 mg/m² i.v.

Capecitabine: 1250 mg/m² p.o.

Cytarabin: 200 mg/m² i.v.

Cytarabin: 3000 mg/m² i.v. high dose therapy

Gemcitabine: 800-1250 mg/m² i.v.

Hydroxyurea: 800-4000 mg/m² p.o.

Pemetrexed 250-500 mg/m² i.v.

Antimitotic agents and Vincristine 1.5-2 mg/m i.v. Plant-derived agents: Vinblastine 4-8 mg/m² i.v.

Vindesine 2-3 mg/m² i.v.

Etoposide (VP 16) 100-200 mg/m² i.v.

Etoposide (VP 16) 100 mg p.o.

Teniposide (VM26) 20-30 mg/m² i.v.

Paclitaxel (Taxol) 175-250 mg/m² i.v.

Docetaxel (Taxotere) 100-150 mg/m² i.v.

Antibiotics: Actinomycin D 0.6 mg/m2 i.v.

Daunorubicin 45-6.0 mg/m² i.v.

Doxorubicin 45-60 mg/m² i.v.

Epirubicin 60-80 mg/m² i.v.

Idarubicin 10-12 mg/m² i.v.

Idarubicin 35-50 mg/m² p.o.

Mitoxantron 10-12 mg/m² i.v.

Bleomycin 10-15 mg/m² i.v., i.m., s.c.

Mitomycin C 10-20 mg/² i.v.

Irinotecan (CPT -ll) 350 mg/m² i.v.

Topotecan 1.5 mg/m i.v.

Alkylating Agents: Mustargen 6 mg/m² i.v.

Estramustinphosphate 150-200 mg/m² i.v.

Estramustinphosphate 480-550 mg/m² p.o.

Melphalan 8-10 mg/m² i.v.

Melphalan 15 mg/m² i.v. Chlorambucil 3-6 mg/m² i.v.

Prednimustine 40-100 mg/m² p.o.

Cyclophosphamide 750-1200 mg/m² i.v.

Cyclophosphamide 50-100 mg/m² p.o.

Ifosfamide 1500-2000 mg/m² i. v.

Trofosfamide 25-200 mg/m² p.o.

Busulfan 2-6 mg/m² p.o.

Treosulfan 5000-8000 mg/m² i.v.

Treosulfan 750-1500 mg/m² p.o.

Thiotepa 12-16 mg/m² i.v.

Carmustin (BCNU) 100 mg/m² i.v.

Lomustin (CCNU) 100-130 mg/m² p.o.

Nimustin (ACNU) 90-100 mg/m² i.v.

Dacarbazine (OTIC) 100-375 mg/m² i.v.

Procarbazine 100 mg/m² p.o.

Cisplatin 20-120 mg/m² i.v.

Carboplatin 300-400 mg/m² i.v.

Hormones, Cytokines Interferon-a 2-10 x 10⁶ IU/m² and Vitamins: Prednisone 40-100 mg/m² p.o.

Dexamethasone 8-24 mg p.o.

G-CSF 5-20 μg/kg BW s.c. all-trans Retinoic Acid 45 mg/m²

Interleukin-2 18 x l0⁶ rU/m²

GM-CSF 250 mg/m²

Erythropoietin 150 IU/kg tiw

hi particular aspects, Cetuximab (e.g., Erbitux™; hnClone Systems Inc., Branchburg, NJ/Bristol-Myers Squibb Co., Princeton, NJ) can be combined with a histone deacetylase (HDAC) inhibitor such as suberoylanilide hydroxamic acid (SAHA), and optionally, further combined with one or more anti-cancer agents such as Fluorouracil, Leucovorin, and Oxaliplatin (e.g., FOLFOX). Cetuximab can be administered at an initial dose at or about 400 mg/m² initial dose, followed by weekly doses of at or about 250 mg/m². hi other aspects, Bevacizumab (e.g., Avastin ; Genentech, Inc., San Francisco, CA) can be combined with a histone deacetylase (HDAC) inhibitor such as suberoylanilide hydroxamic acid (SAHA), and optionally, further combined with one or more anti-cancer agents such as Fluorouracil, Leucovorin, and Oxaliplatin (e.g., FOLFOX). Bevacizumab can be administered at a dose at or about 5 mg/kg, for example, as an intravenous infusion, e.g., once every 14 days.

The dosage regimens utilizing the anti-cancer agents described herein (or any pharmaceutically acceptable salts or hydrates of such agents, or any free acids, free bases, or other free forms of such agents) can follow the exemplary dosages herein, including those provided for HDAC inhibitors. The dosage can be selected in accordance with a variety of factors including type, species, age, weight, sex, and the type of disease being treated; the severity (i.e., stage) of the disease to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. A dosage regiment can be used, for example, to treat, for example, to prevent, inhibit (fully or partially), or arrest the progress of the disease. In particular embodiments, an antimetabolic agent (e.g., Fluorouracil) is administered in combination with SAHA. As an antimetabolic agent, Fluorouracil (e.g., Fluorouracil Injection or Adrucil®), Fluorouracil can be used in bolus and/or infusion regimens. Fluorouracil can be administered by bolus at a dose at or about 200 mg/m² to at or about 500 mg/m², at or about 200 mg/m², at or about 250 mg/m², at or about 300 mg/m², at or about 400 mg/m², or at or about 500 mg/m². Fluorouracil can be administered by infusion at a total dose at or about 1800 mg/m² to at or about 3000 mg/m², at or about 1800 mg/m², at or about 2000 mg/m², at or about 2200 mg/m², at or about 2400 mg/m², at or about 2600 mg/m², at or about 2800 mg/m², or at or about 3000 mg/m². In specific aspects, Fluorouracil can be administered by bolus at a dose at or about 400 mg/m², followed by an infusion at a dose at or about 2400 mg/m² over about 1-2 days, e.g., up to 46 to 48 hours. In particular aspects,

Fluorouracil is administered by bolus at a dose of up to 600 mg/m², e.g., once per week, or up to 425 mg/m², e.g., once per day, for example, for 5 days every 4-5 weeks. In additional aspects, Fluorouracil is administered by infusion at a dose of up to 2400 mg/m² to 3000 mg/m² over about 2 days (for example, 46 hours), e.g., once every 2 weeks. In other infusion regimens, Fluorouracil is administered at a dose at or about 2000 mg/m² to at or about 2600 mg/m² over about 1 day (for example, 22 hours), e.g., once per week. This regimen can be combined with Leucovorin and with or without bolus administration of Fluorouracil. In other aspects, Fluorouracil can be administered by extended infusion at a dose at or about 200 to at or about 300 mg/m²/day for monotherapy or in combination with radiation therapy. In certain embodiments, Fluorouracil for injection is administered only intravenously, using care to avoid extravasation. Dosages can be based on the patient's actual weight or body surface area. However, the estimated lean body mass (dry weight) can be used if the patient is obese or if there has been a spurious weight gain due to edema, ascites or other forms of abnormal fluid retention. As examples, patients can receive from 9 to 45 courses of treatment during periods which range from 12 to 60 months. In specific aspects, Fluorouracil can be coadministered with one or more other anti-cancer agents, e.g., SAHA, Leucovorin, and Oxaliplatin. As examples, SAHA (e.g., Vorinostat) can be administered at a total daily dose of up to 400 mg or 600 mg, and Fluorouracil can be administered at a total daily dose up to 1600 mg/m².

In particular embodiments, an alkylating agent (e.g., Oxaliplatin) is administered in combination with SAHA. As an alkylating agent, Oxaliplatin can be administered (e.g., via injection of Eloxatin®) at a dose at or about 45 mg/m² to at or about 130 mg/m², at or about 45 to at or about 55 mg/m², at or about 55 to at or about 65 mg/m², at or about 65 to at or about 75 mg/m², at or about 75 to at or about 85 mg/m², at or about 85 to at or about 95 mg/m², at or about 95 mg/m² to at or about 100 mg/m², or at or about 100 mg/m² to at or about 130 mg/m². In a particular embodiment, Oxaliplatin is administered at a dose at or about 85 mg/m², for example, by a 2-hour infusion. In particular embodiments, at or about 85 mg/m² Oxaliplatin is administered as an IV infusion in about 250-500 mL D5W (5% dextrose in water). The administration can be continued once per cycle for 1-, 2-, or 3-week cycles up to 6 months (e.g., once every 2 weeks for 12 cycles, or once every 3 weeks). More than 4, 7, or 10 cycles can be carried out. As alternate embodiments, the infusion times for Oxaliplatin can be extended to 6 hours and/or the dosage can be reduced to at or about 75 mg/m or at or about 65 mg/m . In specific aspects, Oxaliplatin can be co-administered with one or more other anti-cancer agents, e.g., SAHA, Leucovorin, and Fluorouracil. As examples, SAHA (e.g., Vorinostat) can be administered at a total daily dose of up to 400 mg or 600 mg, and Oxaliplatin can be administered at a total daily dose of up to 85 mg/m².

In particular embodiments, an adjunctive agent (e.g., Leucovorin) is administered in combination with SAHA. As examples, Leucovorin can be administered (e.g., via intravenous of Leucovorin calcium) at a dose at or about 20 mg/m to at or about 400 mg/m , at or about 20 mg/m² to at or about 50 mg/m², at or about 50 mg/m² to at or about 100 mg/m², at or about 100 mg/m² to at or about 200 mg/m², at or about 200 mg/m² to at or about 250 mg/m², at or about 250 mg/m² to at or about 400 mg/m², or at or about 400 mg/m² to at or about 500 mg/m². In particular, Leucovorin can be administered at a dose at or about 400 mg/m², e.g., by 2 hour infusion. In the presence of gastrointestinal toxicity, nausea, or vomiting, Leucovorin can be administered parenterally. Leucovorin rescue can be administered for 24 hour intervals (e.g., total 14 doses over 84 hours) in subsequent courses of therapy. In specific aspects, Leucovorin can be co-administered with one or more other anti-cancer agents, e.g., SAHA, Fluorouracil, and Oxaliplatin. As examples, SAHA (e.g., Vorinostat) can be administered at a total daily dose of up to 400 mg or 600 mg, and Leucovorin can be administered at a total daily dose of up to 400 mg/m². In a particular combination of SAHA with FOLFOX, SAHA can be administered twice daily, e.g., by mouth, at a dose at or about 100 mg, at or about 200 mg, at or about 300 mg, or at or about 400 mg (per dose), for example, for at least one treatment period of 7 out of 14 days (e.g., 7 consecutive days or Days 1-7 of a 14 day cycle). The Leucovorin component of FOLFOX can be administered by infusion at a dose at or about 400 mg/m² (e.g., over about 2 hours) for at least one treatment in a treatment period of 14 days (e.g., Day 4 of a 14 day cycle). The Oxaliplatin component of FOLFOX can be administered by infusion at a dose at or about 55 mg/m², at or about 65 mg/m², or at or about 85 mg/m² (e.g., over about 2 hours) for at least one treatment in a treatment period of 14 days (e.g., Day 4 of a 14 day cycle). The Fluorouracil component of FOLFOX can be administered by infusion, and can be preceded, optionally, by Fluorouracil bolus. The Fluorouracil bolus can be administered at a dose at or about 300 mg/m² or at or about 400 mg/m², for at least one treatment in a treatment period of 14 days (e.g., Day 4 of a 14 day cycle). The Fluorouracil infusion can be administered at a total dose at or about 1800 mg/m², at or about 2000 mg/m², or at or about 2400 mg/m² over about 2 days (e.g., 46 hours) for at least one treatment in a treatment period of 14 days (e.g., Day 4 of a 14 day cycle). FOLFOX can be administered, for example, for at least one treatment period of 14 days (e.g., Day 4 of a 14 day cycle), to include Leucovorin infusion at or about 200 mg/m² and Oxaliplatin infusion at or about 85 mg/m² over about 2 hours, followed by Fluorouracil bolus at or about 400 mg/m² and Fluorouracil infusion at or about 600 mg/m² over 46 hours. In specific combinations, SAHA can be administered up to 3 days prior to FOLFOX at a dose at or about 200 mg or at or about 300 mg twice daily, by mouth, for at least one treatment period of 7 out of 14 days (e.g., Days 1-7 of a 14 day cycle), and FOLFOX can be administered for at least one treatment period of 14 days (e.g., Day 4 of a 14 day cycle), to include Leucovorin infusion at or about 400 mg/m² and Oxaliplatin infusion at or about 85 mg/m² over about 2 hours, followed by Fluorouracil bolus at or about 400 mg/m² and Fluorouracil infusion at or about 2400 mg/m² over 46 hours. Combination Administration In accordance with the invention, HDAC inhibitors and anti-cancer agents can be used in the treatment of a wide variety of cancers, including but not limited to solid tumors (e.g., tumors of the head and neck, lung, breast, colon, colon/rectum, esophagus, prostate, bladder, rectum, brain, gastric tissue, bone, ovary, thyroid, or endometrium), hematological malignancies (e.g., leukemias, lymphomas, myelomas), adenocarcinomas (e.g., advanced or metastatic adenocarcinomas), carcinomas (e.g. bladder carcinoma, renal carcinoma, breast carcinoma, colorectal carcinoma), neuroblastoma, or melanoma. Non-limiting examples of these cancers include diffuse large B-cell lymphoma (DLBCL), T-cell lymphomas or leukemias, e.g., cutaneous T-cell lymphoma (CTCL), noncutaneous peripheral T-cell lymphoma, lymphoma associated with human T-cell lymphotrophic virus (HTLV), adult T- cell leukemia/lymphoma (ATLL), as well as acute lymphocytic leukemia, acute nonlymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, myeloma, multiple myeloma, mesothelioma, childhood solid tumors, brain neuroblastoma, retinoblastoma, glioma, Wilms' tumor, bone cancer and soft-tissue sarcomas, common solid tumors of adults such as head and neck cancers (e.g., oral, laryngeal, and esophageal), genitourinary cancers (e.g., prostate, bladder, renal, uterine, ovarian, testicular, rectal, and colon), lung cancer (e.g., small cell carcinoma and non-small cell lung carcinoma, including squamous cell carcinoma and adenocarcinoma), breast cancer, pancreatic cancer, melanoma and other skin cancers, basal cell carcinoma, metastatic skin carcinoma, squamous cell carcinoma of both ulcerating and papillary type, stomach cancer, brain cancer, liver cancer, adrenal cancer, kidney cancer, thyroid cancer, medullary carcinoma, osteosarcoma, soft-tissue sarcoma, Ewing's sarcoma, veticulum cell sarcoma, and Kaposi's sarcoma. Also included are pediatric forms of any of the cancers described herein.

In various aspects of the invention, the treatment procedures are performed sequentially in any order, simultaneously, or any combination thereof. For example, one treatment procedure, e.g., administration of an HDAC inhibitor, can take place prior to the other procedure, e.g., administration of the anti-cancer agent, or alternatively, after the treatment with the anticancer agent, at the same time as the treatment with the anticancer agent, or any combination thereof.

In one aspect of the invention, a total treatment period can be decided for the HDAC inhibitor. The anti-cancer agent can be administered prior to onset of treatment with the HDAC inhibitor or following treatment with the HDAC inhibitor. In addition, the anti-cancer agent can be administered during the period of HDAC inhibitor administration but does not need to occur over the entire HDAC inhibitor treatment period. Similarly, the HDAC inhibitor can be administered prior to onset of treatment with the anti-cancer agent or following treatment with the anti-cancer agent. In addition, the HDAC inhibitor can be administered during the period of anti-cancer agent administration but does not need to occur over the entire anti-cancer agent treatment period. Alternatively, the treatment regimen includes pre-treatment with one agent, either the HDAC inhibitor or the anti-cancer agent, followed by the addition of the other agent(s) for the duration of the treatment period.

In a particular embodiment, the combination of the HDAC inhibitor and anti-cancer agent is additive, i.e., the combination treatment regimen produces a result that is the additive effect of each constituent when it is administered alone. In accordance with this embodiment, the amount of HDAC inhibitor and the amount of the anti-cancer together constitute an effective amount to treat cancer.

In another embodiment, the combination of the HDAC inhibitor and anti-cancer agent is considered therapeutically synergistic when the combination treatment regimen produces a significantly better anti-cancer result (e.g., cell growth arrest, apoptosis, induction of differentiation, cell death) than the additive effects of each constituent when it is administered alone at a therapeutic dose. Standard statistical analysis can be employed to determine when the results are significantly better. For example, a Mann- Whitney Test or some other generally accepted statistical analysis can be employed.

In one particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one antimetabolic agent such as Fluorouracil In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one antimetabolic agent such as Fluorouracil and at least one other HDAC inhibitor. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one antimetabolic agent such as Fluorouracil and at least one alkylating agent. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one antimetabolic agent such as Fluorouracil and at least one antibiotic agent. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one antimetabolic agent such as Fluorouracil and at least one other antimetabolic agent. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one antimetabolic agent such as Fluorouracil and at least one hormonal agent. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one antimetabolic agent such as Fluorouracil and at least one plant-derived agent. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one antimetabolic agent such as Fluorouracil and at least one anti-angiogenic agent.

In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one antimetabolic agent such as Fluorouracil and at least one differentiation inducing agent. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one antimetabolic agent such as Fluorouracil and at least one cell- growth arrest inducing agent. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one antimetabolic agent such as Fluorouracil and at least one apoptosis inducing agent. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one antimetabolic agent such as Fluorouracil and at least one cytotoxic agent. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one antimetabolic agent such as Fluorouracil and at least one tyrosine kinase inhibitor. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one antimetabolic agent such as Fluorouracil and at least one adjunctive agent. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one antimetabolic agent such as Fluorouracil and at least one biologic agent.

In one particular embodiment, the HDAC inhibitor (e.g., SAHA) can be administered in combination with an alkylating agent such as Oxaliplatin. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one alkylating agent such as Oxaliplatin. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one alkylating agent such as Oxaliplatin and at least one other HDAC inhibitor. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one alkylating agent such as Oxaliplatin and at least one other alkylating agent. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one alkylating agent such as Oxaliplatin and at least one antibiotic agent. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one alkylating agent such as

Oxaliplatin and at least one other antimetabolic agent. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one alkylating agent such as Oxaliplatin and at least one hormonal agent. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) cai be administered in combination with at least one alkylating agent such as Oxaliplatin and at least one plant-derived agent. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one alkylatin « agent such as Oxaliplatin and at least one anti-angiogenic agent.

In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one alkylating agent such as

Oxaliplatin and at least one differentiation inducing agent. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one alkylating agent such as Oxaliplatin and at least one cell-growth arrest inducing agent. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one a alkylating agent such as Oxaliplatin and at least one apoptosis inducing agent, hi another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one alkylating agent such as Oxaliplatin and at least one cytotoxic agent, hi another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one alkylating agent such as Oxaliplatin and at least one tyrosine kinase inhibitor. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one alkylating agent such as Oxaliplatin and at least adjunctive agent. In another particular embodiment of the present invention, the HDAC inhibitor (e.g., SAHA) can be administered in combination with at least one alkylating agent such as Oxaliplatin and at least one biologic agent.

In the preferred embodiment of the present invention, patients receive oral SAHA twice daily on days 1-7. Patients also receive oxaliplatin IV over 2 hours and leucovorin calcium IV over 2 hours on day 4 followed by fluorouracil IV over 46 hours on days 4-5. Courses repeat every 14 days in the absence of disease progression or unacceptable toxicity. The combination therapy can act through the induction of cancer cell differentiation, cell growth arrest, and/or apoptosis. The combination of therapy is particularly advantageous, since the dosage of each agent in a combination therapy can be reduced as compared to monotherapy with the agent, while still achieving an overall anti-tumor effect.

Pharmaceutical Compositions As described above, the compositions comprising the HDAC inhibitor and/or the anticancer agent can be formulated in any dosage form suitable for oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, transbuccal, intranasal, liposomal, via inhalation, vaginal, or intraocular administration, for administration via local delivery by catheter or stent, or for subcutaneous, intraadiposal, intraarticular, intrathecal administration, or for administration in a slow release dosage form.

The HDAC inhibitor and the anti-cancer agent can be formulated in the same formulation for simultaneous administration, or they can be in two separate dosage forms, which may be administered simultaneously or sequentially as described above.

The invention also encompasses pharmaceutical compositions comprising pharmaceutically acceptable salts of the HDAC inhibitors and/or the anti-cancer agents.

Suitable pharmaceutically acceptable salts of the compounds described herein and suitable for use in the method of the invention, are conventional non-toxic salts and can include a salt with a base or an acid addition salt such as a salt with an inorganic base, for example, an alkali metal salt (e.g., lithium salt, sodium salt, potassium salt, etc.), an alkaline earth metal salt (e.g., calcium salt, magnesium salt, etc.), an ammonium salt; a salt with an organic base, for example, an organic amine salt (e.g., triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolaniine salt, dicyclohexylamine salt, N,N'- dibenzylethylenediamine salt, etc.) etc.; an inorganic acid addition salt (e.g., hydrochloride, hydrobromide, sulfate, phosphate, etc.); an organic carboxylic or sulfonic acid addition salt (e.g., formate, acetate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, etc.); a salt with a basic or acidic amino acid (e.g., arginine, aspartic acid, glutamic acid, etc.) and the like. The invention also encompasses pharmaceutical compositions comprising hydrates of the HDAC inhibitors and/or the anti-cancer agents.

In addition, this invention also encompasses pharmaceutical compositions comprising any solid or liquid physical form of SAHA or any of the other HDAC inhibitors. For example, the HDAC inhibitors can be in a crystalline form, in amorphous form, and have any particle size. The HDAC inhibitor particles may be micronized, or may be agglomerated, particulate granules, powders, oils, oily suspensions or any other form of solid or liquid physical form.

For oral administration, the pharmaceutical compositions can be liquid or solid. Suitable solid oral formulations include tablets, capsules, pills, granules, pellets, and the like. Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils, and the like.

Any inert excipient that is commonly used as a carrier or diluent may be used in the formulations of the present invention, such as for example, a gum, a starch, a sugar, a cellulosic material, an acrylate, or mixtures thereof. The compositions may further comprise a disintegrating agent and a lubricant, and in addition may comprise one or more additives selected from a binder, a buffer, a protease inhibitor, a surfactant, a solubilizing agent, a plasticizer, an emulsifier, a stabilizing agent, a viscosity increasing agent, a sweetener, a film forming agent, or any combination thereof. Furthermore, the compositions of the present invention may be in the form of controlled release or immediate release formulations. The HDAC inhibitors can be administered as active ingredients in admixture with suitable pharmaceutical diluents, excipients, or carriers (collectively referred to herein as "carrier" materials or "pharmaceutically acceptable carriers") suitably selected with respect to the intended form of administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference.

For liquid formulations, pharmaceutically acceptable carriers may be aqueous or nonaqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions, or suspensions, including saline and buffered media. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil. Solutions or suspensions can also include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.

Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Solid carriers/diluents include, but are not limited to, a gum, a starch (e.g., corn starch, pregelatinized starch), a sugar (e.g., lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g., microcrystalline cellulose), an acrylate (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

In addition, the compositions may further comprise binders (e.g., acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g., cornstarch, potato starch, alginic acid, silicon dioxide, eroscarmellose sodium, crospovidone, guar gum, sodium starch glycolate, Primogel), buffers (e.g., tris-HCI, acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g., sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., glycerol, polyethylene glycerol), a glidant (e.g., colloidal silicon dioxide), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosity increasing agents (e.g., carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g., sucrose, aspartame, citric acid), flavoring agents (e.g., peppermint, methyl salicylate, or orange flavoring), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g., colloidal silicon dioxide), plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g., ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

It is especially advantageous to formulate oral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

The preparation of pharmaceutical compositions that contain an active component is well understood in the art, for example, by mixing, granulating, or tablet-forming processes. The active therapeutic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. For oral administration, the active agents are mixed with additives customary for this purpose, such as vehicles, stabilizers, or inert diluents, and converted by customary methods into suitable forms for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic, or oily solutions and the like as detailed above.

The amount of the compound administered to the patient is less than an amount that would cause toxicity in the patient. In the certain embodiments, the amount of the compound that is administered to the patient is less than the amount that causes a concentration of the compound in the patient's plasma to equal or exceed the toxic level of the compound. In particular embodiments, the concentration of the compound in the patient's plasma is maintained at about 10 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 25 nM. hi another embodiment, the concentration of the compound in the patient's plasma is maintained at about 50 nM. hi another embodiment, the concentration of the compound in the patient's plasma is maintained at about 100 nM. hi another embodiment, the concentration of the compound in the patient's plasma is maintained at about 500 nM. hi another embodiment, the concentration of the compound in the patient's plasma is maintained at about 1,000 nM. hi another embodiment, the concentration of the compound in the patient's plasma is maintained at about 2,500 nM. hi another embodiment, the concentration of the compound in the patient's plasma is maintained at about 5,000 nM. The optimal amount of the compound that should be administered to the patient in the practice of the present invention will depend on the particular compound used and the type of cancer being treated.

The percentage of the active ingredient and various excipients in the formulations may vary. For example, the composition may comprise between 20 and 90%, or specifically between 50-70% by weight of the active agent.

For IV administration, Glucuronic acid, L-lactic acid, acetic acid, citric acid or any pharmaceutically acceptable acid/conjugate base with reasonable buffering capacity in the pH range acceptable for intravenous administration can be used as buffers. Sodium chloride solution wherein the pH has been adjusted to the desired range with either acid or base, for example, hydrochloric acid or sodium hydroxide, can also be employed. Typically, a pH range for the intravenous formulation can be in the range of from about 5 to about 12. A particular pH range for intravenous formulation comprising an HDAC inhibitor, wherein the HDAC inhibitor has a hydroxamic acid moiety, can be about 9 to about 12.

Subcutaneous formulations can be prepared according to procedures well known in the art at a pH in the range between about 5 and about 12, which include suitable buffers and isotonicity agents. They can be formulated to deliver a daily dose of the active agent in one or more daily subcutaneous administrations. The choice of appropriate buffer and pH of a formulation, depending on solubility of the HDAC inhibitor to be administered, is readily made by a person having ordinary skill in the art. Sodium chloride solution wherein the pH has been adjusted to the desired range with either acid or base, for example, hydrochloric acid or sodium hydroxide, can also be employed in the subcutaneous formulation. Typically, a pH range for the subcutaneous formulation can be in the range of from about 5 to about 12. A particular pH range for subcutaneous formulation of an HDAC inhibitor a hydroxamic acid moiety, can be about 9 to about 12. The compositions of the present invention can also be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, or course, be continuous rather than intermittent throughout the dosage regime. The present invention also provides in vitro methods for selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells, thereby inhibiting proliferation of such cells, by contacting the cells with an amount of suberoylanilide hydroxamic acid (SAHA) or a pharmaceutically acceptable salt or hydrate thereof, and an amount of an anti-cancer agent, wherein the amounts together can comprise an amount effective to induce terminal differentiation, cell growth arrest of apoptosis of the cells.

Although the methods of the present invention can be practiced in vitro, it is contemplated that a particular embodiment for the methods of selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells will comprise contacting the cells in vivo, i.e., by administering the compounds to a subject harboring neoplastic cells or tumor cells in need of treatment.

As such, the present invention also provides methods for selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells, thereby inhibiting proliferation of such cells in a subject by administering to the subject a first amount of suberoylanilide hydroxamic acid (SAHA) or a pharmaceutically acceptable salt or hydrate thereof, in a first treatment procedure, and a second amount of an anti-cancer agent in a second treatment procedure, wherein the first and second amounts together comprise an amount effective to induce terminal differentiation, cell growth arrest of apoptosis of the cells. The invention is illustrated in the examples that follow. This section is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to limit in any way the invention as set forth in the claims which follow thereafter.

EXAMPLES The examples are presented in order to more fully illustrate the various embodiments of the invention. These examples should in no way be construed as limiting the scope of the invention recited in the appended claims.

EXAMPLE l: Synthesis of SAHA SAHA can be synthesized according to the method outlined below, or according to the method set forth in US Patent 5,369,108, the contents of which are incorporated by reference in their entirety, or according to any other method.

Synthesis of SAHA Step 1 - Synthesis of Suberanilic acid

hi a 22 L flask was placed 3,500 g (20.09 moles) of suberic acid, and the acid melted with heat. The temperature was raised to 175⁰C, and then 2,040 g (21.92 moles) of aniline was added. The temperature was raised to 190°C and held at that temperature for 20 minutes. The melt was poured into a Nalgene tank that contained 4,017 g of potassium hydroxide dissolved in 50 L of water. The mixture was stirred for 20 minutes following the addition of the melt. The reaction was repeated at the same scale, and the second melt was poured into the same solution of potassium hydroxide. After the mixture was thoroughly stirred, the stirrer was turned off, and the mixture was allowed to settle.

The mixture was then filtered through a pad of Celite (4,200 g). The product was filtered to remove the neutral by-product from attack by aniline on both ends of suberic acid. The filtrate contained the salt of the product, and also the salt of unreacted suberic acid. The mixture was allowed to settle because the filtration was very slow, taking several days. The filtrate was acidified using 5 L of concentrated hydrochloric acid; the mixture was stirred for one hour, and then allowed to settle overnight. The product was collected by filtration, and washed on the funnel with deionized water (4 x 5 L). The wet filter cake was placed in a 72 L flask with 44 L of deionized water, the mixture heated to 5O⁰C, and the solid isolated by a hot filtration (the desired product was contaminated with suberic acid which is has a much greater solubility in hot water. Several hot triturations were done to remove suberic acid. The product was checked by NMR [D₆DMSO] to monitor the removal of suberic acid). The hot trituration was repeated with 44 L of water at 50°C. The product was again isolated by filtration, and rinsed with 4 L of hot water. It was dried over the weekend in a vacuum oven at 65°C using a Nash pump as the vacuum source (the Nash pump is a liquid ring pump ^' (water) and pulls a vacuum of about 29 inch of mercury. An intermittent argon purge was used to help carry off water); 4,182.8 g of suberanilic acid was obtained.

The product still contained a small amount of suberic acid; therefore the hot trituration was done portionwise at 65°C, using about 300 g of product at a time. Each • portion was filtered, and rinsed thoroughly with additional hot water (a total of about 6 L). This was repeated to purify the entire batch. This completely removed suberic acid from the product. The solid product was combined in a flask and stirred with 6 L of methanol/water (1 :2), and then isolated by filtration and air dried on the filter over the week end. It was placed in trays and dried in a vacuum oven at 65⁰C for 45 hours using the Nash pump and an argon bleed. The final product has a weight of 3,278.4 g (32.7% yield).

Step 2 -Synthesis of Methyl Suberanilate

To a 50 L flask fitted with a mechanical stirrer, and condenser was placed 3,229 g of suberanilic acid from the previous step, 20 L of methanol, and 398.7 g of Dowex 50WX2-400 resin. The mixture was heated to reflux and held at reflux for 18 hours. The mixture was filtered to remove the resin beads, and the filtrate was taken to a residue on a rotary evaporator.

The residue from the rotary evaporator was transferred into a 50 L flask fitted with a condenser and mechanical stirrer. To the flask was added 6 L of methanol, and the mixture heated to give a solution. Then 2 L of deionized water was added, and the heat turned off. The stirred mixture was allowed to cool, and then the flask was placed in an ice bath, and the mixture cooled. The solid product was isolated by filtration, and the filter cake was rinsed with 4 L of cold methanol/water (1:1). The product was dried at 45°C in a vacuum oven using a Nash pump for a total of 64 hours to give 2,850.2 g (84% yield) of methyl suberanilate.

To a 50 L flask with a mechanical stirrer, thermocouple, and inlet for inert atmosphere was added 1,451.9 g of hydroxylamine hydrochloride, 19 L of anhydrous methanol, and a 3.93 L of a 30% sodium methoxide solution in methanol. The flask was then charged with 2,748.0 g of methyl suberanilate, followed by 1.9 L of a 30% sodium methoxide solution in methanol. The mixture was allowed to stir for 16 hr and 10 minutes. Approximately one half of the reaction mixture was transferred from the reaction flask (flask 1) to a 50 L flask (flask 2) fitted with a mechanical stirrer. Then 27 L of deionized water was added to flask 1 and the mixture was stirrer for 10 minutes. The pH was taken using a pH meter; the pH was 11.56. The pH of the mixture was adjusted to 12.02 by the addition of 100 ml of the 30% sodium methoxide solution in methanol; this gave a clear solution (the reaction mixture at this time contained a small amount of solid. The pH was adjusted to give a clear solution from which the precipitation the product would be precipitated). The reaction mixture in flask 2 was diluted in the same manner; 27 L of deionized water was added, and the pH adjusted by the addition of 100 ml of a 30 % sodium methoxide solution to the mixture, to give apH of 12.01 (clear solution).

The reaction mixture in each flask was acidified by the addition of glacial acetic acid to precipitate the product. Flask 1 had a final pH of 8.98, and Flask 2 had a final pH of 8.70. The product from both flasks was isolated by filtration using a Buchner funnel and filter cloth. The filter cake was washed with 15 L of deionized water, and the funnel was covered and the product was partially dried on the funnel under vacuum for 15.5 hr. The product was removed and placed into five glass trays. The trays were placed in a vacuum oven and the product was dried to constant weight. The first drying period was for 22 hours at 60°C using a Nash pump as the vacuum source with an argon bleed. The trays were removed from the vacuum oven and weighed. The trays were returned to the oven and the product dried for an additional 4 hr and 10 minutes using an oil pump as the vacuum source and with no argon bleed. The material was packaged in double 4-mill polyethylene bags, and placed in a plastic outer container. The final weight after sampling was 2633.4 g (95.6%).

Step 4 - Recrvstallization of Crude SAHA

The crude SAHA was recrystallized from methanol/water. A 50 L flask with a mechanical stirrer, thermocouple, condenser, and inlet for inert atmosphere was charged with the crude SAHA to be crystallized (2,525.7 g), followed by 2,625 ml of deionized water and 15,755 ml of methanol. The material was heated to reflux to give a solution. Then 5,250 ml of deionized water was added to the reaction mixture. The heat was turned off, and the mixture was allowed to cool. When the mixture had cooled sufficiently so that the flask could be safely handled (28°C), the flask was removed from the heating mantle, and placed in a tub for use as a cooling bath. Ice/water was added to the tub to cool the mixture to -5°C. The mixture was held below that temperature for 2 hours. The product was isolated by filtration, and the filter cake washed with 1.5 L of cold methanol/water (2:1). The funnel was covered, and the product was partially dried under vacuum for 1.75 hr. The product was removed from the funnel and placed in 6 glass trays. The trays were placed in a vacuum oven, and the product was dried for 64.75 hr at 60°C using a Nash pump as the vacuum source, and using an argon bleed. The trays were removed for weighing, and then returned to the oven and dried for an additional 4 hours at 60°C to give a constant weight. The vacuum source for the second drying period was an oil pump, and no argon bleed was used. The material was packaged in double 4-mill polyethylene bags, and placed in a plastic outer container. The final weight after sampling was 2,540.9 g (92.5%).

In other experiments, crude SAHA was crystallized using the following conditions:

Table 1 : SAHA Crystallization Conditions

All these reaction conditions produced SAHA Polymorph I.

EXAMPLE 2: Generation of Wet-Milled Small Particles in 1:1 Ethanol/Water The SAHA Polymorph I crystals were suspended in 1 : 1 (by volume) EtOH/water solvent mixture at a slurry concentration ranging from 50 mg/gram to 150 mg/gram (crystal/solvent mixture). The slurry was wet milled with IKA- Works Rotor-Stator high shear homogenizer model T50 with superfine blades at 20-30 m/s, until the mean particle size of SAHA was less than 50 μm and 95% less than 100 μm, while maintaining the temperature at room temperature. The wet-milled slurry was filtered and washed with the 1 : 1 EtOH/water solvent mixture at room temperature. The wet cake was then dried at 4O⁰C. The final mean particle size of the wet-milled material was less than 50 μm as measured by the Microtrac method below.

Particle size was analyzed using an SRA-150 laser diffraction particle size analyzer, manufactured by Microtrac Inc. The analyzer was equipped with an ASVR (Automatic Small Volume Recirculator). 0.25 wt% lecithin in ISOPAR G was used as the dispersing fluid. Three runs were recorded for each sample and an average distribution was calculated. Particle size distribution (PSD) was analyzed as a volume distribution. The mean particle size and 95%< values based on volume were reported.

EXAMPLE 2A: Large Scale Generation of Wet-Milled Small Particles in 1:1

Ethanol/Water

56.4 kg SAHA Polymorph I crystals were charged to 610 kg (10.8 kg solvent per kg SAHA) of a 50% vol/vol solution of 200 proof punctilious ethanol and water (50/50 EtOH/Water) at 20-25°C. The slurry (~ 700 L) was recirculated through an IKA Works wet- mill set with super-fine generators until reaching a steady-state particle size distribution. The conditions were: DR3-6, 23 m/s rotor tip speed, 30-35 Lpm, 3 gen, ~ 96 turnovers (a turnover is one batch volume passed through one gen), ~ 12 hrs. Approx. M . ,i.l„l T,,ι.me _n(hr x) = 96 x Batch Volume ( ^vL)

Natural Draft of Mill (Lpni) x # of Generators x 60

The wet cake was filtered, washed 2X with water (total 6 kg/kg, ~ 340 kg) and vacuum dried at 40-45°C. The dry cake was then sieved (595 μm screen) and packed as Fine API.

EXAMPLE 3: Growth of Large Crystals of Mean Particle Size 150 urn in 1:1

EthanolAVater 25 grams of SAHA Polymorph I crystals and 388 grams of 1 : 1 Ethanol/water solvent mixture were charged into a 500 ml jacketed resin kettle with a glass agitator. The slurry was wet milled to a particle size less than 50 μm at room temperature following the steps of Example 2. The wet-milled slurry was heated to 65°C to dissolve ~ 85% of the solid. The heated slurry was aged at 65°C for 1-3 hours to establish a ~ 15 % seed bed. The slurry was mixed in the resin kettle under 20 psig pressure, and at an agitator speed range of 400-700 rpm.

The batch was then cooled slowly to 5⁰C: 65 to 55°C in 10 hours, 55 to 45⁰C in 10 hours, 45 to 5°C in 8 hours. The cooled batch was aged at 5°C for one hour to reach a target supernatant concentration of less than 5 mg/g, in particular, 3 mg/g. The batch slurry was filtered and washed with 1 : 1 EtOH/water solvent mixture at 5⁰C. The wet cake was dried at 4O⁰C under vacuum. The dry cake had a final particle size of- 150 μm with 95% particle size < 300 μm according to the Microtrac method.

EXAMPLE 4: Growth of Large Crystals with Mean Particle Size of 140 μm in 1 :1 EthanolAVater

7.5 grams of SAHA Polymorph I crystals and 70.7 grams of 1:1 EtOH/water solvent mixture were charged into a seed preparation vessel (500-ml jacketed resin kettle). The seed slurry was wet milled to a particle size less than 50 μm at room temperature following the steps of Example 2 above. The seed slurry was heated to 63-67⁰C and aged over 30 minutes to 2 hours.

In a separate crystallizer (1 -liter jacketed resin kettle), 17.5 grams of SAHA Polymorph I crystals and 317.3 grams of 1:1 EtOH/water solvent mixture were charged. The crystallizer was heated to 67-7O⁰C to dissolve all solid SAHA crystals first, and then was cooled to 60-65⁰C to keep a slightly supersaturated solution.

The seed slurry from the seed preparation vessel was transferred to the crystallizer. The slurry was mixed in the resin kettle under 20 psig pressure, and at an agitator speed range similar to that in Example 3. The batch slurry was cooled slowly to 5⁰C according to the cooling profile in Example 3. The batch slurry was filtered and washed with 1:1 EtOH/water solvent mixture at 5⁰C. The wet cake was dried at 4O⁰C under vacuum. The dry cake had a final particle size of about 140 μm with 95% particle size < 280 μm.

EXAMPLE 4A: Large Scale Growth of Large Crystals in 1:1 EthanoIAVater

21.9 kg of the Fine API dry cake from Example 2 A (30% of total) and 201 kg of 50/50 EtOH/Water solution (2.75 kg solvent/kg total SAHA) was charged to Vessel #1 - the Seed Preparation Tank. 51.1 kg of SAHA Polymorph I crystals (70% of total) and 932 kg 50/50 EtOH/Water (12.77 kg solvent/kg total SAHA) was charged to Vessel #2 - the Crystallizer. The Crystallizer was pressurized to 20-25 psig and the contents heated to 67- 70°C while maintaining the pressure to fully dissolve the crystalline SAHA. The contents were then cooled to 61-63°C to supersaturate the solution. During the aging process in the Crystallizer, the Seed Prep Tank was pressurized to 20-25 psig, the seed slurry was heated to 64°C (range: 62-66°C), aged for 30 minutes while maintaining the pressure to dissolve ~ ¹A of the seed solids, and then cooled to 61-63⁰C.

The hot seed slurry was rapidly transferred from the Seed Prep Tank to the Crystallizer (no flush) while maintaining both vessel temperatures. The nitrogen pressure in the Crystallizer was re-established to 20-25 psig and the batch was aged for 2 hours at 61- 63°C. The batch was cooled to 5°C in three linear steps over 26 hours: (1) from 62°C to 55°C over 10 hours; (2) from 55°C to 45°C over 6 hours; and (3) from 45°C to 5°C over 10 hours. The batch was aged for 1 hr and then the wet cake was filtered and washed 2X with water (total 6 kg/kg, ~ 440 kg), and vacuum dried at 40-45°C. The dry cake from this recrystallization process is packed-out as the Coarse API. Coarse API and Fine API were blended at a 70/30 ratio.

EXAMPLE 5: Generation of Wet-milled Small Particles Batch 288 SAHA Polymorph I crystals were suspended in ethanolic aqueous solution (100% ethanol to 50% ethanol in water by volume) at a slurry concentration ranging from 50 mg/gram to 150 mg/gram (crystal/solvent mixture). The slurry was wet milled with IKA- Works Rotor-Stator high shear homogenizer model T50 with superfine blades at 20-35 m/s, until the mean particle size of SAHA was less than 50 μm and 95% less than 100 μm, while maintaining the temperature at room temperature. The wet-milled slurry was filtered and 5 washed with EtOH/water solvent mixture at room temperature. The wet cake was then dried at 4O⁰C. The final mean particle size of the wet-milled material was less than 50 μm as measured by the Microtrac method as described before.

EXAMPLE 6: Growth of Large Crystals Batch 283

10_. , 24 grams of SAHA Polymorph I crystals and 205 ml of 9:1 Ethanol/water solvent mixture were charged into a 500 ml jacketed resin kettle with a glass agitator. The slurry was . wet milled to a particle size less than 50 μm at room temperature following the steps of Example 1. The wet-milled slurry was heated to 65⁰C to dissolve ~ 85% of the solid. The heated slurry was aged at 64-65⁰C for 1-3 hours to establish a ~ 15 % seed bed. The slurry 15 was mixed at an agitator speed range of 100 - 300 rpm.

The batch was then cooled to 2O⁰C with one heat-cool cycle: 65⁰C to 55⁰C in 2 hours, 55⁰C for 1 hour, 55⁰C to 65⁰C over ~ 30 minutes, age at 65⁰C for 1 hour, 65⁰C to 40⁰C in 5 hours, 40⁰C to 3O⁰C in 4 hours, 3O⁰C to 2O⁰C over 6 hours. The cooled batch was aged at , 2O⁰C for one hour. The batch slurry was filtered and washed with 9:1 EtOH/water solvent 0 mixture at 2O⁰C. The wet cake was dried at 4O⁰C under vacuum. The dry cake had a final particle size of ~ 150 μm with 95% particle size < 300 μm per Microtrac method.

30% of the batch 288 crystals and 70% of the batch 283 crystals were blended to produce capsules containing about 100 mg of suberoylanilide hydroxamic acid; about 44.3 mg of microcrystalline cellulose; about 4.5 mg of croscarmellose sodium; and about 1.2 mg 5 of magnesium stearate.

EXAMPLE 7; A Phase I Study of SAHA in Combination with FOLFOX in Advanced Colorectal Cancer (CRC)

A phase I study of oral SAHA plus FOLFOX (5-Fluorouracil, Leucovorin, and 0 Oxaliplatin) is ongoing in patients with advanced colorectal cancer (CRC) to determine the recommended dose of this combination.

This study is designed to evaluate the pharmacokinetics of SAHA (Vorinostat) when administered in combination with FOLFOX (5-Fluorouracil, Leucovorin, and Oxaliplatin) in patients with colorectal cancer. The study is also designed to determine the maximum tolerated dose (MTD) of oral SAHA when administered in combination with FOLFOX in patients with relapsed or refractory colorectal cancer. In addition, the study is designed to assess the safety and tolerability of this combination regimen and estimate response rate to SAHA among patients with colorectal cancer when administered in combination with FOLFOX.

First analysis: The pharmacokinetics profile of SAHA in the patients is evaluated for comparability to that of patients enrolled in parallel studies, to permit further evaluation. Second analysis: Administration of SAHA in combination with FOLFOX is evaluated for safety and tolerance sufficient to permit further study.

Study Design and Duration: An open-label, dose-escalating, multi-center trial is ongoing for SAHA combination therapy with FOLFOX in patients who failed first-line treatment for colorectal cancer. Patients are enrolled in 2-week treatment cycles of SAHA and FOLFOX, and treated until progression on this protocol. Patients are restaged with computed tomography (CT) every 4 cycles. If > 20% decrease is observed in sum of the greatest diameters, treatment is discontinued. Patients are evaluated for safety (laboratory tests, adverse event assessment, physical exam) and efficacy associated with the treatment. For the discontinued patients, a post-treatment follow-up visit is conducted within 4 weeks after the last study drug dose or prior to the initiation of new treatment. Patient Sample: Approximately 10 patients are enrolled, and up to 4 dose levels of

SAHA are planned. The study is designed to include a total of about 21 to 30 patients, with a minimum of 3 patients and a maximum of 6 patients enrolled at each dose level. The MTD (maximum tolerated dose) is defined as the dose preceding that at which 2 of 3 or 2 of 6 patients experience dose-limiting toxicity. Once the MTD has been established, an additional 4 patients are planned for enrollment at that MTD to study the pharmacokinetics, safety, and efficacy of the combination therapy. Eligibility criteria include: >18 years with colorectal cancer, histological diagnosis of colorectal cancer; life expectancy > 3 months; Eastern Cooperative Oncology Group (ECOG) Performance Status of 0 to 2; > 4 weeks from prior therapies; and adequate hematologic, hepatic, and renal function. Patients with following conditions are excluded: history of prior treatment with any HDAC inhibitor, gastrointestinal bleeding, gastroduodenal ulcers, HIV infection, viral hepatitis, pregnancy, or breast feeding. Dosage/Dosage Form, Route, and Dose Regimen: This is a dose-escalation study of suberoylanilide hydroxamic acid (SAHA). Patients receive treatment in cycles repeated every 2 weeks. Treatment is continued until evidence of progression, depending on the patient's tolerance of the treatment. SAHA is administered during the first 7 days of the cycle, followed by 7 days of rest. The starting dose of SAHA includes 100 mg p.o. BID (by mouth, twice daily). If no DLT (dose-limiting toxicity) is observed during the first 2 cycles, dose-escalation is continued. If one DLT is observed, 3 more patients are planned for enrollment at the same dose level. If none of the next 3 patients have DLT, dose-escalation is continued. If two or more DLT are observed at one dose level, dose-escalation is not continued. Once MTD is identified, 4 additional patients are planned for enrollment at the MTD. The escalated dose levels include 200 mg BID and 300 mg BID SAHA. An additional escalated dose level includes 400 mg BID SAHA. SAHA is administered 3 days prior to FOLFOX at the dosages set forth below.

Table 2: Summary of Treatment Cycles and Dose Levels

Pharmacokinetics Measurements: Plasma and urine samples for pharmacokinetic measurements are obtained on Day 3, Day 7, and Day 14 of the first two cycles. In addition, in the event that treatment is interrupted, additional samples are requested, at the beginning of the interruption and at the end of the interruption.

Efficacy Measurements: The efficacy assessment is focused on objective response rates (ORR), including complete response (CR) or partial response (PR), based on computed tomography (CT) scan findings. Patients are required to have one site of measurable disease defined as tumor that can be accurately measured in at least one dimension of > 2 cm by conventional CT scan or > 1 cm by PET. Other efficacy measurements include response duration, time to progression, and time to response.

Safety Measurements: Vital signs, physical examinations, ECOG performance status, electrocardiograms (ECGs), and laboratory safety tests, including CBC (complete blood count), comprehensive chemistry panel, APTT (activated partial thromboplastin time), PTYINR (prothrombin time/international normalized ratio), urinalysis, liver function, thyroid function, lipid levels, are collected prior to drug administration and at designated intervals throughout the study. To begin a cycle, ANC (absolute neutrophil count) must be > 1500/μl and platelets > 100,000/ μl. The cycle is delayed by up to 2 weeks if these values are not met on Day 1 of cycle.

Dose Modifications and Management of Toxicity: In the second part of the study, with 12 patients enrolled at MTD of SAHA, FOLFOX chemotherapy is dose reduced if certain toxicities occur.

Guideline for FOLFOX Dose Modifications: Each treatment course is started when hematologic parameters recover to neutrophils > 1500/μL and platelets > 75,000/μL. Prior to the start of the next course of treatment, the patient should show resolution of stomatitis/pharyngitis and diarrhea to Grade 1 or less. Paresthesias/dysesthesias should be Grade 2 or less and skin toxicity should be Grade 1 or less prior to the next course. CBC and chemistries are assessed on the first day of SAHA administration for each cycle to determine if the patient can proceed with treatment.

Dose modification for hematological toxicity: The table below details the dose level modifications for FOLFOX for neutropenia and thrombocytopenia.

FOLFOX dose de-escalation: In the case of any Grade 3 or 4 neutropenia or thrombocytopenia during the cycle, or any Grade 2 neutropenia or thrombocytopenia prior to (within 4 days prior to) the next scheduled FOLFOX cycle, FOLFOX is reduced by one dose level. No dose reductions are allowed below Dose Level -3.

Dose modifications for non-hematological toxicities (except neuropathy): In the case of Grade 3 or above toxicity secondary to FOLFOX, treatment is held until toxicities recover to Grade 1 or less. Any Grade 3 or above non-hematological toxicity (except neuropathy and nausea) attributed to FOLFOX requires dose reduction by one dose level. Prolonged Grade 2 toxicities such as diarrhea and mucositis may also be managed with a reduction of FOLFOX by one dose level. Dose modification for neuropathy: Only Oxaliplatin will be reduced for sensory neuropathy. Oxaliplatin dose modifications are shown in the table, below.

*Further dose reduction in Oxaliplatin to 55 mg/m² is allowed in the case of a worsening persistent Grade 2 neuropathy after first reduction or recurrent transient Grade 3 neuropathy despite first dose reduction. No dose reduction below 55 mg/m² Oxaliplatin is allowed.

Data Analysis: Summary statistics on duration, intensity, and the time to onset of toxicity by dose are used to assess the adverse effects of the combination therapy. Time to response, response duration, and time to progression are assessed. Summary statistics of PK (pharmacokinetic) parameters are determined for SAHA and FOLFOX at the MTD.

EXAMPLE 8: Preliminary Results From Phase I Study of SAHA in Combination with FOLFOX in Advanced Colorectal Cancer (CRC)

A phase I study of oral SAHA plus FOLFOX (5-Fluorouracil, Leucovorin, and Oxaliplatin) was initiated in patients with advanced colorectal cancer (CRC) to determine the recommended dose of this combination.

Methods: SAHA dosage was escalated in a standard 3+3 design. FOLFOX was administered at a fixed dose every 2 weeks. Leucovorin 400 mg/m² and Oxaliplatin 85 mg/m² were administered over 2 hours followed by 5-Fluorouracil bolus 400 mg/m² and 5- Fluorouracil infusion 2400 mg/m² over 46 hours. SAHA was started 3 days prior to FOLFOX and was given by mouth (PO) twice daily (BED) for 1 week followed by 1 week without dosage. Reduced dose levels included Leucovorin 400 mg/m² and Oxaliplatin 65 mg/m administered over 2 hours followed by 5-Fluorouracil bolus 300 mg/m and 5- Fluorouracil infusion 2000 mg/m over 46 hours. Further reduced dose levels included Leucovorin 400 mg/m² and Oxaliplatin 55 mg/m² administered over 2 hours followed by 5- Fluorouracil infusion 1800 mg/m² over 46 hours. Investigated dose levels of SAHA (BID) included 100 mg, 200 mg, and 300 mg. For particular patients, treatment cycles were continued 6 to 12 months, or longer. Tumor biopsies were obtained from all patients with an accessible liver metastasis before and on Day 4 of SAHA treatment, prior to FOLFOX, to assess thymidylate synthase (TS) expression. TS is the main target of 5-Fluorouracil. For TS analysis, mouse anti-human monoclonal antibody (TS 106 from Novus Biologicals, Littleton, CO) at 3μg/ml concentration was applied overnight at 4°C. The biotinylated secondary goat anti-mouse antibody (Jackson ImmunoResearch Labs., West Grove, PA) was applied for 30 min. The secondary detection system was used (streptavidin complex; Zymed Lab. Inc., South San Francisco, CA). Human tonsil samples were used as well-known positive (germinal centers) and negative (lymphocytes) controls.

Results: The study included 9 patients enrolled (M/F (male/female): 8/1; median age: 57, ECOG (Eastern Cooperative Oncology Group score) 0/1 : 5/4). All patients had failed prior FOLFOX, Irinotecan, and Cetuximab therapy. One patient at Dose Level 1 could not be evaluated due to rapid clinical progression. No dose-limiting toxicities were noted among the 8 evaluated patients. No Grade 3 toxicities were noted on the first cycle of treatment within 2 weeks after first FOLFOX administration, and accrual continued on Dose Level 3. Two patients at Dose Level 3 completed Cycle 1 without any dose-limiting toxicities. Cycle 1 toxicities were attributed to FOLFOX and consisted of one Grade 2 neutropenia, one Grade 2 mucositis, and two Grade 2 nausea/vomiting. Responses were evaluated in 6 patients. One patient on Dose Level 1 with peritoneal carcinomatosis had stable disease for 6 months, along with a stable CEA (carcinoembryonic antigen). Three patients at Dose Level 2 had stable disease at 2 months along with declining CEA in 2 out of 3 patients. One patient showed CEA levels of 9.4 ng/ml reduced to 5.4 ng/ml. The other patient showed CEA levels of 333 ng/ml reduced to 125 ng/ml. The decline in CEA in these two patients was confirmed by multiple blood draws. Two patients at Dose Level 1 with liver metastases biopsies showed a substantial decrease in TS expression by immunohistochemical assay after 4 days of SAHA treatment. Exemplary results from one patient are shown in FIGS. 1 A-IB. Based on the preliminary results from this study, it is concluded that SAHA at 100- 200 mg PO BED for 1 week out of 2 weeks in combination with FOLFOX is well tolerated. Even the lowest dose level of SAHA (100 mg PO BED) is associated with down-regulation of TS. Highly refractory patients are stabilized by treatment, indicating that this approach is promising and warrants future application of this regimen in the first or second-line treatment of metastatic CRC.

While this invention has been particularly shown and described with references to specific embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the meaning of the invention described. The scope of the invention encompasses the claims that follow.

Claims

WHAT IS CLAIMED IS:

1. A method of treating colorectal cancer in a subject in need thereof comprising administering to the subject: i) SAHA (suberoylanilide hydroxamic acid), represented by the structure:

or a pharmaceutically acceptable salt or hydrate thereof; ii) 5-fluoro-2,4 (\H,3H)- pyrimidinedione, or a pharmaceutically acceptable salt or hydrate thereof; iii) N-[4- [[(2-amino-5-formyl-l,4,5,6,7,8-hexahydro-4-oxo-6- pteridinyl)methyl]amino]benzoyl]-L-glutamic acid, or a pharmaceutically acceptable salt or hydrate thereof; and iv) cis-[(li?,2i?)-l,2-cyclohexanediamine-N,N'] [oxalato(2-)-O,O'J platinum, or a pharmaceutically acceptable salt or hydrate thereof; wherein the SAHA, 5-fluoro-2,4 (lH,3H)-pyrimidinedione, N-[4-[[(2-amino-5- formyl-l,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid and cis-[(li?,2i?)-l,2-cyclohexanediamine-N,N'] [oxalato(2-)-O,O'J platinum, or pharmaceutically acceptable salts or hydrates thereof, are administered in amounts effective for treating the cancer.

2. The method of claim 1, wherein the SAΗA or pharmaceutically acceptable salt or hydrate thereof is administered orally.

3. The method of claim 1, wherein the i) 5-fluoro-2,4 (lH,3H)-pyrirnidinedione; ii) N-[4- [[(2-amino-5-formyl- 1 ,4,5,6,7,8-hexahydro-4-oxo-6- pteridinyl)methyl]amino]benzoyl]-L-glutamic acid; and iii) cis-[(li?,2i?)-l,2- cyclohexanediamine-N,N'] [oxalato(2-)-O,O'] platinum, or pharmaceutically acceptable salts or hydrates thereof, are administered intravenously.

4. The method of claim 3, wherein the SAΗA or pharmaceutically acceptable salt or hydrate thereof is administered twice daily at a dose of 100 mg intermittently.

5. The method of claim 3, wherein the SAHA or pharmaceutically acceptable salt or hydrate thereof is administered twice daily at a dose of 100 mg for at least one treatment period of 7 out of 14 days.

6. The method of claim 3, wherein the SAHA or pharmaceutically acceptable salt or hydrate thereof is administered twice daily at a dose of 200 mg intermittently.

7. The method of claim 3, wherein the SAHA or pharmaceutically acceptable salt or hydrate thereof is administered twice daily at a dose of 200 mg for at least one treatment period of 7 out of 14 days.

8. The method of claim 3, wherein the SAHA or pharmaceutically acceptable salt or hydrate thereof is administered twice daily at a dose of 300 mg intermittently.

9. The method of claim 3, wherein the SAHA or pharmaceutically acceptable salt or hydrate thereof is administered twice daily at a dose of 300 mg for at least one treatment period of 7 out of 14 days.

10. The method of any one of claims 4 to 9, wherein the 5-fluoro-2,4 (\H,3H)- pyrimidinedione or pharmaceutically acceptable salt or hydrate thereof is administered by infusion at a dose of 1800 to 3000 mg/m².

11. The method of any one of claims 4 to 9, wherein the 5-fluoro-2,4 (IH,3H)- pyrimidinedione or pharmaceutically acceptable salt or hydrate thereof is administered by infusion at a dose of 2400 mg/m² over about 2 days for at least one treatment in a treatment period of 14 days.

12. The method of claim 10, wherein prior to infusion, the 5-fluoro-2,4 (IH,3H)- pyrimidinedione or pharmaceutically acceptable salt or hydrate thereof is administered by bolus at a dose of 200 to 500 mg/m².

13. The method of claim 10, wherein prior to infusion, the 5-fluoro-2,4 (1H,3H)- pyrimidinedione or pharmaceutically acceptable salt or hydrate thereof is administered by bolus at a dose of 400 mg/m².

14. The method of claims 4 to 9, wherein the N-[4-[[(2-amino-5-formyl-l,4,5,6,7,8- hexahydro-4-oxo-6-pteridinyl)methyl] amino]benzoyl] -L-glutamic acid or pharmaceutically acceptable salt or hydrate thereof is administered at a dose of 20-400 mg/m².

15. The method of claims 4 to 9, wherein the N-[4-[[(2-amino-5-formyl- 1,4,5,6,7,8- hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid or pharmaceutically acceptable salt or hydrate thereof is administered once daily at a dose of 400 mg/m for at least one treatment in a treatment period of 14 days.

16. The method of claims 4 to 9, wherein the cis-[(l i?,2i?)- 1 ,2-cyclohexanediamine-N,N'] [oxalato(2-)-O,O'] platinum or pharmaceutically acceptable salt or hydrate thereof is administered once daily at a dose of 45 to 130 mg/m².

17. The method of claims 4 to 9, wherein the cis-[(l i?,2i?)-l ,2-cyclohexanediamine-N,N'] [oxalato(2-)-O,O'] platinum or pharmaceutically acceptable salt or hydrate thereof is administered once daily at a dose of 85 mg/m² for at least one treatment in a treatment period of 14 days.

18. The method of claim 3, wherein i) the S AHA or pharmaceutically acceptable salt or hydrate thereof is administered twice daily at a dose of 300 mg for days 1-7 of a 14 day treatment cycle; ii) the 5-fluoro-2,4 (lH,3H)-pyrimidinedione or a pharmaceutically acceptable salt or hydrate thereof is administered on day 4 by bolus at 400 mg/m² and infusion at 2400 mg/m² over 46 hours ; iii) the N-[4-[[(2-amino-5- formyl-l₅4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid or a pharmaceutically acceptable salt or hydrate thereof is administered at 400 mg/m² on day 4; and iv) cis-[(lR,2i?)-l,2-cyclohexanediamine-N,N'] [oxalato(2-)- O₅O'] platinum or pharmaceutically acceptable salt or hydrate thereof is administered at 85 mg/m² on day 4.

19. The method of any one of claims 1-18, wherein: i) SAHA (suberoylanilide hydroxamic acid); ii) 5-Fluorouracil (5-fluoro-2,4 (lH,3H)-pyrimidinedione); iii) Leucovorin (L-Glutamic acid, N-[4-[[(2-amino-5-formyl-l,4,5,6,7,8-hexahydro-4~ oxo-6-pteridinyl)-methyl]amino]benzoyl]-, calcium salt); and iv) Oxaliplatin (cis- [(li?,2i?)-l,2-cyclohexanediamine-N,N'] [oxalato(2-)-O,O'] platinum) are administered.

20. A pharmaceutical composition comprising: i) SAHA (suberoylanilide hydroxamic acid), represented by the structure:

or a pharmaceutically acceptable salt or hydrate thereof; ii) 5-fluoro-2,4 (1//,3H)- pyrirnidinedione, or a pharmaceutically acceptable salt or hydrate thereof; iii) N-[4- [[(2~ammo-5-formyl-l,4,5,6,7,8-hexahydro-4-oxo-6- pteridinyl)methyl]amino]benzoyl]-L-glutamic acid, or a pharmaceutically acceptable salt or hydrate thereof; and iv) cis-[(li?,2#)-l,2-cyclohexanediamine-N,N'] [oxalato(2-)-O,O'] platinum, or a pharmaceutically acceptable salt or hydrate thereof.

21. The pharmaceutical composition of claim 20, wherein the composition is formulated for oral or intravenous administration.

22. The pharmaceutical composition of claim 20, which comprises: i) SAHA (suberoylanilide hydroxamic acid); ii) 5-Fluorouracil (5-fluoro-2,4 (IH,3H)- pyrirnidinedione); iii) Leucovorin (L-Glutamic acid, N-[4-[[(2-amino-5-formyl- l,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)-methyl]amino]benzoyl]-, calcium salt); and iv) Oxaliplatin (cis-[(li?,2i?)-l,2-cyclohexanediamine-N,N'J [oxalato(2-)-O,O'] platinum).